Turk et al.

Annals of Microbiology (2021) 71:8

https://doi.org/10.1186/s13213-020-01615-3
Annals of Microbiology

ORIGINAL ARTICLE Open Access

Assessing the genetic impact of

Enterococcus faecalis infection on gastric
cell line MKN74
Seyhan Turk1, Can Turk2*, Elif Sena Temirci3, Umit Yavuz Malkan4, Gulberk Ucar1 and Sukru Volkan Ozguven2

Abstract
Purpose: Enterococcus faecalis (E. faecalis) is an important commensal microbiota member of the human gastrointestinal
tract. It has been shown in many studies that infection rates with E. faecalis in gastric cancer significantly increase. It has been
scientifically proven that some infections develop during the progression of cancer, but it is still unclear whether this
infection factor is beneficial (reduction in metastasis) or harmful (increase in proliferation, invasion, stem cell-like phenotype)
of the host. These opposed data can significantly contribute to the understanding of cancer progress when analyzed in
detail.
Methods: The gene expression data were retrieved from Array Express (E-MEXP-3496). Variance, t test and linear regression
analysis, hierarchical clustering, network, and pathway analysis were performed.
Results: In this study, we identified altered genes involved in E. faecalis infection in the gastric cell line MKN74 and the
relevant pathways to understand whether the infection slows down cancer progression. Twelve genes corresponding 15
probe sets were downregulated following the live infection of gastric cancer cells with E. faecalis. We identified a network
between these genes and pathways they belong to. Pathway analysis showed that these genes are mostly associated with
cancer cell proliferation.
Conclusion: NDC80, NCAPG, CENPA, KIF23, BUB1B, BUB1, CASC5, KIF2C, CENPF, SPC25, SMC4, and KIF20A genes were found
to be associated with gastric cancer pathogenesis. Almost all of these genes are effective in the proliferation of cancer cells,
especially during the infection process. Therefore, it is hypothesized that downregulation of these genes may affect gastric
cancer pathogenesis by reducing cell proliferation. And, it is predicted that E. faecalis infection may be an important factor
for gastric cancer.
Keywords: Gastric cancer, Enterococcus faecalis infection, E. faecalis, Proliferation, Cell cycle Abbreviations, E. faecalis
Enterococcus faecalis, ROS Reactive oxygen species, CGP Cancer Genome Project, DAVID Database for Annotation,
Visualization and Integrated Discovery, GSEA Gene set enrichment analysis, ES Enrichment score, NES Normalized enrichment
score, NOM p value Nominal p value, FDR q value False discovery rate q value, FWER Familywise error rate p value

Introduction ulcer, diet, smoking, and alcohol use, but the main risk
According to the 2018 GLOBOCAN data, gastric cancer factor is bacterial infections (Rawla and Barsouk 2019).
has a death rate of 8.2% among ten common cancers Infections are very important in the chemotherapeutic
and a 5.7% new case rate among five common cancers response because of their effects on various mechanisms.
(Bray et al. 2018). Risk factors include genetics, gastric Due to weakened immune systems and various treat-
ment procedures, cancer patients are more likely to be-
* Correspondence: can.turk@lokmanhekim.edu.tr
2
come infected from the flora.
Department of Medical Microbiology, Faculty of Medicine, Lokman Hekim
Most cases of stomach cancer are associated with bac-
University, Ankara, Turkey
Full list of author information is available at the end of the article terial infections, particularly Helicobacter pylori gastritis,
© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Turk et al. Annals of Microbiology (2021) 71:8 Page 2 of 10

and have been found to increase the likelihood of stom- benefit or harm the host. Although there are studies
ach cancer by about 6 times within 10 years after infec- reporting genetic alterations in gastric cancer caused by
tion. However, the data on the roles of bacterial E. faecalis that may affect the chemotherapeutic re-
infections in cancer are definitely in two different direc- sponse and patients’ prognosis, withal their roles, related
tions. Some researchers suggest that bacterial infections pathways, and mechanisms remains unclear.
play a protective role, while others highlight the harmful In the current study, we aimed to determine the E. fae-
effects associated with the infection. calis-related altered genes, the network relations of these
Enterococcus is a part of the normal intestinal flora of genes with each other and to relevant pathways using all
humans and animals (Dubin and Pamer 2014). Entero- genome profiles of gastric cancer cells. The results can
coccus faecalis (E. faecalis) has a fundamental role in the provide important contribution how prognosis can be af-
development of intestinal immunity in the early stages of fected in the presence of infection.
life in the human gastrointestinal system and plays a role
as a protective agent in the regulation of colon homeo- Materials and methods
stasis in newborn babies (Fanaro et al. 2003; Are et al. Microarray gene expression data
2008; Wang et al. 2014). Moreover, due to its anti- The gene expression data was obtained from the Cancer
inflammatory potential, it is considered to be probiotic Genome Project (CGP) database (http://www.
in the treatment of certain diseases, such as chronic si- cancerrxgene.org/). Transcription profile data of human
nusitis, bronchitis, or infant acute diarrhea (Habermann MKN74 gastric epithelial cells treated with Enterococcus
et al. 2001; Huycke et al. 2002; Nueno-Palop and Narbad faecalis were obtained from Array Express (E-MEXP-
2011; Gong et al. 2017). Besides its beneficial effects it is 3496) (Strickertsson et al. 2014).
also known to play important roles in the formation of
systemic infections and translocations (Khan et al. 2018; Processing and normalization of data
Ben Braïek and Smaoui 2019). It can easily colonize in The raw data from Array Express were normalized with
extra-intestinal and unusual areas and can cause infec- the Affy package in the R software. Normalized tran-
tions throughout certain predisposing conditions such as scription profile data consists of 23,344 different genes/
long-term hospitalizations and some immune compro- 54,675 probe sets. The data contains control day 1, con-
mised conditions. trol day 5, E. faecalis infected day 1, and E. faecalis in-
It has been reported in many studies that the increase of fected day 5 groups’ whole genome expression data, and
E. faecalis infections in various cancer types is very com- they were triplicated.
mon. An increase in E. faecalis-related infections has been
noted in cancerous lesions of patients with oral and oro- Variance, t test, and linear regression analysis
pharyngeal squamous cell carcinoma (Boonanantanasarn Among the groups, significant genes with a standard de-
et al. 2012). The increased levels of E. faecalis infection viation value above 1 were identified. In order to group
and leading to chromosomal imbalance have been shown the identified genes more specifically, Pearson’s
in colon cancer (Khan et al. 2018). It triggers poor progno- correlation absolute P value based on the correlation co-
sis by causing invasion in bladder cancer cells (Horsley efficient was calculated and genes above 0.05 were se-
et al. 2013). It has been shown that cells that interact with lected. In addition, Pearson correlation coefficient value
E. faecalis trigger the production of reactive oxygen spe- (R value) was calculated and genes above 0.99 were se-
cies (ROS), and the mitochondrial genome is impaired in lected. Genes with a P value less than 0.01 were selected
gastric cancer (Strickertsson et al. 2013). by comparing the expression values of the genes with
However, uncontrolled overproduction of ROS can the control groups by two-tailed t test analysis.
lead to carcinogenic, cytotoxic results. On the contrary, Analyses were done using GraphPad Prism 5.0
it was found that the colorectal cancer cell line HCT- (Graphpad Prism 5 Software, San Diego, CA, USA). To
116-cocultured with E. faecalis downregulated the ex- determine the difference between groups two tail t test
pression of the fasting-induced adipose factor (FIAF) were performed. Genes with a t test P value less than
gene, which is normally associated with the development 0.01 were selected. P value less than 0.05 were selected
of some cancer types (Grootaert et al. 2011). It has been in Pearson analysis.
suggested that E. faecalis strains isolated from human
breast milk may be candidates for the prevention and Hierarchical clustering
treatment of cancer when they inhibit the proliferation Genes determined in linear regression analysis were
of breast cancer cells (Hassan et al. 2016). hierarchically clustered with mean standardized gene ex-
These opposed data can contribute significantly to un- pression values with the Euclidean Gene Cluster 3.0 pro-
derstanding of cancer progression when analyzed in gram. The data was standardized after cluster analysis,
more detail whether the interaction of the E. faecalis will and the standardized data were viewed using Treeview.
Turk et al. Annals of Microbiology (2021) 71:8 Page 3 of 10

Hierarchical clustering was performed by clustering both the importance of ES calculation. Therefore, NOM P
genes and arrays using Euclidian distance as similarity value directly correlated with ES as well as NES value.
metric and complete linkage as clustering method. Increase of NOM P value show critical role of ES. On
the other hand, FWER P value indicates false positives
Network analysis probability of NES and so, lower FWER P value directly
To generate a network based on coexpression and gen- and significantly correlated with correctness of NES cal-
etic interactions, the GeneMANIA software were used culation. Moreover, FDR q value is the most vital value
and genes in similar pathways were identified with of this analysis. This value needs to be lower than 0.25
Cytoscape (Shannon et al. 2003). Twelve differentially and even, when this value become smaller, the enrich-
expressed genes from the 138 genes/165 probe sets were ment of gene sets is more meaningful.
used as input. The datasets have been integrated, ana-
lyzed, and visualized to find out if they have functionally Results
similar genes associated with each other and to identify Time-dependent gene expression alterations in the
related functions for different gene groups in the net- MKN74 gastric cancer cell line treated with E. faecalis
work. Thus, the network relationship of these genes was Whole genome expression data were analyzed by linear
determined. The software scores each gene (node). The regression to determine gene expression alterations be-
size of nodes directly correlated with the score. For ex- tween control day 1, infected day 1, and infected day 5
ample, NDC80 gene has virtually 0.56 score and CENPT groups of human gastric carcinoma MKN74 cells. Ac-
has 0.05 score. This directly correlated with the size of cording to the results, 138 genes corresponding to 165
the nodes. Higher scores indicate genes that are more probe sets with a standard deviation value above 1.0, P
likely to be functionally related and more likely has co- value below 0.05, and Pearson R value above 0.99
expression potential. showed statistically significant expression alteration. For
further analyses, we focused on these genes that altered
Pathway enrichment analysis expression diversity between groups. In the time-
To understand biological linkage behind these genes, dependent E. faecalis-treated groups, 10 probe sets were
“Database for Annotation, Visualization and Integrated positively correlated and upregulated, and 155 probe sets
Discovery” (DAVID) software was used. The pathways were negatively correlated and downregulated (Supple-
associated with our genes were identified. mentary Table 1).
Hierarchical cluster analysis demonstrated gene alter-
Gene set enrichment analysis (GSEA) ations between 3 certain groups; control day 1, infected
The gene set enrichment analysis (GSEA) was carried day 1, and infected day 5. Results defining as 155 of the
out in concordance with GSEA guideline procedure probe sets were negatively correlated, highly expressed
(http://software.broadinstitute.org/gsea/ in the control day 1 group, and expression decreased at
docGSEAUserGuideFrame.html). E-MEXP-3496 data infected day 1 group and so on at infected day 5 group.
was used in order to perform the analysis. This data in- And conversely 10 of the probe sets were positively cor-
cludes 54,675 probe sets (23,344 different genes). Ana- related, low expressed in the control day 1 group, and
lysis was performed between the control day 5 group expression increased at infected day 1 and so on at in-
and the infected day 5 group to understand the pattern fected day 5 group. Top 50 genes were visualized in the
of E. Faecalis infection among these groups. The main figure and the rest were given as supplementary data
purpose of this analysis is to determine which gene sig- (Fig. 1) (Supplementary Figures 1, 2, 3). So, there were
nificantly enriched in which gene set that belongs to the significantly altered genes between control and treated
GSEA as well as to understand which gene set enriched groups.
in which groups.
GSEA calculate the enrichment score (ES), normalized Gene alterations due to treatment with E. faecalis
enrichment score (NES), nominal P value (NOM P In order to determine whether this expression change was
value), false discovery rate q value (FDR q value), and caused by E. faecalis or cancer itself, variance analyses and t
familywise error rate P value (FWER). The ES value indi- test were performed between control day 5 groups’ and in-
cates gene’s maximum deviation in gene sets; in other fected day 5 groups’ 138 genes/165 probe set expression data.
words, this score helps to find most upregulated genes. To better analyze gene expression alteration, long range
NES value represents the connection or difference be- group “infected day 5” group was selected for variance ana-
tween gene sets and gene expression. The higher NES lysis and t test. Thus, 12 statistically significant genes
value shows the elevation of permutations. Hence, (NDC80, NCAPG, CENPA, KIF23, BUB1B, BUB1, CASC5,
higher NES value increase significance of gene sets. In KIF2C, CENPF, SPC25, SMC4, KIF20A) corresponding 15
addition to ES and NES values, NOM P value evaluates probe sets with a standard deviation above 1.0 and a t test P
Turk et al. Annals of Microbiology (2021) 71:8 Page 4 of 10

Fig. 1 Hierarchical clustering of 138 statistically significant genes/165

probe sets in the three groups specified. The analysis reveals sensitive
low expressions (green), intermediate (black), and high expressions
(red) of 138 genes for control day 1, infected day 1, and infected day 5.
Designated groups are clearly classified. Top 50 genes were given in
the figure

value less than 0.01 were determined (Table 1) (Supplemen-

tary Figure 4).
In linear regression analysis, all of these 12 genes were
found to be negatively correlated and downregulated.
Figure 2 shows the expression alterations of these 12
genes time-based between control day 1, infected day 1,
and infected day 5 (Fig. 2). Gene expressions have been
shown to decrease as the infection process progresses in
all these 12 genes.

Network construction and identification of key candidate

genes using gene-gene interaction
Network analysis was performed with Cytoscape to bet-
ter demonstrate the biological linkage of these 12 genes
whose expression decreased from day 1 to day 5 during
infection. As can be seen from the figure, it has been
shown that the 12 genes we identified have a strong net-
work relationship between each other and with other
candidate genes (Fig. 3). The linking line between genes
illuminates the network of these genes. The thickness of
the linking line determines the power of connection of
the related genes. For instance, the linking line between

Table 1 The list of 12 genes (15 probe sets) which have the
most alterations in expression. These genes have standard
deviation above 1.0 and t test value less than 0.01 between 5
days control group and 5 days treated group. These significant
values indicate that the change occurred due to infection.
Probe Set Gene Symbol St Dev T-Test
204162_at NDC80 1.76016363 0.00153695
218663_at NCAPG 1.74829872 0.0002009
204962_s_at CENPA 1.5109911 0.00039232
218662_s_at NCAPG 1.46733952 0.00062378
204709_s_at KIF23 1.42573476 0.00046214
203755_at BUB1B 1.36025723 1.7849E-05
215509_s_at BUB1 1.30655845 0.00019534
209642_at BUB1 1.26836983 0.0001898
228323_at CASC5 1.19910534 4.6314E-05
211519_s_at KIF2C 1.17437172 0.00024729
207828_s_at CENPF 1.13683208 8.8436E-05
209891_at SPC25 1.13377039 0.00260856
201664_at SMC4 1.05623047 0.00770427
218755_at KIF20A 1.02361512 0.0033544
209408_at KIF2C 1.02269745 0.00046939
Turk et al. Annals of Microbiology (2021) 71:8 Page 5 of 10

Fig. 2 Twelve genes/15 probe sets that show significant downregulation after treatment with live infection. Statistically significant alterations were
detected between control day 1, infected day 1, and infected day 5 for all probe sets

NCAPG and SMC4 represent one of the thickest lines. important pathways have been identified such as cell
This indicates the link formed between these genes has cycle, pyrimidine metabolism, mismatch repair, and
been determined to be stronger by studying more P53 signaling pathway which are associated with 12
clearly. Additionally, the black nodes indicate the target genes during the exposure to E. faecalis. The relation-
genes giving by authors. On the other hand, the gray ship of these pathways with cancer development, such
nodes demonstrate the genes which associated genes de- as the importance of cell cycle in gastric cancer cells,
termined by GeneMANIA application. is very clear (Table 2).
The significantly enriched gene sets and their ES,
Functional enrichment of genes and correlations with NES, NOM P value, FWER P value, and FDR q value
pathways represented in Supplementary Table 2. According to
Pathway analysis of biological processes was done GSEA, the cell cycle checkpoint as well as the mitotic
with DAVID software to reveal the relationship of cell cycle checkpoint gene set was found as signifi-
these 12 genes with cellular functions and pathways cantly correlated with the genes that are present in
and to understand their new meaning. The four the data. These gene sets even correlated with
Turk et al. Annals of Microbiology (2021) 71:8 Page 6 of 10

Fig. 3 Network analysis of 12 genes that are statistically significant. The figure shows that these 12 genes have a strong network connection
(Cytoscape). The linking line between genes illuminates the network of the genes. The thickness of the linking line determines the power of
connection of the related genes. This indicates the link formed between these genes has been determined to be stronger by studying more
clearly. Additionally, the black nodes indicate the target genes giving by authors. On the other hand, the gray nodes demonstrate the genes
which associated genes determined by GeneMANIA application. The size of nodes directly correlated with score

pathway analysis in Table 2 and other emphasized

studies. The enriched genes were NCD80, BUB1,
BUB1B, and CENPF in these two gene sets.
Also, Fig. 4 demonstrated the cell cycle checkpoint
gene set plot. This graph showed genes with enriched
Table 2 Pathways related to the genes are linked. It is seen that
NCD80, BUB1, BUB1B, and CENPF in the control day 5 important pathways in cancer progression are related to our
group, also called positively correlated. On the other genes. Most of these genes are linked to the cell cycle pathway
hand, the same genes were downregulated and nega- from the database for annotation, visualization, and integrated
tively correlated in the infected day 5 group. The same discovery (DAVID)
results were obtained for the mitotic cell cycle check- Category Pathways Count P value Benjamini
point gene set (Fig. 5). KEGG_PATHWAY Cell cycle 6*1 1.3E−3 9.0E−2
2
KEGG_PATHWAY Pyrimidine metabolism 2* 3.1E−3 1.1E−1
Discussion
KEGG_PATHWAY p53 signaling pathway 4*3 1.0E−2 1.7E−1
In recent years, the association between intestinal flora-
4
related microbiota and gastric cancer has increased the KEGG_PATHWAY Mismatch repair 3* 9.1E−3 2.0E−1
interest to its roles in human health with crucial find- *1NDC80, NCAPG, BUB1B, BUB1, CENPA, KIF23
*2BUB1B, BUB1
ings. E. faecalis infections have become important due *3KIF2C, SPC25, KIF20A, CENPF
to their potential to affect cell cycle which can alter the *4SMC4, CENPA, NCAPG
Turk et al. Annals of Microbiology (2021) 71:8 Page 7 of 10

Fig. 4 The cell cycle checkpoint enrichment plot was represented. The black straight line refers the enriched genes in the groups. Red part
contains the genes that positively correlated in control day 5 group and were upregulated in control day 5. On contemporarily, the blue line
includes downregulated or in other words negatively regulated genes that belong to infected day 5

response to chemotherapy. E. faecalis is a part of the day 5 were enriched in the specified biological gene sets.
normal intestinal flora that can pass through the gut and This enrichment supports that these genes have import-
cause systemic infection (Khan et al. 2018). If the barrier ant effects on the cycles of cells. In addition, these ef-
is violated, the intestinal flora such as E. faecalis can fects trigger cancer development by causing critical
translocate and cause infection (Ramphal 2004). In this changes in cell function, as indicated.
study, it is aimed to identify E. faecalis-related gene ex- NDC80 is a kinetocol protein that combines with
pression alterations, their network relationships, and as- other proteins to form a microtubule binding site. It has
sociated pathways using all genome expression data of important roles in the cell cycle and an abnormality can
MKN74 gastric cancer cells. lead to apoptotic cell death. Thus, downregulation of
NDC80, NCAPG, CENPA, KIF23, BUB1B, BUB1, this gene may cause abnormalities in the cell cycle of
CASC5, KIF2C, CENPF, SPC25, SMC4, and KIF20A gastric cells after E. faecalis infection. In this case, cells
gene expression alterations were identified depending on can escape from proliferation and go into apoptosis.
time after treatment with E. faecalis of gastric cell line High expression of the NDC80 gene in pancreatic and
MKN70. These genes were identified as downregulated breast cancer cells is associated with poor prognosis and
in gastric cancer cells, and the treated and untreated plays important roles in tumor formation in pancreatic,
groups (P < 0.05) were hierarchically forming a very dis- breast, and stomach cancer (Meng et al. 2015; Tang and
tinct cluster, as expected. Toda 2015; Liu et al. 2018). NCAPG is the mitotic gene
Gene set enrichment analysis supported the results that plays important roles in the cell cycle. High expres-
that some crucial genes function effectively in the cell sion of NCAPG is associated with proliferation in gastric
cycle as a result of E. Faecalis infection. NDC80, BUB1B, cancer (Song et al. 2018). A downregulation of this gene
BUB1, and CENPF genes showed a statistically signifi- may cause a decrease in proliferation of gastric cancer
cant difference in cell cycle checkpoint and mitotic cell cells. CENP-A and CENP-F are histone variants that en-
cycle checkpoint gene sets associated with cell cycle. sure kinetochores and centromeres to form and function
Genes found statistically significant as a result of the properly. High expressions are related with cancer pro-
comparison between the control day 5 and the infected gress and have an important role in cell division (Sun
Turk et al. Annals of Microbiology (2021) 71:8 Page 8 of 10

Fig. 5 The mitotic cell cycle checkpoint enrichment plot was represented. The black straight line refers the enriched genes in the groups. Red
part contains the genes that positively correlated in control day 5 group and were upregulated in control day 5. On contemporarily, the blue line
includes downregulated or in other words negatively regulated genes that belongs to infected day 5

et al. 2016). High expression can be predicted as a bio- chromosome 2 (SMC2) gene encodes condensin com-
marker in the malignant progression and poor prognosis plexes and responsible from chromosomal stability (Je
of many types of cancer. The decrease in the expression et al. 2014). It is showed that SMC2 gene has low ex-
of this gene can cause irregularity in the regulation of pression in gastric cancer, and it is a risk loci for breast
the centromere, causing genome imbalance in gastric and ovarian cancer (Je et al. 2014; Murakami-Tonami
cancer. Kinesin superfamily proteins (KIFs) are et al. 2014; Kar et al. 2016). The SPC25 gene encodes a
microtubule-dependent motor proteins and function as protein that may be involved in kinetochore-microtubule
oncogenes in cancer cells. It was shown that KIF overex- interaction, and it is shown that upregulation of SPC25
pression is associated with poor prognosis and plays im- promotes breast cancer (Wang et al. 2019).
portant roles in cancer progression and metastasis (Yang It seems that all genes often play a role in the progres-
et al. 2019). BUB1 is a mitotic spindle checkpoint pro- sion of cancer by affecting cell proliferation and espe-
tein (budding uninhibited by benzimidazoles 1). It has cially the cell cycle. An irregularity in the cell cycle
been suggested that high expression of BUB gene family pathway is an important factor that could lead gastric
members in gastric cancer correlates strongly with tumor formation. Some cyclin-related kinases with bac-
tumor proliferation (Stahl et al. 2017). Its expression was terial infection are known to upregulate gastric cancer
inversely correlated with the residual tumor stage, and cells. Activation and regular functioning of these cyclin-
low BUB1 expression was associated with shorter sur- depended kinases occur due to the progress of the cell
vival (Grabsch et al. 2003; Stahl et al. 2017) (P < 0.001). cycle (Molaei et al. 2018). Cyclin-depended kinases play
CASC5 (cancer susceptibility candidate 5) is required for an important role in the G1/S phase transition in the cell
creation of kinetochore-microtubule attachments and cycle by targeting E2F transcription factors. An abnor-
chromosome segregation. It has been shown to play im- mality occurring in this molecular mechanism causes
portant roles in various types of cancer and has been damage to this connection and causes the development
shown to be a new oncogene in lung adenocarcinoma of gastric cancer cells.
(Cui et al. 2020). Its downregulation in gastric cancer It has been reported that most of identified genes in
may be important. Structural maintenance of this study are highly expressed in various cancers and
Turk et al. Annals of Microbiology (2021) 71:8 Page 9 of 10

are associated with a poor prognosis. Thus, cancer pro- expressions (red) of 138 genes for control day 1, infected day 1 and
gression may also be expected to decrease due to E. fae- infected day 5. Designated groups are clearly classified.
calis infection. The relationship of some of these genes Additional file 6: Supplementary Figure 4. The 12 genes (15 Probe
with gastric cancer was first shown in this study. As a re- Sets) which have alterations in expression. These genes have standard
deviation above 1.0 and T-test value less than 0.01 between 5 day control
sult of network and pathway analysis, it was determined group and 5 day infected group. These significant values indicate that
that these genes are associated with important pathways the change occurred due to infection. Top 50 genes were visualized in
in cancer progression. These pathways are cell cycle, the figure.
pyrimidine metabolism, mismatch repair, P53 signaling
pathway, endocytosis, and spliceosome. It is obvious that Acknowledgements
Not applicable
most of these pathways are associated with cancer pro-
gression and proliferation. And this allows us to posi- Authors’ contributions
tively intervene in the progression of gastric cancer. The Sukru Volkan Ozguven and Can Turk conceived and conducted the study.
Can Turk, Seyhan Turk, Umit Yavuz Malkan, and Elif Sena Temirci performed
limitation of this study is lack of incorporation of in vivo the statistical analysis. Can Turk and Seyhan Turk wrote the manuscript.
experiments. Sukru Volkan Ozguven and Gulberk Ucar revised the manuscript. Sukru
Volkan Ozguven and Gulberk Ucar supervised the study. The authors offered
intellectual contribution for the manuscript and approved its final form.
Conclusion Funding
In this study, it has been shown that E. faecalis infection No funding was received.
may have important effects on the proliferation of cells
Availability of data and materials
in gastric cancer. The results are important indicators A majority of data generated or analyzed during this study included in this
that infections caused by E. faecalis directly or indirectly published article. The datasets used and/or analyzed during the current
have an effect on the progression of gastric cancer, and study available from the corresponding author on reasonable request. The
gene expression data was obtained from the Cancer Genome Project (CGP)
this effect may favor the host in fighting with cancer. database (http://www.cancerrxgene.org/), and transcription profile data were
Most of the genes we identified have functions that slow obtained from Array Express (E-MEXP-3496).
down cell proliferation. More in vitro and in vivo studies
Ethics approval and consent to participate
are needed to reveal which structural or functional com-
Ethics approval was not necessary because the used in silico data were
ponents are responsible for this adverse effect on gastric obtained from Array Express (E-MEXP-3496).
cancer cells.
Consent for publication
Not applicable.

Supplementary Information Competing interests

The online version contains supplementary material available at https://doi. The authors declared no competing interests.
org/10.1186/s13213-020-01615-3.
Author details
1
Additional file 1: Supplementary Table 1. The list of genes that are Department of Biochemistry, Faculty of Pharmacy, Hacettepe University,
significantly expressed as a result of statistical comparison with Pearson Ankara, Turkey. 2Department of Medical Microbiology, Faculty of Medicine,
analysis. 138 genes (165 Probe sets) are expressed significantly between Lokman Hekim University, Ankara, Turkey. 3Department of Molecular Biology
control day 1, infected day 1 and infected day 5 groups as a result of and Genetics, Faculty of Science, Bilkent University, Ankara, Turkey.
4
linear regression analysis. Pearson r correlation analysis with all genom Department of Hematology, Dışkapı Yıldırım Beyazıt Training and Research
expression data, SD value above 1, r value above 0.99 and p value less Hospital, Ankara, Turkey.
than 0.05 were selected.
Received: 28 September 2020 Accepted: 18 December 2020
Additional file 2: Supplementary Table 2. Gene set enrichment
analysis (GSEA). GSEA was performed between control day 5 group and
infected day 5 group.
References
Additional file 3: Supplementary Figure 1. Hierarchical clustering of Are A, Aronsson L, Wang S, Greicius G, Lee YK, Gustafsson JA, Pettersson S,
rest of the 138 statistically significant genes / 165 probe sets in the three Arulampalam V (2008) Enterococcus faecalis from newborn babies regulate
groups specified. Rest of the genes in the Fig 1. The analysis reveals endogenous PPARgamma activity and IL-10 levels in colonic epithelial cells.
sensitive low expressions (green), intermediate (black) and high Proc Natl Acad Sci U S A 105:1943–1948
expressions (red) of 138 genes for control day 1, infected day 1 and Ben Braïek O, Smaoui S (2019) Enterococci: between emerging pathogens and
infected day 5. Designated groups are clearly classified. potential probiotics. BioMed Res Int 2019:5938210
Additional file 4: Supplementary Figure 2. Hierarchical clustering of Boonanantanasarn K, Gill AL, Yap Y, Jayaprakash V, Sullivan MA, Gill SR (2012)
rest of the 138 statistically significant genes / 165 probe sets in the three Enterococcus faecalis enhances cell proliferation through hydrogen
groups specified. Rest of the genes in the Fig. 1. The analysis reveals peroxide-mediated epidermal growth factor receptor activation. Infect
sensitive low expressions (green), intermediate (black) and high Immun 80:3545–3558
expressions (red) of 138 genes for control day 1, infected day 1 and Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global
infected day 5. Designated groups are clearly classified. cancer statistics 2018: GLOBOCAN estimates of incidence and mortality
Additional file 5: Supplementary Figure 3. Hierarchical clustering of worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68:394–424
rest of the 138 statistically significant genes / 165 probe sets in the three Cui Y, Zhang C, Ma S, Guo W, Cao W, Guan F (2020) CASC5 is a potential tumour
driving gene in lung adenocarcinoma. Cell Biochem Funct 38(6):733-742.
groups specified. Rest of the genes in the Fig. 1. The analysis reveals
sensitive low expressions (green), intermediate (black) and high Dubin K, Pamer EG (2014) Enterococci and their interactions with the intestinal
microbiome. Microbiol Spectr 5(6):10.
Turk et al. Annals of Microbiology (2021) 71:8 Page 10 of 10

Fanaro S, Chierici R, Guerrini P, Vigi V (2003) Intestinal microflora in early infancy: Liu X, Wu J, Zhang D, Bing Z, Tian J, Ni M, Zhang X, Meng Z, Liu S (2018)
composition and development. Acta Paediatr Suppl 91:48–55 Identification of potential key genes associated with the pathogenesis and
Gong J, Bai T, Zhang L, Qian W, Song J, Hou X (2017) Inhibition effect of prognosis of gastric cancer based on integrated bioinformatics analysis. Front
Bifidobacterium longum, Lactobacillus acidophilus, Streptococcus Genet 9:265–265
thermophilus and Enterococcus faecalis and their related products on Meng Q-C, Wang H-C, Song Z-L, Shan Z-Z, Yuan Z, Zheng Q, Huang X-Y (2015)
human colonic smooth muscle in vitro. PLoS One 12:e0189257 Overexpression of NDC80 is correlated with prognosis of pancreatic cancer
Grabsch H, Takeno S, Parsons WJ, Pomjanski N, Boecking A, Gabbert HE, Mueller W and regulates cell proliferation. Am J Cancer Res 5:1730–1740
(2003) Overexpression of the mitotic checkpoint genes BUB1, BUBR1, and BUB3 Molaei F, Forghanifard MM, Fahim Y, Abbaszadegan MR (2018) Molecular
in gastric cancer--association with tumour cell proliferation. J Pathol 200:16–22 signaling in tumorigenesis of gastric cancer. Iran Biomed J 22:217–230
Grootaert C, Van de Wiele T, Van Roosbroeck I, Possemiers S, Vercoutter-Edouart Murakami-Tonami Y, Kishida S, Takeuchi I, Katou Y, Maris JM, Ichikawa H, Kondo
AS, Verstraete W, Bracke M, Vanhoecke B (2011) Bacterial monocultures, Y, Sekido Y, Shirahige K, Murakami H, Kadomatsu K (2014) Inactivation of
propionate, butyrate and H2O2 modulate the expression, secretion and SMC2 shows a synergistic lethal response in MYCN-amplified neuroblastoma
structure of the fasting-induced adipose factor in gut epithelial cell lines. cells. Cell Cycle (Georgetown, Tex) 13:1115–1131
Environ Microbiol 13:1778–1789 Nueno-Palop C, Narbad A (2011) Probiotic assessment of Enterococcus faecalis
Habermann W, Zimmermann K, Skarabis H, Kunze R, Rusch V (2001) The effect of CP58 isolated from human gut. Int J Food Microbiol 145:390–394
a bacterial immunostimulant (human Enterococcus faecalis bacteria) on the Ramphal R (2004) Changes in the etiology of bacteremia in febrile neutropenic
occurrence of relapse in patients with. Arzneimittelforschung 51:931–937 patients and the susceptibilities of the currently isolated pathogens. Clin
Hassan Z, Mustafa S, Rahim RA, Isa NM (2016) Anti-breast cancer effects of live, Infect Dis 39(Suppl 1):S25–S31
heat-killed and cytoplasmic fractions of Enterococcus faecalis and Rawla P, Barsouk A (2019) Epidemiology of gastric cancer: global trends, risk
Staphylococcus hominis isolated from human breast milk. In Vitro Cell Dev factors and prevention. Prz Gastroenterol 14:26–38
Biol Anim 52:337–348 Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski
Horsley H, Malone-Lee J, Holland D, Tuz M, Hibbert A, Kelsey M, Kupelian A, Rohn B, Ideker T (2003) Cytoscape: a software environment for integrated models of
JL (2013) Enterococcus faecalis subverts and invades the host urothelium in biomolecular interaction networks. Genome Res 13:2498–2504
patients with chronic urinary tract infection. PloS One 8:e83637–e83637 Song B, Du J, Song DF, Ren JC, Feng Y (2018) Dysregulation of NCAPG, KNL1,
Huycke MM, Abrams V, Moore DR (2002) Enterococcus faecalis produces miR-148a-3p, miR-193b-3p, and miR-1179 may contribute to the progression
extracellular superoxide and hydrogen peroxide that damages colonic of gastric cancer. Biol Res 51:44
epithelial cell DNA. Carcinogenesis 23:529–536 Stahl D, Braun M, Gentles AJ, Lingohr P, Walter A, Kristiansen G, Gütgemann I
Je EM, Yoo NJ, Lee SH (2014) Mutational and expressional analysis of SMC2 gene (2017) Low BUB1 expression is an adverse prognostic marker in gastric
in gastric and colorectal cancers with microsatellite instability. Apmis 122: adenocarcinoma. Oncotarget 8:76329–76339
499–504 Strickertsson JA, Desler C, Martin-Bertelsen T, Machado AM, Wadstrom T, Winther
Kar SP, Beesley J, Amin Al Olama A, Michailidou K, Tyrer J, Kote-Jarai Z, O, Rasmussen LJ, Friis-Hansen L (2013) Enterococcus faecalis infection causes
Lawrenson K, Lindstrom S, Ramus SJ, Thompson DJ, Kibel AS, Dansonka- inflammation, intracellular oxphos-independent ROS production, and DNA
Mieszkowska A, Michael A, Dieffenbach AK, Gentry-Maharaj A, Whittemore damage in human gastric cancer cells. PLoS One 8:e63147
AS, Wolk A, Monteiro A, Peixoto A, Kierzek A, Cox A, Rudolph A, Gonzalez- Strickertsson JA, Rasmussen LJ, Friis-Hansen L (2014) Enterococcus faecalis
Neira A, Wu AH, Lindblom A, Swerdlow A, Ziogas A, Ekici AB, Burwinkel B, infection and reactive oxygen species down-regulates the miR-17-92 cluster
Karlan BY, Nordestgaard BG, Blomqvist C, Phelan C, McLean C, Pearce CL, in gastric adenocarcinoma cell culture. Genes (Basel) 5:726–738
Vachon C, Cybulski C, Slavov C, Stegmaier C, Maier C, Ambrosone CB, Sun X, Clermont PL, Jiao W, Helgason CD, Gout PW, Wang Y, Qu S (2016) Elevated
Hogdall CK, Teerlink CC, Kang D, Tessier DC, Schaid DJ, Stram DO, Cramer expression of the centromere protein-A(CENP-A)-encoding gene as a prognostic
DW, Neal DE, Eccles D, Flesch-Janys D, Edwards DR, Wokozorczyk D, Levine and predictive biomarker in human cancers. Int J Cancer 139:899–907
DA, Yannoukakos D, Sawyer EJ, Bandera EV, Poole EM, Goode EL, Tang NH, Toda T (2015) MAPping the Ndc80 loop in cancer: a possible link between
Khusnutdinova E, Hogdall E, Song F, Bruinsma F, Heitz F, Modugno F, Hamdy Ndc80/Hec1 overproduction and cancer formation. Bioessays 37:248–256
FC, Wiklund F, Giles GG, Olsson H, Wildiers H, Ulmer HU, Pandha H, Risch HA, Wang Q, Zhu Y, Li Z, Bu Q, Sun T, Wang H, Sun H, Cao X (2019) Up-regulation of
Darabi H, Salvesen HB, Nevanlinna H, Gronberg H, Brenner H, Brauch H, SPC25 promotes breast cancer. Aging 11:5689–5704
Anton-Culver H, Song H, Lim HY, McNeish I, Campbell I, Vergote I, Gronwald Wang S, Hibberd ML, Pettersson S, Lee YK (2014) Enterococcus faecalis from
J, Lubinski J, Stanford JL, Benitez J, Doherty JA, Permuth JB, Chang-Claude J, healthy infants modulates inflammation through MAPK signaling pathways.
Donovan JL, Dennis J, Schildkraut JM, Schleutker J, Hopper JL, Kupryjanczyk J, PLoS One 9:e97523
Park JY, Figueroa J, Clements JA, Knight JA, Peto J, Cunningham JM, Pow- Yang Z, Li C, Yan C, Li J, Yan M, Liu B, Zhu Z, Wu Y, Gu Q (2019) KIF14 promotes tumor
Sang J, Batra J, Czene K, Lu KH, Herkommer K, Khaw KT, Matsuo K, Muir K, progression and metastasis and is an independent predictor of poor prognosis in
Offitt K, Chen K, Moysich KB, Aittomaki K, Odunsi K, Kiemeney LA, Massuger human gastric cancer. Biochim Biophys Acta Mol Basis Dis 1865:181–192
LF, Fitzgerald LM, Cook LS, Cannon-Albright L, Hooning MJ, Pike MC, Bolla
MK, Luedeke M, Teixeira MR, Goodman MT, Schmidt MK, Riggan M, Aly M, Publisher’s Note
Rossing MA, Beckmann MW, Moisse M, Sanderson M, Southey MC, Jones M, Springer Nature remains neutral with regard to jurisdictional claims in
Lush M, Hildebrandt MA, Hou MF, Schoemaker MJ, Garcia-Closas M, published maps and institutional affiliations.
Bogdanova N, Rahman N, Le ND, Orr N, Wentzensen N, Pashayan N,
Peterlongo P, Guenel P, Brennan P, Paulo P, Webb PM, Broberg P, Fasching
PA, Devilee P, Wang Q, Cai Q, Li Q, Kaneva R, Butzow R, Kopperud RK,
Schmutzler RK, Stephenson RA, MacInnis RJ, Hoover RN, Winqvist R, Ness R,
Milne RL, Travis RC, Benlloch S, Olson SH, McDonnell SK, Tworoger SS, Maia
S, Berndt S, Lee SC, Teo SH, Thibodeau SN, Bojesen SE, Gapstur SM, Kjaer SK,
Pejovic T, Tammela TL, Dork T, Bruning T, Wahlfors T, Key TJ, Edwards TL,
Menon U, Hamann U, Mitev V, Kosma VM, Setiawan VW, Kristensen V, Arndt
V, Vogel W, Zheng W, Sieh W, Blot WJ, Kluzniak W, Shu XO, Gao YT,
Schumacher F, Freedman ML, Berchuck A, Dunning AM, Simard J, Haiman
CA, Spurdle A, Sellers TA, Hunter DJ, Henderson BE, Kraft P, Chanock SJ,
Couch FJ, Hall P, Gayther SA, Easton DF, Chenevix-Trench G, Eeles R, Pharoah
PD, Lambrechts D (2016) Genome-wide meta-analyses of breast, ovarian, and
prostate cancer association studies identify multiple new susceptibility loci
shared by at least two cancer types. Cancer Discov 6:1052–1067
Khan Z, Siddiqui N, Saif MW (2018) Enterococcus faecalis infective endocarditis
and colorectal carcinoma: case of new association gaining ground.
Gastroenterol Res 11:238–240