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Master of Science in Food Science and Biotechnology
Influence of Pathogen Contamination
on Beef Microbiota under
Different Storage Temperatures
식중독균 오염 여부와 보관 온도에 따른
소고기 마이크로바이옴 분석
February, 2020
HyeLim Choi
Department of Agricultural Biotechnology
College of Agriculture and Life Sciences
Seoul National University
석사학위논문
Influence of Pathogen Contamination
on Beef Microbiota under
Different Storage Temperatures
지도교수 최 상 호
이 논문을 석사학위논문으로 제출함
2020년 2월
서울대학교 대학원
농생명공학부
최 혜 림
최혜림의 석사학위논문을 인준함
2020년 2월
위원장 강 동 현 (인)
부위원장 최 상 호 (인)
위원 이 도 엽 (인)
I
Abstract
Outbreaks of food poisoning due to the consumption of
contaminated beef from fast-food chains are becoming more
frequent. Pathogen contamination in beef influences its spoilage as
well as the development of foodborne illness. Thus, the influence of
pathogen contamination on beef microbiota should be analyzed to
evaluate food safety. We analyzed the influence of pathogen
contamination on the shift in microbiota and the interactions
between the pathogen and indigenous microbes in beef stored under
different conditions. Sixty beef samples were stored at 25 °C and
4 °C for 24 h, and the shifts in microbiota were analyzed using the
MiSeq system. The influence of pathogen contamination on
microbiota was analyzed by artificial contamination experiments
with Escherichia coli FORC_044, which was isolated from the stool
of a food poisoning patient in Korea. The bacterial amounts and the
proportion of Escherichia were higher when the beef was stored at
25 °C. Artificially introduced Escherichia positively correlated with
the indigenous microbes such as Pseudomonas, Brochothrix,
Staphylococcus, Rahnella, and Rhizobium as determined by co-
occurrence network analyses. Carnobacterium, a potential spoilage
microbe, was negatively correlated with other microbes, including
Escherichia. The predicted functions of altered microbiota showed
that the pathways related to the process of spoilage including
II
biosynthesis of acetic acid and lactic acid increased over time. The
shift in pathways was more pronounced in contaminated beef stored
at 25 °C. Carnobacterium, Lactobacillus, and Escherichia were the
main genera contributing to the shift in the relative abundance of
functional genes involved in the various spoilage pathways. Our
results indicated that pathogen contamination could influence beef
microbiota and mediate spoilage. This study extends our
understanding of the beef microbiota and provides insights into the
role of pathogen and storage conditions in meat spoilage.
Keywords: Metagenomics, Microbiota, Beef, Spoilage
microorganism, Escherichia coli, Contamination, Microbial
interactions, Food safety
Student Number: 2018-22519
III
Contents
Abstract∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙Ⅰ
Contents∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙Ⅲ
List of Figures∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙Ⅴ
List of Tables∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙Ⅵ
Ⅰ. INTRODUCTION∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1
Ⅱ. MATERIALS AND METHODS∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙4
Sample preparation and artificial Escherichia coli contamination∙∙∙∙∙∙4
Metagenomic DNA extraction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙6
Quantitative real-time polymerase chain reaction (PCR) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙7
MiSeq sequencing∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙9
Sequence data analysis∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙10
Ⅲ. RESULTS AND DISCUSSION∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙15
Comparison of bacterial amounts between samples∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙15
IV
Comparison of Shannon diversity index between samples∙∙∙∙∙∙∙∙∙∙∙∙∙∙17
Shift in beef microbiota contaminated with E. coli under different
storage conditions∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙25
Comparison of co-occurrence networks∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙31
Shifts in predicted pathways in the microbiota under different
storage conditions∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙37
Shifts in predicted functional genes in the microbiota under different
storage conditions∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙43
OTU contribution to the shift in functional genes∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙50
Validation of OTU contribution using quantitative real-time PCR∙∙56
Ⅳ. CONCLUSIONS∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙61
Ⅴ. REFERENCES∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙63
Ⅵ. 국문초록∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙71
V
List of Figures
Figure 1. Comparison of bacterial cell numbers among samples∙∙∙∙∙16
Figure 2. Comparison of Shannon diversity index among samples∙∙24
Figure 3. Shift in beef microbiota composition∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙29
Figure 4. Co-occurrence network of microbiota in beef samples
following experimental contamination with E. coli and storage under
different conditions∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙34
Figure 5. Shifts in predicted pathways in beef microbiota under
different storage conditions∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙40
Figure 6. Shifts in predicted functional genes of microbiota under
different storage conditions∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙44
Figure 7. OTUs contributing to the shift in functional genes∙∙∙∙∙∙∙∙∙∙∙52
Figure 8. Validation of OTUs contributing to the shift in predicted
functional genes using quantitative real-time PCR∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙59
VI
List of Tables
Table 1. ANI score between E. coli FORC_044 and other EHEC
strains∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙12
Table 2. Primers used in this study∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙13
Table 3. Summary of diversity indices obtained from Illumina
Miseq∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙18
VII
1
Ⅰ. INTRODUCTION
Beef is one of the most popular meats and is consumed in
large quantities around the world. According to the Organization for
Economic Cooperation and Development (OECD) Agriculture
Statistics, the United States consumes over 27 kilograms of beef
per capita, ranking it second in the world, and South Korea
consumes over 10 kilograms of beef per capita, ranking it the 16th
(OECD/FAO, 2018) in the world. Outbreaks of food poisoning in the
US due to the consumption of contaminated beef has been reported
several times since the outbreak of Escherichia coli O157:H7 in
1982 (Rangel, J. M., 2005). E. coli O157:H7, Shiga toxin-producing
E. coli (STEC), has been reported to cause food poisoning (Nataro,
J. P., & Kaper, J. B., 1998). An estimated 265,000 STEC infections
are reported each year in the US (CDC, 2018). Over 51 cases of
pathogenic E. coli outbreak and over 2,600 patients were reported
in South Korea in the past three years (Ministry of Food and Drug
Safety, 2019). Recently, a case of a young child who lost 90
percent of her kidney function due to the consumption of E. coli-
contaminated hamburgers was reported in South Korea. It was
reported that the child suffered from hemolytic uremic syndrome
(HUS), which is commonly caused by an E. coli O157 infection
(Rangel, J. M., 2005).
2
Several studies have reported the relationship between
pathogenic E. coli contamination and spoilage microorganisms in
beef products. Contamination with E. coli O157:H7 in ground beef
has been reported to be related to spoilage (Koutsoumanis, K.,
2009). This study showed a positive correlation between E. coli
O157:H7 and pseudomonads during retail storage of beef using
kinetic modeling of spoilage bacteria and exposure assessment.
They concluded that spoilage could affect the growth of pathogens
and thus should be considered as a risk factor in foods. Another
study showed the negative correlation between E. coli O157:H7 and
lactic acid bacteria after spiking E. coli in beef (Vold, L., 2000).
However, a comprehensive study to evaluate the effect of
pathogenic E. coli contamination on the indigenous beef microbiota
and its role in spoilage has not been attempted.
Ideal storage conditions are essential to prevent pathogen
contamination and spoilage of meat products. Fresh meat, including
beef, poultry, and seafood should be kept at or below 4 °C (39 °F)
according to the HACCP (Hazard Analysis and Critical Control
Points) Plan (USDA, 1999). These products are recommended to
be stored between -2 and 10 °C in Korea (Food, K., & Drug
Association., 2005). The Center for Disease Control and Prevention
(CDC) recommends that raw beef be refrigerated or frozen within 2
h of purchase and be consume within 1-2 days, even if it is stored
in the refrigerator (CDC, 2019). Meat products might be exposed to
3
higher temperatures and could get contaminated with foodborne
pathogens during transportation and delivery (Mercier, S., 2017), in
particular, during loading and unloading. Furthermore, it is difficult
to maintain the inner temperature of the transport vehicle below
4 °C throughout the distribution process. Therefore, a
comprehensive analysis of microbiota and pathogen contamination in
beef stored at different conditions is necessary to reduce the
potential risk of foodborne illnesses.
This study aimed (1) to investigate the influence of
pathogen contamination and storage temperature on the beef
microbiota, (2) to analyze microbial interaction in the beef
microbiota under different storage conditions over time after
contamination, and (3) to understand the effects of contamination
and temperature on the spoilage of meat. To evaluate the influences
and interactions of pathogens with indigenous microbes, artificial
contamination was induced using E. coli FORC_044 isolated from a
food poisoning patient. Results from this study can extend our
understanding of the influence of pathogen contamination on the
indigenous microbiota in beef and its effect on food safety.
4
Ⅱ. MATERIALS AND METHODS
Sample preparation and artificial Escherichia coli contamination
A total of 60 beef samples were collected from the
Livestock Packing Center (LPC) in Um-Seung in October 2018.
The Um-Seung LPC was selected as they are the largest supplier
of livestock (cattle) to wholesale markets, and over 25% of beef
distributed in Korea is from this LPC (Baek Jong-Ho, 2019).
Ground beef was used in this study as it is commonly used for
hamburger patties and is the most frequently consumed raw meat in
Korea. The influence of pathogen contamination on the indigenous
microbiota of beef was analyzed by artificial contamination with E.
coli FORC_044 under different storage conditions. The FORC_044
strain is an Enterohemorrhagic E. coli (EHEC) that was isolated
from a food poisoning patient by the National Culture Collection for
Pathogens (NCCP) in March 2015. The serotype of FORC_044 is
O157:H7, which has been frequently detected in numerous beef-
related food poisoning outbreaks. The sequenced genome of the
FORC_044 strain was similar to other well-known O157:H7 strains
isolated from beef, including the EDL933 strain (Perna, N. T., 2001)
in Average Nucleotide Identity (ANI) analysis (Table S1). The
FORC_044 strain was cultivated at 37 °C in Luria-Bertani (LB)
medium overnight. The number of contaminant cells was adjusted to
5
10 cell/g, which was the infective dose of EHEC reported by the
United States Department of Agriculture (USDA) (Schmid-Hempel,
2007). The FORC_044 strain was then evenly sprayed on ground
beef and homogenized thoroughly. Samples were stored in sterile
containers at 4 °C or 25 °C and collected at five different time
points (0 h, 4 h, 8 h, 12 h, and 24 h). The contaminated samples at
0 h were acquired immediately after introducing the E. coli and
before the storage process. The samples at 0 h were used to
evaluate the shift in each storage conditions with time. Since the
CDC guide recommends the consumption of beef within one day and
beef tended to decompose at 25 °C after 24 h, we analyzed the
microbiota until 24 h. Samples (25 g) collected at different time
points were mixed with 225 mL buffered peptone water (BPW).
Bacterial cells were detached from meat using a spindle
(microorganism homogenizer, Korea patent registration: 10-2010-
0034930). The samples were homogenized by rotation and
vibration using a direct drive motor in a stomach bag in the spindle.
Bacterial cells were stored at -80 °C before metagenomic DNA
extraction.
Carnobacterium divergens KCTC 3675, Lactobacillus sakei
KCTC 3603, Staphylococcus saprophyticus KCTC 3345, and E. coli
K12 W3110 were cultured as controls for quantitative real-time
PCR. C. divergens KCTC 3675 was cultivated at 37 °C in tryptic soy
6
broth (TSB) medium with 3% yeast extract, and L. sakei KCTC
3603 was cultivated at 30 °C in De Man, Rogosa, and Sharpe (MRS)
medium. S. saprophyticus KCTC 3345 was cultivated at 37 °C in
brain heart infusion (BHI) medium, and E. coli K12 W3110 was
cultivated at 37 °C in LB medium. These four strains were grown
until they attained an optical density (OD) of 1.0 at 600 nm. The
bacteria were collected by centrifugation and then stored at -80 °C
before DNA extraction.
Metagenomic DNA extraction
Metagenomic DNA was extracted from the samples using
the phenol DNA extraction method, as described in previous studies
(Lee, Lee, Chung, Choi & Kim, 2016; Naravaneni & Jamil, 2005).
Briefly, bacterial cells in 225 mL BPW were filtered through a
sterilized gauze filter and centrifuged. The pellets were dissolved in
10 mL TES buffer (10 mM Tris-HCl, pH 8.0, 1 mM
ethylenediaminetetraacetic acid (EDTA), 0.1 M NaCl), and then
centrifuged. The pellets were suspended in 400 μL TE buffer (10
mM Tris-HCl, pH 8.0, 1 mM EDTA) and then, were treated with 50
μL lysozyme solution (100 mg/mL) and 200 μL Proteinase K
mixture (140 μL 0.5 M EDTA, 20 μL 20 mg/mL Proteinase K, 40 μL
10% sodium dodecyl sulfate) and incubated for 1 h at 37 °C. After
7
that, 100 μL 5 M NaCl and 80 μL CTAB/NaCl solution were added
to the pellet and incubated for 10 min at 65 °C. One milliliter of
phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v) was added to the
pellet and mixed well followed by centrifugation at 4 °C. The upper
phase was transferred to 3 μL RNase A (100 mg/mL) and 80 μL 3
M sodium acetate solution was added. One milliliter of 100% ethanol
was then added to the mixture. It was washed again with 70%
ethanol, and the DNA pellet was resuspended in 100 μL TE buffer
and incubated at 55 °C for 1 h. The extracted metagenomic DNA
was purified using the PowerClean DNA Clean-up kit (Mo Bio
Laboratories, Carlsbad, CA, USA) and confirmed by 1% agarose gel
electrophoresis. DNA from the cultured control strains was
extracted similarly.
Quantitative real-time polymerase chain reaction (PCR)
The total amount of bacteria in the sample was determined
using quantitative real-time (qRT) PCR of the 16S rRNA genes.
The rRNA gene was amplified using the primers 340F (5′-
TCCTACGGGAGGCAGCAG-3′) and 518R (5′-
ATTACCGCGGCTGCTGG-3′) with the BioRad CFX96 Real-Time
System (Biorad, CA, United States). Triplicate reactions were
8
performed for each sample with a final volume of 20 μL,
compromising 10 μl SYBR Green Supermix (Biorad), 1 μM each
primer, and 1 μL DNA template (ten-fold diluted DNA) or distilled
water (negative control). The conditions for the reaction were as
follows: initial denaturation at 95 °C for 30 s; 40 cycles of
denaturation at 95 °C for 5 s and extension at 60 °C for 30 s; and
dissociation at 72 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s.
Standard curves were generated from parallel PCRs with serial
log-concentrations (1 × 102–1 × 108) of the copy number of the
16S rRNA from E. coli K12 w3110. Regression coefficients (r2) for
all standard curves were higher than 0.987.
The amount of contaminant E. coli FORC_044 was
determined by the expression level of the stxI gene, which encodes
the most significant virulence factor (Shiga-like toxins I) in EHEC
strains (Watterworth, L., 2005) (Table S2). Triplicate reactions of
each sample were conducted using a BioRad CFX96 Real-Time
System, as described above. Standard curves were generated from
parallel PCRs of serial log-concentrations (1 × 102–1 × 108) of the
E. coli FORC_044 strain. Regression coefficients (r2) for all
standard curves were higher than 0.995.
The expression levels of functional genes, acetate kinase
(ackA), and menaquinone-specific isochorismate synthase (menF),
9
in the samples was calculated using ackA- and menF-targeted
primers for Carnobacterium, Lactobacillus, Staphylococcus, and
Escherichia (Table S2). Triplicate reactions of each sample were
conducted as described above. Standard curves were generated
from parallel PCRs of serial log-concentrations (1 × 102–1 × 108)
for each strain. Regression coefficients (r2) for all standard curves
were higher than 0.980.
MiSeq sequencing
The extracted metagenomic DNA was amplified using
primers (targeting the V1-V3 region of the 16S rRNA gene). PCR
amplification was performed by following the protocol for preparing
a 16S metagenomic sequencing library using the MiSeq system
(Illumina, Inc., San Diego, CA, USA). Briefly, the first amplification
was performed under the following conditions – initial denaturation
at 95 °C for 3 min; 25 cycles of denaturation at 95 °C for 30 s,
annealing at 55 °C for 30 s, and extension at 72 °C for 30 s; and a
final extension at 72 °C for 5 min. The amplicons were verified by
1.5% agarose gel electrophoresis, and purification and size selection
were performed using the Agencourt AMPure XP beads (Beckman
Coulter, Indianapolis, IN, USA). The index PCR was performed
using 5 μL of the initial PCR product in a final volume of 50 μL using
10
the Nextera XT Index Kit (Illumina, Inc.). The index PCR was
performed under the following conditions – initial denaturation at
95 °C for 3 min; 8 cycles of denaturation at 95 °C for 30 s,
annealing at 55 °C for 30 s, and extension at 72 °C for 30 s; and
final extension at 72 °C for 5 min. The amplicons of each sample
were purified again using Agencourt AMPure XP beads (Beckman
Coulter). The library was quantified using a BioRad CFX96 Real-
Time System. Equimolar concentrations of each library from the
different samples were pooled and sequenced using an Illumina
MiSeq system (300 bp-paired ends) according to the
manufacturer's instructions.
Sequence data analysis
Sequences obtained from the Illumina MiSeq sequencer
were sorted by index, and low-quality sequences were removed
using the USEARCH tool (Edgar, RC, 2010). Trimmed sequences
were clustered with 97% identity using the CLC genomic
workbench (ver. 8.5.1) with the Microbial Genomics Module
(Qiagen, Redwood City, CA, USA). The representative sequence in
each cluster was classified based on their taxonomic position using
the EzTaxon-e database (Yoon, S.H. et al., 2017). Various read
numbers in samples were normalized by random sub-sampling, and
the diversity indices were calculated using MOTHUR (Schloss et al.,
11
2009). Spearman coefficient was used to evaluate the correlation
between genera, and a network was constructed using the criteria,
threshold = 0.6 with FDR <0.05. The correlation values and FDR
values were calculated using SAS software. Co-occurrence
networks were visualized using Cytoscape, and genera, with a
relative abundance <10-3 within the network, were excluded as
their amount was considered negligible. Shifts in the potential
pathways and functional genes were predicted using PICRUSt2
(Douglas, G. M., 2019). Pathways that had over 2 log2 fold change
compared to 0 h and P-value <0.05 were selected, and the shift in
predicted pathways was analyzed using heatmaps in R. STAMP
(Parks, D. H., 2014) was used for further statistical tests of
predicted functional profiles and Welch’s t-test with Benjamini-
Hochberg FDR was conducted. The differences among samples
were analyzed using Welch’s t-test in R and GraphPad. The results
with P-values or FDR values less than 0.05 were considered
statistically significant.
12
Table 1. ANI score between E. coli FORC_044 and other EHEC
strains
Strain ANI score (%)
FORC_044 -
EDL933 99.97
Sakai 99.97
Xuzhou21 99.97
TW14359 99.86
EC4115 99.84
MG1655 98.01
K-12_ER3440 98
FORC_082 97.98
FORC_081 97.94
FORC_031 97.88
FORC_042 97.82
VR50 97.71
120009 97.7
FORC_041 97.7
13
Table 2. Primers used in this study
Strain Primer
Name
Primer Sequence Length
(bp)
Tm
(℃)
GC
(%)
Product
size (bp)
Carnobacterium divergens
KCTC 3675
ackA_F TTGTCACCTAGGAAACGGCG 20 60.32 55 116
ackA_R TATCGCCAGAACGAGTTCCC 20 59.54 55
menF_F AAGCCATGCATCCAACTCCA 20 59.96 50 215
menF_R ACCGGCTACTAAGCCACAAC 20 60.04 55
Escherichia coli K12 w3110
ackA_F ATCCGGCGATCATCTTCCAC 20 59.97 55 274
ackA_R GCGGCATTTTCACCGATACC 20 59.97 55
menF_F ACCCGCAATTCTACTGGCAA 20 59.96 50 99
menF_R GAAAACGTTGTGCCTGGTCC 20 59.97 55
14
Escherichia coli FORC_044
stxI_F ACCTCACTGACGCAGTCTGTGG 22 65.9 59 350
stxI_R TCTGCCGGACACATAGAAGGAAA 23 62.9 48
Lactobacillus sakei
KCTC 3603
ackA_F CGCTACTACCAGGTGTGCCC 20 62.57 65 299
ackA_R CCCAGCCAGTGGGGTAAAAC 20 60.9 60
Staphylococcus saprophyticus
KCTC 3345
menF_F TGAATTCGGTACGCGTGGAT 20 59.83 50 139
menF_R CACAATGCCACAACCAGCAA 20 59.9 50
15
Ⅲ. RESULTS AND DISCUSSION
Comparison of bacterial amounts between samples
The amounts of total bacteria and FORC_044 strain were
estimated using qRT-PCR of 16S rRNA and stx1 gene (Fig. 1A, B).
The total amounts of bacteria in beef stored at 25 °C (non-
contaminated: 2.55 x 109 cells/g, contaminated: 3.61 x 109 cells/g)
were higher than those stored at 4 °C (non-contaminated: 1.72 x
107 cells/g, contaminated: 1.61 x 108 cells/g) (P <0.001). The
bacterial amounts in the contaminated samples were higher than
those in non-contaminated samples at both 4 °C (P <0.001) and
25 °C (P <0.01) storage conditions. Furthermore, the amounts of
contaminated FORC_044 were significantly lower in samples stored
at 4 °C (4.94 x 104 cells/g) than those stored at 25 °C (1.39 x 107
cells/g) (P <0.001). These results indicated that contamination and
storage temperature influenced the abundance of bacteria in beef.
16
(A) (B)
0 h 4 h 8 h 1 2 h 2 4 h
5
6
7
8
9
1 0
S to ra g e t im e
To
tal
am
ou
nts
of
ba
cte
ria
(lo
g1
0c
ell
/g)
** *
* * *
*
* * *
*
0 h 8 h 1 2 h 2 4 h
0
1
4
5
6
7
8
S to ra g e t im e
Th
e a
mo
un
ts o
fE
. c
oli
(lo
g1
0c
ell
/g)
** *
Figure 1. Comparison of bacterial cell numbers among samples. (A) Total bacterial amounts in beef plotted against
time under storage. (B) The amount of E. coli FORC_044 in contaminated samples over time. Total bacterial amounts
and E. coli were estimated by quantitative real-time PCR. * P <0.05, ** P <0.01, *** P <0.001.
Non-contaminated_25℃
Non-contaminated_4℃
Contaminated_4℃
Contaminated_25℃
17
Comparison of Shannon diversity index between samples
A total of 3,841,637 sequence reads after trimming were
analyzed (Table S3). The number of reads in each sample was
normalized to 12,200 by random sub-sampling. The diversity of
microbiota was compared among different conditions, and it was
significantly different after 8 h of storage (Fig. 2). The highest
diversity was observed in contaminated samples stored at 4 °C after
12 h (3.29 ± 0.29), and the lowest was detected in contaminated
samples stored at 25 °C after 8 h (1.11 ± 0.10). The diversity of
microbiota in the contaminated samples stored at 4 °C (3.19 ± 0.53
after 8 h and 3.29 ± 0.29 after 12 h) was higher than those stored
at 25 °C (1.11 ± 0.10 after 8 h and 1.24 ± 0.21 after 12 h) (P <0.01
and P <0.001, respectively). The diversity of microbiota in the
non-contaminated samples stored at 4 °C (2.44 ± 0.11) was also
higher than those stored at 25 °C (1.63 ± 0.22) after 12 h storage
(P <0.01). The microbial diversity in the contaminated samples was
higher than that in the non-contaminated samples stored at 4 °C
after 12 h (P <0.01), while the microbial diversity in the non-
contaminated samples was higher than that in the contaminated
samples stored at 25 °C after 8 h (P <0.01).
18
Table 3. Summary of diversity indices obtained from Illumina Miseq
Sampling
temperature
Sampling
time Sampling group
Sample Analyzed
reads
Normalized
reads
Estimated
OTUs (Chao1)
Shannon
diversity index
4℃
0h
Non-contaminated
rc.111 61866 12,200 207.6471 1.467724
rc.112 112572 12,200 313.4286 1.502521
rc.113 62571 12,200 226 1.562666
Contaminated
r.111 60403 12,200 475.5714 2.283696
r.112 56391 12,200 496.6364 2.293656
r.113 52380 12,200 418.2857 2.448299
4h
Non-contaminated
rc.121 39339 12,200 702.0789 2.69688
rc.122 68699 12,200 590.5789 2.389502
19
rc.123 64217 12,200 3 0.012031
Contaminated
r.121 102414 12,200 330.7778 1.517648
r.122 107080 12,200 408.8485 1.569155
r.123 76081 12,200 377.2 1.477902
8h
Non-contaminated
rc.131 23660 12,200 669.6792 2.993406
rc.132 34852 12,200 680.5 2.644146
rc.133 164521 12,200 1083.886 3.405391
Contaminated
r.131 56241 12,200 973.08 3.460266
r.132 16038 12,200 592.4872 2.662675
r.133 52848 12,200 841.88 3.458732
12h Non-contaminated rc.141 83242 12,200 809.0652 2.546459
20
rc.142 41086 12,200 620.4103 2.409403
rc.143 32616 12,200 525.875 2.361775
Contaminated
r.141 26782 12,200 658.25 2.996108
r.142 117643 12,200 1149.821 3.49857
r.143 116216 12,200 1158.857 3.376143
24h
Non-contaminated
rc.151 42902 12,200 716.52 2.944889
rc.152 25194 12,200 727.2273 3.034855
rc.153 56511 12,200 643.0625 2.487126
Contaminated
r.151 97662 12,200 352.7576 2.269347
r.152 56859 12,200 4.5 0.003299
r.153 29923 12,200 4 0.001706
21
25℃
0h
Non-contaminated
rc.211 44889 12,200 562 2.361746
rc.212 36807 12,200 344.6154 2.136071
rc.213 93378 12,200 796.25 2.618868
Contaminated
r.211 91938 12,200 378.2609 1.713709
r.212 51310 12,200 264 1.724152
r.213 92954 12,200 467.129 2.009535
4h
Non-contaminated
rc.221 38082 12,200 425.5833 2.036611
rc.222 135952 12,200 620.2258 2.430011
rc.223 76605 12,200 370.5 2.260002
Contaminated
r.221 34112 12,200 369 1.884932
r.222 94346 12,200 573.0638 2.535436
22
r.223 73598 12,200 591.2273 2.015475
8h
Non-contaminated
rc.231 20437 12,200 603.4 2.752212
rc.232 33458 12,200 592.7885 2.633175
rc.233 54662 12,200 972.2581 3.021465
Contaminated
r.231 25828 12,200 160.3529 1.031955
r.232 51543 12,200 203.5357 1.213556
r.233 34027 12,200 187.05 1.088009
12h
Non-contaminated
rc.241 112554 12,200 331.5357 1.501244
rc.242 109640 12,200 323.5625 1.544121
rc.243 37017 12,200 324.5 1.847059
Contaminated r.241 66237 12,200 264.0909 1.249603
23
r.242 137229 12,200 311.4 1.449259
r.243 50744 12,200 170 1.029922
24h
Non-contaminated
rc.251 39604 12,200 496.7143 2.252539
rc.252 28728 12,200 482.9333 2.673846
rc.253 43855 12,200 209.3529 1.963034
Contaminated
r.251 97990 12,200 310.619 2.140346
r.252 47175 12,200 229 2.213802
r.253 48129 12,200 262.9375 2.069714
24
0 h 4 h 8 h 1 2 h 2 4 h
0
1
2
3
4
S to ra g e t im e
Sh
an
no
n d
ive
rsit
y i
nd
ex
* **** **
**
***
Figure 2. Comparison of Shannon diversity index among samples. * P <0.05, ** P <0.01, *** P <0.001.
Non-contaminated_25℃
Non-contaminated_4℃
Contaminated_4℃
Contaminated_25℃
25
Shift in beef microbiota contaminated with E. coli under different
storage conditions
The shift in beef microbiota composition at different storage
conditions was analyzed at the phylum and genus levels (Fig. 3).
Firmicutes (average 76.32% of all microbiota) and Proteobacteria
(23.47%) were the dominant phyla in all samples (Fig. 3A). The
relative abundances of Firmicutes were lower in samples stored at
4 °C than those stored at 25 °C (P <0.01), whereas Proteobacteria
were higher in samples stored at 4 °C. Carnobacterium,
Lactobacillus, Pseudomonas, and Bacillus were the dominant genera
in all samples (Fig. 3B). Carnobacterium, Lactobacillus,
Staphylococcus, Lactococcus, and Bacillus belong to Firmicutes and
have been reported as spoilage-causing bacteria (Stellato, G.,
2016).
Under 4 °C storage, the proportion of Carnobacterium
increased in the contaminated samples from 4 h to 12 h (P <0.0001,
P <0.05, and P <0.01, respectively), whereas Pseudomonas
increased in the non-contaminated samples over time and was
significantly high at 12 h (P <0.001). The proportion of Escherichia
increased after 8 h and 12 h (P <0.01) in the contaminated samples.
Under 25 °C storage, Carnobacterium was the predominant genus
over time in both non-contaminated and contaminated samples. The
proportions of Bacillus (P <0.05) and Staphylococcus (P <0.05)
26
increased, and that of Pseudomonas decreased in the non-
contaminated samples over time (P <0.05). In contrast, the
proportion of Lactobacillus (P <0.05) and Escherichia increased in
the contaminated samples.
The proportion of Carnobacterium was higher in non-
contaminated samples stored at 25 °C than those in the non-
contaminated samples stored at 4 °C, over time (P <0.01). The
proportions of Lactobacillus and Staphylococcus were higher in
non-contaminated samples stored at 25 °C compared to those in the
non-contaminated samples stored at 4 °C after 8 h. However, the
proportion of Pseudomonas was higher in the non-contaminated
samples stored at 4 °C than that in the non-contaminated samples
stored at 25 °C (P <0.01). In the contaminated samples,
Carnobacterium was the dominant genus over time under both 4 °C
and 25 °C storage. However, the proportion of Carnobacterium
decreased at 4 °C after 24 h (P <0.01), and Pseudomonas and
Bacillus were dominant in these samples. The proportion of
Lactobacillus in samples stored at 25 °C was also higher than those
stored at 4 °C. The proportions of Pseudomonas and Escherichia
were higher in samples stored at 4 °C than those stored at 25 °C (P
<0.05). Although the proportion of Escherichia was higher in the
27
contaminated samples stored at 4 °C than that in samples stored at
25 °C, the cell numbers of Escherichia were higher in samples at
25 °C than those at 4 °C (Fig. 1B). This could be due to the higher
amounts of total bacteria in samples at 25 °C.
Carnobacterium, which is known to be potential spoilage
bacteria in chilled meat products, was the most abundant genus in
all samples. Another dominant genus, Pseudomonas, gradually
increased its proportion when stored at 4 °C (non-contaminated:
47.3%, contaminated: 30.8%), while its proportion decreased when
stored at 25 °C (non-contaminated: 2.2%, contaminated: 0.1%).
Pseudomonas spp. are also known to cause spoilage in beef as they
have proteolytic properties even at low temperatures and cause
undesirable changes (Jay, J. M., 1967). Lactobacillus was also
detected in all samples; Carnobacterium and Lactobacillus are the
frequently found lactic acid bacteria (LAB) in meat products
(Leisner, J. J. et al., 2007; Stiles, M. E., 1996; Zagorec, M. et al.,
2017). Escherichia and Rahnella of the Enterobacteriaceae family
were detected at relatively low proportions in all samples.
Enterobacteriaceae are widespread in the environment, and many
mesophilic species contaminate food in low numbers (Lindberg, A.
M. et al., 1998). Carnobacterium, Pseudomonas spp., Lactobacillus,
and the majority of Enterobacteriaceae are psychrotrophic bacteria
that can grow even at refrigeration temperatures. However, the
28
relative abundances of these genera were significantly higher when
the samples were stored at 25 °C and even higher with E. coli
contamination.
Due to the predominance of Carnobacterium in samples
stored at 25 °C, the microbial diversity was higher in samples
stored at 4 °C than at 25 °C (Fig. 2). Carnobacterium was the
predominant genus in all samples, but the relative abundances were
higher in samples stored at 25 °C than in samples at 4 °C. However,
the relative abundances of other genera, including Pseudomonas,
Rhizobium, Rahnella, and Photobacterium, were higher in samples
stored at 4 °C. These results indicated that the dominant genera
were overgrown in samples stored at 25 °C and that the minor
genera could be influenced by the overgrowth of the dominant
genera under these conditions. Therefore, the diversity decreased
even with an increase in the total bacterial count in samples at
25 °C.
29
(A)
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
4 C
S to ra g e t im e
Re
lati
ve
ab
un
da
nc
e
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
2 5 C
S to ra g e t im e
Re
lati
ve
ab
un
da
nc
e
other
Firmicutes
Proteobacteria
30
(B)
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
4 C
S to ra g e t im e
Re
lati
ve
ab
un
da
nc
e
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
2 5 C
S to ra g e t im e
Re
lati
ve
ab
un
da
nc
e
Figure 3. Shift in beef microbiota composition. Shifts in beef microbiota composition at the (A) phylum and (B) genus
levels following experimental contamination with E. coli and storage under different conditions.
Rhizobium Photobacterium
other
Carnobacterium
Lactobacillus
Rahnella
Escherichia
Bacillus
Staphylococcus Pseudomonas
Lactococcus
Vibrio
31
Comparison of co-occurrence networks
The correlation between microbes over storage time was
analyzed to understand the shift in microbiota under each condition.
The proportion of Escherichia was the highest in contaminated
samples at 4 °C after 8 h. Thus the correlations between microbes
and Escherichia were determined at 8 h in both 4 °C and 25 °C
conditions (Fig. 4). The co-occurrence network showed that the
dominant bacteria in beef microbiota coexist and interact with each
other. The predominant genus, Carnobacterium, was negatively
correlated with genera whose proportions were decreased over
time in non-contaminated samples at both 4 °C and 25 °C and
contaminated samples stored at 4 °C. This suggested that
Carnobacterium could be a critical microbe in the shift in microbiota
over time.
Escherichia was present at 0 h (0.05% of microbiota) in
non-contaminated samples stored at 4 °C and was positively
correlated with Brochothrix, Rhizobium, and Pseudomonas after 4 h
(Fig. 4A, E). However, it was negatively correlated with
Carnobacterium, which was the predominant genus in beef
microbiota; and the proportion of Escherichia decreased after 8 h
(0.5% at 8 h and 0.065% at 24 h). Contaminated Escherichia was
positively correlated with Citrobacter and Pseudocitrobacter and
negatively correlated with Carnobacterium which can be related to
32
the decreased proportion of Escherichia after 8 h at 4 °C (Fig. 4B).
Carnobacterium was positively correlated with Staphylococcus and
negatively correlated with Pseudomonas in non-contaminated
samples stored at 25 °C (Fig. 4C). Thus, the relative abundance of
Staphylococcus increased after 8 h, and that of Pseudomonas
decreased after 8 h (Fig. 3B). Only one negative correlation –
between Staphylococcus and Rahnella – was significant in the
contaminated samples stored at 25 °C (Fig. 4D). Contaminated
Escherichia was not significantly correlated with indigenous
microbes at 25 °C after 8 h, but it was positively correlated with
Pseudomonas after 24 h (Fig. 4F).
These results indicated that the artificially introduced
Escherichia interacted with the dominant Carnobacterium at 4 °C,
and with Pseudomonas at 25 °C with time. These correlations
between Escherichia and other genera are consistent with previous
studies (Koutsoumanis, K., 2009 and Vold, L., 2000). Furthermore,
since Carnobacterium influenced the growth of Escherichia at 4 °C,
it may be assumed that the indigenous microbiota of beef could
influence microbial contamination. Besides, the significant increase
in Escherichia at 25 °C without significant interactions until 24 h
indicates that temperature is also a key factor for its growth in
33
addition to the interactions between microbes. The correlations
between microbes were different at each time point; and, the
composition of the microbiota changed with storage time.
34
(A) 4℃_NC_8h
(C) 25℃_NC_8h (D) 25℃_C_8h
(B) 4℃_C_8h
Proteobacteria
Firmicutes
positive negative
Bacteroidetes
Actinobacteria
35
(E) 4℃_NC_4h (F) 25℃_C_24h
36
Figure 4. Co-occurrence network of microbiota in beef samples following experimental contamination with E. coli and
storage under different conditions. Networks for (A) non-contaminated samples and (B) contaminated samples
storage at 4 °C after 8 h. Networks for (C) non-contaminated samples and (D) contaminated samples storage at
25 °C after 8 h. Spearman coefficient was used to evaluate the correlation between genera (> 0.1% in microbiota),
and the network was constructed using the criteria, threshold = 0.6; Q-value < 0.05. Green line indicates positive
correlation, and red line indicates negative correlation. Circle size represents the proportion of each genus. NC: Non-
contaminated samples, C: Contaminated samples.
37
Shifts in predicted pathways in the microbiota under different storage
conditions
The shifts in microbiota could be related to the different
roles of microbiota under different storage conditions. Thus,
functions of microbiota were predicted using the PICRUSt2 program
and compared among different conditions. A total of 392 pathways
were predicted, and the significantly changed pathways compared to
the 0 h samples were analyzed using heatmaps (Fig. 5A). The
changes in the predicted functions were greater in samples stored
at 25 °C than those at 4 °C, and changes were more significant in
contaminated samples than non-contaminated samples. The
changes in the predicted pathways were more significant in
contaminated samples stored at 4 °C after 4 h, but the changes were
relatively decreased with increasing storage times. However, the
changes in predicted pathways increased over time in the
contaminated samples stored at 25 °C. Moreover, the non-
contaminated samples stored at 4 °C showed lower activation levels
of pathways that were activated under other conditions. This
suggests that temperature and contamination affected the microbial
functions in the beef microbiota.
Twenty pathways were categorized into five major
metabolic pathways (spoilage metabolism, ubiquinone biosynthesis,
nucleotide metabolism, allantoin degradation, and amino acid
38
metabolism), according to the MetaCyc database (Fig. 5A). Six
pathways were grouped under spoilage metabolism, and these were
mainly related to the biosynthesis of acetate and lactate. Four
pathways were involved in ubiquinone biosynthesis; three pathways,
methionine cycle, L-isoleucine biosynthesis, and L-lysine
biosynthesis, were grouped into amino acid metabolism; and five
pathways were grouped under nucleotide metabolism. Two
pathways, which were related to degrading allantoin to CO2 and
glyoxylate, were grouped under allantoin degradation.
Predicted pathways related to spoilage metabolism were
associated with degrading the carbon source to acetic acid and
lactic acid. Acetic acid and lactic acid are common metabolites that
cause off-odor in spoiled beef (Gram, L. et al., 2002; Dainty, R. H.,
1996; Borch, E. et al., 1996). Ubiquinone is an electron transporter
which is essential for the survival of facultative gram-positive
anaerobes and facultative gram-negative anaerobes (Bentley, R., &
Meganathan, R., 1982; Jiang, M. et al., 2007). Allantoin, which is
synthesized by the degradation of nucleic acids, is a marker for
bacterial protein synthesis as it is degraded and recycled as a
nitrogen source (Lamothe, M. et al., 2002; Cusa, E. et al., 1999).
Consequently, nucleotide metabolism and amino acid metabolism
increased along with allantoin degradation. Hence, these shifts in
the predicted pathways and functional genes indicate that spoilage
bacteria grew and survived over time.
39
The changes in the predicted pathways in the samples under
all conditions were significant after 12 h. Therefore, the mean
proportion (%) was determined and compared between samples at 0
h and 12 h under each storage condition (Fig. 5B). The mean
proportions of the twenty pathways increased in all samples after
12 h except in the non-contaminated samples stored at 4 °C. The
differences in the predicted pathways were higher in samples
stored at 25 °C than those stored at 4 °C, and higher in the
contaminated samples than in the non-contaminated samples. The
adenine and adenosine salvage III pathway showed the highest
difference of mean proportions in all conditions (contaminated
samples at 25 °C: 2.37%, contaminated samples at 4 °C: 1.077%,
non-contaminated samples at 25 °C: 0.817%, non-contaminated
samples at 4 °C: 0.391%). This indicates that the microbiota
functions could be altered to a greater extent under 25 °C storage
and that refrigeration could reduce the risks caused by pathway
alteration even with pathogen contamination.
40
(A)
PWY-5837 : 2-carboxy-1,4-naphthoquinol biosynthesis
PWY-922 : mevalonate pathway I PWY-5910 : geranylgeranyldiphosphate biosynthesis I (via mevalonate)
PWY0-41 : allantoin degradation IV PWY-5705 : allantoin degradation to glyoxylate III
PWY-2941 : L-lysine biosynthesis II
PWY-6151 : S-adenosyl-L-methionine cycle I
PWY0-1296 : purine ribonucleosides degradation
PWY-6609 : adenine and adenosine salvage III
PWY-5104 : L-isoleucine biosynthesis IV
ANAGLYCOLYSIS-PWY : glycolysis III (from glucose) ANAEROFRUCAT-PWY : homolactic fermentation
PWY-5100 : pyruvate fermentation to acetate and lactate II PWY-5484 : glycolysis II (from fructose 6-phosphate)
PWY-5863 : superpathway of phylloquinol biosynthesis
PWY-621 : sucrose degradation III (sucrose invertase)
PWY-7199 : pyrimidine deoxyribonucleosides salvage
PWY0-1298 : superpathway of pyrimidine deoxyribonucleosides degradation PWY0-1297 : superpathway of purine deoxyribonucleosides degradation
P161-PWY : acetylene degradation (anaerobic) Spoilage
Metabolism
Nucleotide Metabolism
Amino acid Metabolism
Allantion Degradation
Ubiquinone Biosynthesis
25℃ NC C
4℃ NC C
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
(P-value < 0.05)
log2FoldChange
41
(B)
PWY-5863
PWY-5484 PWY-5705
PWY0-1298 PWY0-41
PWY-621 PWY-5910
PWY-922 PWY0-1297
PWY-6151
PWY-5100 PWY-5104
P161-PWY ANAEROFRUCAT-PWY
PWY-5837
ANAGLYCOLYSIS-PWY
PWY0-1296
PWY-6609 PWY-7199
PWY-2941
Mean proportion (%)
Difference in mean proportions (%)
Mean proportion (%)
Difference in mean proportions (%)
95% confidence intervals 4℃_NC 95% confidence intervals 4℃_C
95% confidence intervals 25℃_C 95% confidence intervals 25℃_NC
Mean proportion (%)
Difference in mean proportions (%)
Mean proportion (%)
Difference in mean proportions (%)
PWY-5863
PWY-5484 PWY-5705
PWY0-1298 PWY0-41
PWY-621 PWY-5910
PWY-922 PWY0-1297
PWY-6151
PWY-5100 PWY-5104
P161-PWY ANAEROFRUCAT-PWY
PWY-5837
ANAGLYCOLYSIS-PWY
PWY0-1296
PWY-6609 PWY-7199
PWY-2941 (P-value < 0.05)
log2FoldChange
12h 0h
42
Figure 5. Shifts in predicted pathways in beef microbiota under different storage conditions. (A) Pathways that had
over 2 log2 fold change compared to 0 h were selected and the shift in the predicted pathways was analyzed using a
heatmap. The pathways showing a significant change (P <0.05) are represented in colors, and the pathways without
any significant changes are shown in gray. (B) Difference in mean proportions (%) of predicted pathways between
samples stored for 0 h and 12 h under different conditions. Twenty pathways that showed a significant increase in
heatmap analysis were further analyzed using an extended error bar plot at 95% confidence intervals. Welch’s t-test
with Benjamini-Hochberg FDR was conducted (Q <0.05). The log2 fold change of samples stored for 12 h compared
to those stored for 0 h (P <0.05) are represented in colors. NC: Non-contaminated samples, C: Contaminated
samples.
43
Shifts in predicted functional genes in the microbiota under different
storage conditions
The metabolic pathways of each category were analyzed at
the functional gene level (Fig. 6). The relative abundance of the
functional genes was higher in the contaminated samples stored at
25 °C than that in other samples. These changes suggest that
pathogen contamination and relatively high temperatures had a
higher impact on microbiota functions. Only five genes, one in
spoilage metabolism and four in amino acid metabolism, decreased
over time at all four storage conditions (Fig. 6A, C).
In the S-adenosyl-L-methionine cycle I pathway (Fig. 6C),
luxS (EC:4.4.1.21), which converts S-ribosyl-L-homocysteine to
autoinducer 2 significantly increased by over 4.5 log2 fold in the
contaminated samples at 25 °C, whereas metE (EC:2.1.1.14)
decreased by over 5 log2 fold. This suggests that this pathway
increased due to the increase in the functional genes involved in the
biosynthesis of autoinducer 2. Autoinducer 2 is a molecule that is
involved in the quorum-sensing system recognized by many
different bacterial species, in particular, E. coli O157:H7. It is also
known to regulate attaching and effacing lesions (Sperandio, V. et
al., 2001; Federle, M. J., 2009). This suggests that the survival and
growth of E. coli could be related to the biosynthesis of autoinducer
2 in the contaminated samples at 25 °C.
44
(A)
(P-value < 0.05)
log2FoldChange
25℃ NC C
4℃ NC C
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
EC Number in Spoilage Metabolism
EC:2.7.2.3
EC:1.1.1.1
EC:5.4.2.11
EC:1.2.1.12
EC:5.3.1.9
EC:4.2.1.11
EC:1.2.1.10
EC:1.1.1.27
EC:4.1.2.13
EC:3.2.1.26 EC:2.7.1.4
EC:2.7.1.11
EC:5.3.1.1
EC:5.4.2.12
EC:2.7.1.40
EC:1.2.7.1
EC:2.7.2.1 EC:2.3.1.8
EC:2.7.1.2
Spoilage Metabolism (biosynthesis of acetate and lactate)
pyruvate
acetyl-CoA
EC:1.2.7.1
acetyl phosphate
acetate
(S)-lactate CO2 EC:1.1.1.27
EC:2.3.1.8
EC:2.7.2.1
acetaldehyde
ethanol EC:1.1.1.1 EC:1.2.1.10
D-glucopyranose 6-phosphate
β-D-fructofuranose 6-phophate
β-D-fructose 1,6-biphophate
EC:5.3.1.9
EC:2.7.1.11
D-glyceraldehyde-3-phosphate
EC:4.1.2.13
3-phospho-D-glyceroyl-phosphate EC:1.2.1.12
3-phospho-D-glycerate EC:2.7.2.3
2-phospho-D-glycerate
phosphoenolpyruvate
D-glucopyranose EC:2.7.1.2
glycerone phosphate
sucrose
EC:2.7.1.4
EC:2.7.1.40
EC:4.2.1.11
EC:5.4.2.12/5.4.2.11
EC:3.2.1.26
β-D-fructofuranose EC:2.7.1.4
EC:5.3.1.1
45
(B)
25℃ NC C
4℃ NC C
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
EC Number in Ubiquinone Biosynthesis
EC:2.7.4.2
EC:5.3.3.2
EC:2.7.1.36
EC:4.2.1.113
EC:2.5.1.1
EC:2.5.1.29
EC:4.1.3.36
EC:2.2.1.9 EC:5.4.4.2
EC:6.2.1.26
EC:2.3.3.10
EC:4.1.1.33
EC:2.5.1.10
EC:4.2.99.20
geranyl diphosphate
chorismate
isochorismate
2-succinyl-5-enolpyruvoyl-6-hydroxyl-3-cyclohexene-1-carboxylate
EC:5.4.4.2
EC:2.2.1.9
(1R,6R)-6-hydroxyl-2-succinylcyclohexa-2,4-diene-1-carboxylate
EC:4.2.1.113
EC:4.2.99.20
4-(2’-carboxyphenyl)-4-oxobutyryl-CoA
EC:6.2.1.26
EC:4.1.3.36
2-succinylbenzoate
1,4-dihydroxy-2-naphthoyl-CoA
isopentenyl diphosphate
acetoacetyl-CoA
(S)-3-hydroxyl-3-methylglutaryl-CoA
EC:2.3.3.10
(R)-mevalonate EC:1.1.1.34
(R)-5-phosphomevalonate
EC:2.7.1.36
(R)-mevalonate diphosphate
EC:2.7.4.2
EC:4.1.1.33
prenyl diphosphate EC:5.3.3.2
(2E,6E)-farnesyl diphosphate
geranylgeranyl diphosphate
EC:2.5.1.1
EC:2.5.1.10
EC:2.5.1.29
Ubiquinone biosynthesis
(P-value < 0.05)
log2FoldChange
46
(C)
(P-value < 0.05)
log2FoldChange
2-oxobutanoate
(S)-2-aceto-2-hydroxybutanoate
(R)-2,3-dihydroxy-3-methylpentanoate
(S)-3-methyl-2-oxopentanoate
L-isoleucine
propanoate
propanoyl-CoA EC:6.2.1.17
EC:1.2.7.7
EC:2.2.1.6
EC:1.1.1.383
EC:4.2.1.9
EC:2.6.1.42
L-asparate
L-aspartyl-4-phosphate
L-aspartate 4-semialdehyde
EC:2.7.2.4
EC:1.2.1.11
(2S,4S)-4-hydroxyl-2,3,4,5-tetrahydrodipicolinate
EC:4.3.3.7
(S)-2,3,4,5-tetrahydrodipicolinate EC:1.17.1.8
L-2-acetamido-6-oxoheptanedioate
EC:2.3.1.89
N-acetyl-L,L-2,6-diaminopimelate
L,L-diaminopimelate EC:3.5.1.47
meso-diaminopimelate EC:5.1.1.7
L-lysine EC:4.1.1.20
S-adenosyl-L-methionine
S-adenosyl-L-homocysteine
S-ribosyl-L-homocysteine
EC:3.2.2.9
L-homocysteine EC:2.1.1.14
L-methionine EC:2.5.1.6
autoinducer 2 EC:4.4.1.21
Amino acid Metabolism
EC:6.2.1.17 EC:2.2.1.6
EC:4.3.3.7 EC:1.2.1.11 EC:2.7.2.4
EC:2.6.1.42 EC:4.2.1.9
EC:4.1.1.20 EC:5.1.1.7
EC:3.5.1.47 EC:2.3.1.89 EC:1.17.1.8
EC:2.1.1.14 EC:4.4.1.21 EC:3.2.2.9
EC:2.5.1.6
EC Number in Amino acid Metabolism
25℃ NC C
4℃ NC C
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
47
(D)
xanthosine
α-D-ribose-1-phosphate EC:2.4.2.1
D-ribose 5-phosphate EC:5.4.2.7
adenosine guanine
inosine hypoxanthine IMP
2’-deoxycytidine
2’-deoxyuridine
dUMP
EC:3.5.4.5
EC:2.7.1.21/2.7.1.145
dTMP EC:2.1.1.45
thymidine EC:2.7.1.21/2.7.1.145
uracil EC:2.4.2.2/2.4.2.3
EC:2.4.2.2/2.4.2.3 2-deoxy-α-D-ribose-1-
phosphate
2-deoxy-D-ribose-5-phosphate
acetaldehyde EC:4.1.2.4
acetyl-CoA EC:1.2.1.10
EC:5.4.2.7
EC:4.1.2.4 D-glyceraldehyde-3-
phosphate
EC:2.4.2.2
2’-deoxyadenosine EC:2.4.2.1
2’deoxyinosine
EC:2.4.2.1 2’-deoxyguanosine
EC:2.4.2.1
Nucleotide Metabolism
EC:2.4.2.1 EC:5.4.2.7
EC:2.7.1.145
EC:2.4.2.3
EC:1.2.1.10 EC:4.1.2.4
EC:2.1.1.45
EC:3.5.4.5 EC:2.7.1.21
EC:2.4.2.2
25℃ NC C
4℃ NC C
4 h
8 h
12 h
24 h
4 h
8h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12h
24 h
EC Number in Nucleotide Metabolism
(P-value < 0.05)
log2FoldChange
48
(E)
(S)-(+)-allantoin
allantoate
(S)-ureidoglycine
EC:3.5.2.5
EC:3.5.3.9
(S)-ureidoglycolate EC:3.5.3.26
N-carbamoyl-2-oxoglycine EC:1.1.1.350
CO2
Allantoin Degradation
EC:3.5.2.5 EC:3.5.3.9
EC:1.1.1.350 EC:3.5.3.26
25℃ NC C
4℃ NC C
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
4 h
8 h
12 h
24 h
EC Number in Allantoin Degradation
(P-value < 0.05)
log2FoldChange
49
Figure 6. Shifts in predicted functional genes of microbiota under different storage conditions. Functional genes that are
involved in the (A) spoilage pathway, (B) ubiquinone biosynthesis, (C) nucleotide metabolism, (D) amino acid
metabolism, and (E) allantoin degradation were further analyzed using a heatmap. A log2 fold change compared to 0 h
(P <0.05) is represented in colors, and those without any significant change are shown in gray. Genes with higher
relative abundances in the contaminated samples are indicated using red arrows, whereas those with higher relative
abundances in the non-contaminated samples are indicated using blue arrows. Genes that decreased over time are
indicated using dotted lines, while those that increased are indicated using solid lines. NC: Non-contaminated samples,
C: Contaminated samples.
50
OTU contribution to the shift in functional genes
In order to determine the genera contributing to the shift in
pathways, the differential abundance of OTUs was identified using
PICRUSt2. One representative functional gene that was significantly
increased in each pathway was analyzed, and the OTU contributing
to its increase was identified. In spoilage metabolism, acetate kinase
(ackA, EC:2.7.2.1), which converts acetyl phosphate to acetate was
selected (Fig. 7A). For ubiquinone biosynthesis, menaquinone-
specific isochorismate synthase (menF, EC:5.4.4.2), which converts
chorismate to isochorismate, the first committed step in the
biosynthesis of menaquinone, was selected (Fig. 7B) (Buss, K. et
al., 2001). Menaquinone is necessary for bacterial vitality and
growth; E. coli, Bacillus subtilis, and Staphylococcus aureus require
menaquinone for their growth (Bentley, R., & Meganathan, R., 1982;
Jiang, M. et al., 2007). Thus, the increase in the biosynthesis of
menaquinone indicates the growth of spoilage-causing bacteria and
foodborne pathogens. Allantoinase (allB, EC:3.5.2.5) was selected
to monitor allantoin degradation, and S-ribosylhomocysteinelyase
(luxS, EC:4.4.1.21) was selected to monitor amino acid metabolism,
and phosphopentomutase (deoB, EC:5.4.2.7) was selected to
monitor nucleotide metabolism (Fig. 7C-E).
The genera contributing to the abundanceof ackA and menF
were compared among samples under different conditions (Fig. 7A,
B). Carnobacterium was the main contributing genus to ackA and
51
menF genes in most samples, except in contaminated samples
stored at 4 °C for 24 h. The most committed genera to the
abundance of ackA were lactic acid bacteria, including
Carnobacterium and Lactobacillus. In contrast, higher normalized
contributions of Escherichia to the abundance of menF compared to
other functional genes were observed. While diverse genera
contributed to the abundance of ackA and menF in the contaminated
samples stored at 4 °C for 24 h, Carnobacterium, Lactobacillus, and
Escherichia were the major contributors to the abundance of ackA
and menF in the contaminated samples stored at 25 °C. The
significant growth of these genera at 25 °C suggests that the
abundances of ackA and menF would be highest in contaminated
samples stored at 25 °C after 24 h.
52
(A) (B)
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
E C :2 .7 .2 .1 ,a c e ta te k in a s e ;a c k A
S to ra g e t im e
Co
ntr
ibu
tio
n c
ou
nt
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
E C :5 .4 .4 .2 ,m e n a q u in o n e -s p e c if ic is o c h o r is m a te s y n th a s e ;m e n F
S to ra g e t im e
Co
ntr
ibu
tio
n c
ou
nt
Brochothrix
Enterobacter
Yersinia
Rouxiella
Serratia
Kosakonia
Rhizobium
other
Carnobacterium
Lactobacillus
Rahnella
Escherichia
Bacillus
Staphylococcus
Vibrio
4℃ 25℃ NC C NC C 4℃ 25℃ NC C NC C
53
(C) (D)
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
E C :4 .4 .1 .2 1 ,S - r ib o s y lh o m o c y s te in e ly a s e ; lu xS
S to ra g e t im e
Co
ntr
ibu
tio
n c
ou
nt
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
E C :5 .4 .2 .7 ,p h o s p h o p e n to m u ta s e ;d e o B
S to ra g e t im e
Co
ntr
ibu
tio
n c
ou
nt
Brochothrix
Enterobacter
Yersinia
Rouxiella
Serratia
Kosakonia
Rhizobium
other
Carnobacterium
Lactobacillus
Rahnella
Escherichia
Bacillus
Staphylococcus
Vibrio
4℃ 25℃ NC C NC C 4℃ 25℃ NC C NC C
54
(E)
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0h
4h
8h
12h
24h
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
E C :3 .5 .2 .5 ,a lla n to in a s e ;a llB
S to ra g e t im e
Co
ntr
ibu
tio
n c
ou
nt
Brochothrix
Enterobacter
Yersinia
Rouxiella
Serratia
Kosakonia
Rhizobium
other
Carnobacterium
Lactobacillus
Rahnella
Escherichia
Bacillus
Staphylococcus
Vibrio
4℃ 25℃ NC C NC C
55
Figure 7. OTUs contributing to the shift in functional genes. OTUs contributing to the shift in(A) acetate kinase (ackA),
menaquinone specific isochorismate (menF), (C) S-ribosylhomocysteine lyase (luxS), (D) phosphopentomutase
(deoB) and (E) allantoinase (allB), were identified using PICRUSt2. NC: Non-contaminated samples, C: Contaminated
samples.
56
Validation of OTU contribution using quantitative real-time PCR
These genera significantly changed their contribution over
time and were selected for validation using qRT-PCR with specific
primers (Table S2) (Fig. 8). The contribution of Staphylococcus to
the abundance of menF was also verified to identify its unique
growth in non-contaminated samples at 25 °C. Change in
ubiquinone biosynthesis was studied as a representative of the
three pathways other than spoilage metabolism, that showed
significant shifts – amino acid metabolism, nucleotide metabolism,
and allantoin degradation as they showed similar shifts in
contribution.
The copy number of ackA and menF in each genus identified
by real-time PCR was used to determine the cell number based on
the genome information in the National Center for Biotechnology
Information (NCBI) database. Consistent with the prediction, both
ackA and menF showed an increase in all samples (Fig. 8). When
beef was stored at 25 °C, ackA was significantly increased in both
non-contaminated samples (average 3.96 x 108 cells/g; P <0.001)
and contaminated samples (2.24 x 108 cells/g; P <0.001). The
abundances of menF were significantly increased at 25 °C in non-
contaminated samples (3.51 x 108 cells/g; P <0.001) and
contaminated samples (1.56 x 108 cells/g; P <0.01). However, the
abundances of ackA and menF genes showed smaller increases in
57
samples stored at 4 °C than samples stored at 25 °C.
Carnobacterium was the predominant genus contributing to
both ackA and menF abundance in the qRT-PCR analysis (Fig. 8).
The proportions of the ackA gene from Lactobacillus increased in
all samples with time (P <0.01) and the Lactobacillus cell number
was exceptionally high in samples at 25 °C (non-contaminated:
1.11 x 108 cells/g, contaminated: 1.20 x 108 cells/g). We also
determined the contribution of Staphylococcus to menF gene
abundance. In the non-contaminated samples stored at 25 °C, the
cell proportion was over 0.05 after 24 h, and the cell number
significantly increased by 1.79 x 107 cells/g from 0 h to 24 h (P
<0.0001) (Fig. 8B). In contrast, Staphylococcus cell number was
significantly low in contaminated samples stored at 25 °C and in
samples at 4 °C, where the cell proportion was less than 0.01 even
after 24 h of storage. This is consistent with the taxonomic
composition results which showed that only non-contaminated
samples stored at 25 °C have a relative abundance of
Staphylococcus over 0.01. This also supports the positive
correlation between Carnobacterium and Staphylococcus in non-
contaminated samples stored at 25 °C. The abundance of
Escherichia increased in contaminated samples stored at 4 °C for 8
h, but decreased after 8 h, whereas the abundance of
58
Carnobacterium gradually increased. This is also consistent with the
shift in taxonomy composition that resulted from the negative
correlation between Carnobacterium and Escherichia at 8 h (Fig.
4B). These qRT-PCR results suggest that the pathway shifts
predicted in this study are reliable.
Together, our results showed that Carnobacterium and
contaminated E. coli interacted with indigenous microbes, and
induced a shift in beef microbiota, which increased the spoilage
metabolism in contaminated samples. The qRT-PCR analysis of
functional genes also showed that the increase in their abundance
was primarily due to the increase in the abundance of
Carnobacterium and Escherichia over time. The relative abundance
of Carnobacterium and Escherichia was especially high in
contaminated samples stored at 25 °C (Carnobacterium: 9.32 x 107
cells/g; ackA, 1.41 × 108 cells/g; menF, Escherichia: 1.07 x 107
cells/g; ackA, 1.40 x 107 cells/g; menF). The alteration of these
microbes in beef with time indicated that the storage temperature
and interactions between microbes are important for maintaining
food quality. Thus, the microbial information of beef distributed
from various regions can be used to predict the spoilage of meat
and provide more detailed guidance to manage those products.
59
(A)
(B)
0h
8h
12h
24h
0 .0
0 .5
1 .0
4
5
6
7
8
9
m e n F
S to ra g e t im e
Ce
ll p
rop
ort
ion lo
g1
0(c
ell/g
)
* * *
0h
8h
12h
24h
0 .0
0 .5
1 .0
4
5
6
7
8
9
m e n F
S to ra g e t im e
Ce
ll p
rop
ort
ion lo
g1
0(c
ell/g
)
* *
0h
8h
12h
24h
0 .0
0 .5
1 .0
4
5
6
7
8
9
m e n F
S to ra g e t im e
Ce
ll p
rop
ort
ion lo
g1
0(c
ell/g
)
* * *
0h
8h
12h
24h
0 .0
0 .5
1 .0
4
5
6
7
8
9
m e n F
S to ra g e t im e
Ce
ll p
rop
ort
ion lo
g1
0(c
ell/g
)
* * * *
0h
8h
12h
24h
0 .0
0 .5
1 .0
4
5
6
7
8
9
a c k A
S to ra g e t im e
Ce
ll p
rop
ort
ion lo
g1
0(c
ell/g
)
*
0h
8h
12h
24h
0 .0
0 .5
1 .0
4
5
6
7
8
9
a c k A
S to ra g e t im e
Ce
ll p
rop
ort
ion lo
g1
0(c
ell/g
)
* *
0h
8h
12h
24h
0 .0
0 .5
1 .0
4
5
6
7
8
9
a c k A
S to ra g e t im e
Ce
ll p
rop
ort
ion lo
g1
0(c
ell/g
)
* * *
0h
8h
12h
24h
0 .0
0 .5
1 .0
4
5
6
7
8
9
a c k A
S to ra g e t im e
Ce
ll p
rop
ort
ion lo
g1
0(c
ell/g
)
* * *
Non-contaminated_25℃
Non-contaminated_4℃
Contaminated_4℃
Contaminated_25℃
Carnobacterium
Lactobacillus
Escherichia
Staphylococcus
60
Figure 8. Validation of OTUs contributing to the shift in predicted functional genes using quantitative real-time PCR.
Bacterial cell number and cell proportion of Carnobacterium, Lactobacillus, and Escherichia in (A) ackA and those of
Carnobacterium, Staphylococcus, and Escherichia in (B) menF were compared between samples under different
conditions. * P <0.05, ** P <0.01, *** P <0.001. NC: Non-contaminated samples, C: Contaminated samples.
61
Ⅳ. CONCLUSIONS
In this study, the influences of storage temperatures and
pathogen contamination on the indigenous microbes in beef were
analyzed. E. coli O157:H7 contamination increased the bacterial cell
number in beef, but refrigeration significantly reduced the growth of
the microbiota. Although the abundances of Escherichia increased in
beef stored at both 4 °C and 25 °C, the cell number of Escherichia
was lower in samples stored at 4 °C. The indigenous beef
microbiota comprised several spoilage-causing microorganisms,
and they had a negative correlation with other genera present in
beef. This resulted in the increased abundance and cell number of
the dominant genera, Carnobacterium, Lactobacillus, and
Escherichia, in the contaminated sample stored at 25 °C. Among
these genera, Carnobacterium was the key microbe that induced the
shift in beef microbiota and influenced the growth of Escherichia at
4 °C. Therefore, spatial and temporal variance among beef
microbiota can indicate different levels of vulnerability of beef
products to the microbial spoilage. Besides, the risk of spoilage,
which is microbe-mediated, was higher when the beef was stored
at 25 °C and pathogen-contaminated conditions. The growth of
spoilage bacteria and foodborne pathogens over time at 25 °C
62
contributed to the increased level of spoilage pathways with a
higher abundance of spoilage associated genes. Thus, storing at
4 °C and avoiding contamination with foodborne pathogens is
essential for the maintenance of beef quality. As E. coli can cause
food poisoning even at very low cell numbers, it was challenging to
determine the effect of E. coli alone on the beef microbiota. This
study provided insights into the effect of E. coli contamination on
beef microbiota and its role in spoilage, under different storage
conditions.
63
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72
Ⅵ. 국문초록
소고기는 세계에서 가장 많이 소비되는 육류 중 하나로 식중독
사고 또한 끊임없이 일어나고 있다. 식중독균은 소고기에 노출이 되었을
때 식중독뿐만 아니라 소고기의 부패를 유발할 수 있다. 따라서,
식품안전을 위해 식중독균이 소고기 마이크로바이옴에 미치는 영향을
연구할 필요가 있다. 본 연구에서는 서로 다른 보관 조건에서
식중독균에의 노출이 소고기 마이크로바이옴과 미생물 종간의
상호작용을 어떻게 변화시키는 지를 살펴보았다. 총 60개의 소고기
샘플을 4℃ 또는 25℃에서 24시간까지 보관하였으며, 마이크로바이옴의
변화는 MiSeq 시스템을 이용해서 분석하였다. 식중독균의 영향은
소고기에 Escherichia coli FORC_044를 인위적으로 노출시킴으로써
확인하였다. FORC_044는 한국에서 식중독환자의 분변에서 분리한
Enterohemorrhagic E. coli (EHEC) 균주이다. 보관 시간에 따른
Escherichia의 균 수와 전체 마이크로바이옴에 대한 비율의 변화를
확인해 보았을 때, 25℃에서 보관하였을 때가 4℃에서 보관하였을
때보다 더욱 많이 증가하는 것을 알 수 있었다. 미생물 종간의 네트워크
분석결과 Escherichia는 Pseudomonas, Brochothrix, Staphylococcus,
Rahnella와 Rhizobium과 같은 소고기 상재균들과 양의 관계를 가지고
있음을 확인하였다. 이와 반대로, 부패 세균 중 하나인
Carnobacterium은 Escherichia를 비롯한 다른 소고기 상재균들과 음의
관계를 가지고 있었다. 보관 시간이 지남에 따라 마이크로바이옴이
나타내는 기능의 변화를 예측해보았을 때, 아세트산과 젖산을 생산하는
반응을 포함하는 부패 과정이 점차 증가하는 것을 확인할 수 있었다.
이러한 변화는 25℃에서 보관한 오염된 소고기에서 가장 크게 나타났다.
73
이와 더불어, Carnobacterium, Lactobacillus와 Escherichia가 이러한
과정에 관여하는 유전자의 변화에 가장 주요한 속임을 확인하였다. 본
연구의 결과는 식중독균이 소고기 마이크로바이옴의 변화와 소고기의
부패에 관여한다는 것을 보여주고 있다. 본 연구는 소고기
마이크로바이옴에 대한 이해를 넓혀줄 것이며, 식중독균과 보관 조건이
소고기의 품질에 어떠한 영향을 미치는 지 알려줄 수 있을 것이다.
주요어: 메타지노믹, 마이크로바이옴, 소고기, 부패 세균, 병원성 대장균,
식중독균 오염, 미생물 상호작용, 식품안전
학번: 2018-22519