Received: 7 March 2020 | Accepted: 10 August 2020

DOI: 10.1002/pros.24063

ORIGINAL ARTICLE

Escherichia coli, a common constituent of benign prostate

hyperplasia‐associated microbiota induces inflammation
and DNA damage in prostate epithelial cells

Sumeet Jain PhD1,2 | Ajit Gopal Samal MS3 | Biswajit Das PhD1,2 |
Biswaranjan Pradhan PhD4 | Nilanjan Sahu MSc5 | Debasish Mohapatra MSc1,6 |
Prativa Kumari Behera MD7 | Partha Sarathi Satpathi MD8 | Akshaya K. Mohanty BSc9 |
Sanghamitra Satpathi MD7,10 | Shantibhusan Senapati BVSc & AH., PhD1

1
Division of Cancer Biology, Tumor
Microenvironment and Animal Models Lab, Abstract
Institute of Life Sciences, Bhubaneswar,
Odisha, India Background: The role of microbiota in the pathophysiology of benign prostate hy-
2
Manipal Academy of Higher Education, perplasia (BPH), especially in creating an inflammatory milieu may not be avoided.
Manipal, Karnataka, India
The major objectives of this study were to investigate the microbial composition of
3
Department of Surgery, Hitech Medical
College, Rourkela, Odisha, India BPH tissues, its association with inflammation and check the effect of clinically
4
School of Basic Sciences, S. K. Dash Center of isolated bacteria on prostate epithelial cells.
Excellence of Biosciences and Engineering &
Methods: The study includes 36 patients with a pathological diagnosis of BPH.
Technology (SKBET), Indian Institute of
Technology, Bhubaneswar, Odisha, India Following strict aseptic measures, tissues were collected after transurethral
5
School of Biological Sciences, National resection of prostate, multiple pieces of the resected tissues were subjected to
Institute of Science Education and Research,
Bhubaneswar, Odisha, India
histopathological analysis, bacterial culture and genomic DNA extraction.
6
School of Biotechnology, KIIT University, Microbial composition was analyzed by culture and/or next‐generation sequencing
Bhubaneswar, Odisha, India methods. Annotation of operational taxonomy unit has been done with an
7
Department of Pathology, Ispat General
in‐house algorithm. The extent of inflammation was scored through histological
Hospital, Rourkela, Odisha, India
8
Department of Microbiology, Midnapore evaluation of tissue sections. The effect of clinical isolates on nuclear factor‐κB
Medical College, Midnapore, West Bengal, (NF‐κB) activity and induction of DNA‐damage in the prostate epithelial cells
India
9
were evaluated.
Infectious Disease Biology Unit, Institute of
Life Sciences, Bhubaneswar, Odisha, India Results: Histopathological analysis of the BPH tissues showed the presence of
10
Department of Pathology, Hitech Medical inflammation in almost all the tissues with a varied level at different regions of
College and Hospital, Rourkela, Odisha, India
the same tissue section and the level of overall inflammation was different from
Correspondence patients to patients. Microbial culture of tissue samples showed the presence of
Sanghamitra Satpathi, MD, Department of
Pathology, Hitech Medical College & Hospital,
live bacteria in 55.5% (20 out of 36) of the patient tissues. Majority of the
Rourkela, Odisha 769004, India. isolates were coagulase‐positive Staphylococcus, E. coli and Micrococcus spp.
Email: sanghamitra.satpathi@gmail.com
Further, V3 16S rRNA sequencing of the DNA isolated from BPH tissues
Shantibhusan Senapati, BVSc & AH., PhD,
Institute of Life Sciences, Nalco Square, showed the presence of multiple bacteria and the most common phylum in the
Bhubaneswar, Odisha 751023, India. BPH tissues were found to be Proteobacteria, Actinobacteria, Firmicutes, and
Email: senapati@ils.res.in
Bacteroidetes. The E. coli, isolated from one of the tissue was able to activate

Sumeet Jain, Ajit Gopal Samal, and Biswajit Das contributed equally to this work.

The Prostate. 2020;1–12. wileyonlinelibrary.com/journal/pros © 2020 Wiley Periodicals LLC | 1

2 | JAIN ET AL.

NF‐κB and induce DNA damage in prostate epithelial cells. Phospho‐histone

γH2A.X staining confirmed the presence of cells with damaged DNA lesion in
BPH tissues and also correlated with the severity of inflammation.
Conclusion: Our study has shown that the BPH tissues do have a divergent
microbial composition including the commonly found E. coli (phylum Proteobacteria),
and these bacteria might contribute to the BPH‐associated inflammation and/or tissue
damage. The BPH‐associated E. coli induced NF‐κB signaling and DNA damage in
prostate epithelial cells in vitro.

KEYWORDS

BPH, DNA damage, inflammation, microbiota, NF‐κB

1 | INTRODUCTION 2 | M A T E R I A L S AN D M E T H O D S

Benign prostatic hyperplasia (BPH) is one of the common urologic
diseases among ageing men.1 Although prostatitis and BPH are
two different clinical conditions, inflammation is reported to be
associated with both prostatitis and BPH. 2 Previous studies have
demonstrated the presence of E. coli and Enterococcus spp. in BPH
and/or prostatitis tissues.3‐5 The chronic inflammation of the
prostate might be a predisposing factor for prostate cancer
6,7
(PC). Studies have emphasized on the association of microbial
population in PC, 8‐10 suggesting the development of PC through
inflammation associated with BPH and prostatitis. However,
comprehensive knowledge about the microbial composition be-
hind the inflammatory environment in BPH and prostatitis and
progression of PC is understudied. Prostatitis is well categorized
into different subtypes, considering the factors and the level of
inflammation involved 11,12; however, the origin and factors in-
volved in the development of BPH associated inflammation are
not clear.
Studies have attempted to dissect the underlying mechanism
behind the initiation and progression of PC in the context of
inflammation.7,13 The inflammatory milieu in association with
the presence of microorganisms in BPH/prostatitis/PC involves
oncogenic mechanisms like oxidative stress10,14 and DNA
damage.15‐19 Inflammatory signaling involving nuclear factor‐κB
(NF‐κB)20‐22 and cyclooxygenase‐2 (COX2)23,24 is also reported
to be intricate in PC pathogenesis. Our earlier study has also
reported that bacterial component lipopolysaccharide (LPS) can
induce NF‐κB activation in PC cells and promote survival and/or
metastasis of these cells.25,26
In this study, we have analyzed the level of inflammation
associated with BPH. Moreover, we have taken a comprehensive
approach in identifying the bacterial population in these tissues
that might have played a role in initiating and/or maintaining the
inflammatory milieu. We also have evaluated the effect of E. coli
isolated from the BPH tissues on prostate epithelial cells in
inducing DNA damage and NF‐κB activity.
2.1 | Patient tissue sample collection and
processing for various studies
Institutional human ethical committee approval was obtained before
the collection of patient samples. Prostate tissues were obtained
from benign prostate hyperplasia patients admitted for transurethral
prostate resection (TURP) surgery. Written consent was obtained
from all participants for this purpose, and available clin-
icopathological characteristics of the patients were provided in
Table S1. Multiple pieces (1‐2 gm) of the resected tissues were divided
into three parts for histopathological analysis, bacterial culture and
genomic DNA extraction. Microbial composition was analyzed by
selective culture and/or next‐generation sequencing methods. Extent
of inflammation was scored through histological evaluation of tissue
sections. Although tissue samples from 37 patients were collected, due
to suspected contamination of one sample, we excluded that sample
from all of our analysis.
2.2 | Microbial culture of prostate tissue
At the time of TURP, after resection of the prostatic urethra, deep ran-
dom tissue specimens (chip) were collected under sterile conditions. By
collecting tissue fragments directly through the retroscope sheath, the
chance of penile urethra contact and subsequent unwanted contamina-
tion of specimens were minimized. Multiple tissue fragments were taken
from each subject (patient) for bacterial culture, genomic DNA extraction
and histological staining. Pieces of prostatic tissue collected at the op-
eration theater were immediately immersed into brain‐heart infusion
(BHI) broth, Laurate broth, and thioglycolate broth and transported to
the microbiology laboratory. All the broth were incubated for 48 hours at
37°C in an incubator. The tissues for BHI and LB were grounded in a
sterilized mortar and pastel. The grounded tissue was inoculated in three
separate 5% sheep blood agar and MacConkey agar media. The media
were then kept in a candle extinction jar, and the whole set was put in a
JAIN ET AL. | 3

37°C incubator for 48 hours. Another set of culture was kept without a
candle extinction jar at 37°C in an incubator. All the media were ob-
served for any growth. If any growth, the colony was subjected to gram
stain and was identified following the standard method. Any disparity in
the three sets of media was not taken as an isolate from the prostate.
For anaerobic culture, we used 5% sheep blood agar with gen-
tamycin media. The prostatic tissue was immediately inoculated into
the media plate, a metronidazole disc placed in the center and in-
cubated in a Gas pack jar maintaining an anaerobic environment for
growth. The jar was kept at 37°C for 7 days. Metronidazole sensitive
strain was processed as anaerobic strain. The growth was identified
following the standard technique.
2.3 | Immunohistochemistry
Slides were deparaffinised in xylene and rehydrated through 100%,
90%, and 70% alcohol, followed by antigen retrieval in acidic pH
citrate buffer (Vector Lab) by incubating slides in a steamer for
20 minutes. Slides were then washed twice with phosphate‐buffered
saline (PBS) for 5 minutes, and endogenous peroxidase quenching was
carried out in 3% H2O2 (SRL) for 15 minutes. Nonspecific binding was
blocked by incubating the slides with horse serum for 30 minutes,
followed by overnight incubation with (anti‐phospho‐histone H2A.X,
dilution 1:400, #9718, Cell Signaling Technologies; anti‐COX2 anti-
body, dilution 1:100, #NB100‐689, Novus Biologicals) primary anti-
body at 4°C in a humidified chamber. Further slides were incubated
with biotinylated anti‐rabbit/mouse immunoglobulin G (IgG) secondary
antibody for 45 minutes. Diaminobenzidine (Vector Lab) was used to
detect the immunoreactivity. Slides were subsequently counterstained
with haematoxylin (Sigma‐Aldrich), dehydrated through sequential
alcohol grading, cleared in xylene, and mounted with permanent
mounting media (Vector Lab). Stained slides were observed under a
Leica DM500 light microscope, and images were captured.
2.4 | Histopathological evaluation
After microscopic examination of hematoxylin and eosin stained
slides, patients with suspected PC were excluded from this analysis.
At first, the presence of different inflammatory cells in the tissues
was looked for. If the cells were neutrophils or eosinophils, then they
were categorized under acute inflammation and if they were lym-
phocytes, plasma cells or macrophages, then chronic inflammation.
Then the extent of inflammation in the whole tissue was visualized.
Scoring of inflammation was done by using a modified version of the
histopathological classification system developed by Nickel et al.27
For each case, the extent (1 = focal, 2 = multifocal, and 3 = diffuse)
and grade of inflammation (1 = mild, 2 = moderate, and 3 = severe)
were calculated for specific anatomical location of the prostate tissue
(Table 1; Table S2). The combination of extent and grade of in-
flammation was obtained by multiplying them separately for acute
and chronic inflammation. Finally, the score for each case was ob-
tained by summing up the multiplied value of both acute and chronic
inflammation. In this scoring system, the minimum and maximum
expected scores are 0 and 54, respectively.
2.5 | Prostate inflammation scoring system
If more than one grade of inflammation is present for a given ana-
tomical location, the dominant grade and the most severe grade was
specified.
Final Score for inflammation (A + B):
Acute inflammation (A = [Luminal, a = extent X grade] + [Epithelial,
b = extent X grade] + [Stromal, c = extent X grade]) + chronic inflammation
(B = [Luminal, i = extent X grade] + [Epithelial, ii = extent X grade] +
[Stromal, iii = extent X grade]).
The overall BPH associated inflammation is categorized into mild,
moderate or severe based on the final score (A + B). Mild inflammation: if

T A B L E 1 Inflammation scoring system

Feature Description
Anatomical location Histological pattern

Luminal Inflammatory cells within the prostatic gland

Epithelial Inflammatory cells infiltrating the glandular epithelium

Stromal Inflammatory cells infiltrating the rest of the tissue besides the glands

Score Extent Tissue area occupied by inflammatory cell infiltrates

1 Focal <10%
2 Multifocal 10%–50%
3 Diffuse >50%

Score Grade Morphological description

1 Mild Individual inflammatory cells, most of which are separated by distinct intervening spaces (<100)
2 Moderate Confluent sheets of inflammatory cells with no tissue destruction or lymphoid nodule/follicle formation (100–500)
3 Severe Confluent sheets of inflammatory cells with tissue destruction or nodule/follicle formation (>500)
4 |
A + B < 5, Moderate inflammation: if A + B = 5 to10, Severe inflammation:
if A + B > 10.
2.6 | Isolation of genomic DNA from prostate tissue
Total genomic DNA was extracted using phenol and chloroform method.
For this purpose, preserved tissues were brought at room temperature
and washed with PBS once, homogenized, dipped in 1 mL lysis buffer
(100 mM Tris‐HCl pH8.0, 0.5 M NaCl, 10% sodium dodecyl sulfate) with
10 mg/mL Proteinase K and incubated at 55°C for overnight. DNA iso-
lation was performed with phenol‐chloroform‐isoamyl alcohol method.
Finally, DNA pellet was dissolved in 100 μL nuclease‐free water and
stored at −20°C until further use. Samples were analyzed qualitatively
and quantitatively by NanoDrop ND 1000 spectrophotometer
(NanoDrop 1000 Spectrophotometer; Thermo Fisher Scientific).
2.7 | V3 16S rRNA gene sequencing, processing and
operational taxonomic units assignment
Genomic DNA was taken to amplify V3 variable region of 16S rRNA
gene. The polymerase chain reaction (PCR) products were purified,
and forward and reverse barcodes were attached and again purified.
Equimolar amounts of DNA per sample were sequenced. Genomic
DNA was subjected to 16S amplification followed by directional se-
quencing covering the variable region V3. The sequencing data were
submitted to NCBI (SRA accession number PRJNA607921).28
After sequencing, raw data were processed for quality control on the
basis of three different parameters, including base quality score dis-
tributions, average base content per read, and GC distribution in the
reads. Nearly 90% of the total reads have a Phred score greater than 30.
Further, a proprietary wet‐lab approach was used to extract 16S rRNA
V3 region of bacteria from Illumina paired‐end sequences. It includes
following sequential steps (a) trimming of the spacer and conserved re-
gion (b) building consensus V3 region from trimmed paired‐end reads (c)
filters to identify high‐quality V3 region sequences. After trimming the
spacer and conserved region from paired‐end reads, ClustalO program
was used to construct consensus V3 region sequence while applying
multiple filters, such as conserved region filter, spacer filter and mismatch
filter to get only high quality V3 region sequences. While making con-
sensus V3 sequence, the passed reads aligned to each other with 0
mismatches with an average contig length of approximately 135 to 165
base pair. Before final analysis chimeras were also removed using the de‐
novo chimera removal method UCHIME implemented in the tool
USEARCH. Further pre‐processed reads from all samples were pooled
and clustered into operational taxonomic units (OTUs) based on their
sequence similarity using UCLUST program. Singleton OTUs were re-
moved to get total OTUs. Annotation of operational taxonomy units has
been done with an in‐house algorithm (https://github.com/nilanjansahu/
NGS_analysis_plot).29 Briefly, the algorithm works as follows; local BLAST
has been done against 16S rRNA bacterial and archaeal sequence data-
base; the taxonomic information of each OTU has been taken from
JAIN ET AL.
Entrez programming utilities.30,31 Phylum, Class, Order, Family, Genus,
and Species level OTU are selected and stripped out followed by nor-
malization over the whole OTU hits. The OTUs having copy numbers
more than equal to 40 were considered for the percentage calculation of
the taxonomic units.
2.8 | Nuclear factor‐κB activity
The NF‐κB activity was checked by using RWPE‐1‐NF‐κB‐Luc cells and
DU145‐NF‐κB‐Luc cells. The RWPE‐1‐NF‐κB‐Luc cells were cultured in
keratinocyte serum‐free medium supplemented (Invitrogen) with bovine
pituitary extract, human recombinant epidermal growth factor, 100 U/mL
penicillin and 100 μg/mL streptomycin. The DU145‐NF‐κB‐Luc cells were
cultured in DMEM media supplemented with 10% fetal bovine serum,
100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were in-
cubated at 37°C in a humidified incubator with 5% CO2. The NF‐κB‐Luc
cells were generated by using ready to transduce‐replication incompetent
lentivirus as reported earlier.25,26 To check the effect of E.coli on NF‐κB
activity, NF‐κB‐Luc cells were cocultured with different multiplicity of
infection (MOI) of E. coli for 6 hours. After 6 hours of treatment, luciferase
activity was checked by adding in vivo grade luciferin (Promega). The
bioluminescence intensity was measured by Varioskan Flash Multimode
Reader (Thermo Fisher Scientific), and values were presented as a
percentage of control. LPS was used as a positive control.
2.9 | Immunofluorescence
To check the effect of E. coli on DNA damage, RWPE‐1 cells cocultured
with E. coli (MOI 1:200) for 4 hours, the medium was removed, and
cells were washed three times with PBS. Cells were then fixed in 4%
paraformaldehyde for 10 minutes at room temperature followed by
permeabilization with 0.1% Tween‐20 for 5 minutes. Cells were then
blocked for 30 minutes with 2% BSA and then incubated with primary
antibody (dilution 1:400, rabbit anti‐phospho‐histone H2A.X MAb;
#9718; Cell Signaling Technologies) for overnight at 4°C. After in-
cubation cells were washed three times with PBS and 1:500 dilution of
the secondary antibody (goat anti‐rabbit IgG, Alexa Fluor 594 con-
jugate) was added (incubated at RT for 45 minutes). Cells were then
washed three times with PBS, mounted with 4′,6‐diamidino‐2‐
phenylindole containing mounting media. Images were captured
using a Leica DMILLED microscope, and images were captured. The
ImageJ software was used to quantify the number of stained cells.
3 | RE SU LTS
3.1 | Incidence and extent of inflammation in BPH
patient samples
Prostate tissues from patients diagnosed with BPH were checked for
the level of inflammation. In this regard, initially, we set a scoring
JAIN ET AL.
system that takes into consideration of localization, extent and grade
of inflammation in resected prostate tissues. Further, a qualitative
estimation of COX‐2 expression through immunohistochemistry
technique in different patient tissue samples was done to validate
our scoring system. Level of total inflammation was calculated by
considering the extent of inflammation present in stroma, epithelium
and lumen of the BPH tissues. Different level of inflammation was
observed in different BPH tissues (Figure 1A). The total inflammation
scores for individual patients are shown in Table S2. Prostate tissues
with a higher level of the inflammatory score were strongly positive
for COX‐2 expression (Figure 1B). Overall, a certain level of
inflammation was evident in almost all tissues.
3.2 | Microbial composition of BPH
The varied inflammation in different BPH tissue could be due to the
level of infection/presence of different microorganisms. To get an idea
about the overall bacterial composition of BPH tissues, the isolated
samples were subjected to bacterial culture and/or genomic analysis.
Out of 36 samples, 20 samples (55.5%) were culture positive, and all of
them were single isolate. Out of all the culture positive cases, 22.2% of
samples were positive for coagulase‐positive Staphylococcus (Phylum‐
Firmicutes), 11.1% of samples were positive for E. coli (Phylum‐
Proteobacteria), 8.3% of samples were positive for Micrococcus spp.
(Phylum‐Actinobacteria), 5.5% of samples were positive for Enterococcus
spp. (Phylum‐ Firmicutes), 2.8% of samples were positive for Serratia spp.
(Phylum‐Proteobacteria), 2.8% of samples were positive for Pseudomonas
aureginosa (Phylum‐Proteobacteria), and 2.8% of samples were positive
for Pantoea spp. (Phylum‐Proteobacteria) (Figure 2A). A higher percen-
tage of E. coli infection in few samples seen in this study matches with
3,10
previous reports available from other parts of the world. To verify
the culture based identification of isolates, some clinical isolates were
subjected to 16S rRNA sequencing, and the sequencing data (data not
shown) supported the identification based on the culture. The in-
flammation score was obtained for all the BPH tissues (36 tissues;
Table S2), which are culture positive (n = 20) or negative (n = 16) for any
microbe. We observed no significant correlation in the inflammation
score with different microbes detected in corresponding tissue samples
(Figure 2B).
The initial 20 samples collected during the study period were pro-
cessed for microbiota analysis through V3 16S rRNA sequencing (NCBI
SRA accession: PRJNA607921).28 For the microbiome analysis, less
abundant and unknown bacteria were excluded. Based on NGS analysis, a
total number of 26 phyla, 83 classes, 180 orders, 453 families, and 1239
genera were detected. Observed dominant phyla in all type of samples
are Proteobacteria, Actinobacteria, and Firmicutes, followed by Bacteroidetes,
Cyanobacteria, and Chloroflexi (Figure 3A). The tissue samples having a
higher percentage of Proteobacteria were found to have a lower percen-
tage of Firmicutes and Actinobacteria (Figure 3A). Similarly, the tissue
samples with a higher percentage of Actinobacteria and Firmicutes were
found to have lower percentage of Proteobacteria (Figure 3A). One sample
(P9) was found to have an unusually higher percentage (29.8%) of
| 5
Cyanobacteria (Figure 3A). For all the culture‐positive samples, the bac-
teria identified through microbial culture were also present in the list of
bacteria identified through V3 16S rRNA sequencing. The mean per-
centage of the abundance of Proteobacteria is higher compared with other
groups of micro‐organisms (Figure 3B) in BPH tissues used in NGS ana-
lysis. When we segregated the BPH tissues with high (>50%) or low
(<50%) abundance of Proteobacteria and compared the inflammation
score, there was a marginally greater score (statistically not significant) in
high abundance group (Figure 3C). However, we observed a significantly
higher percentage of p‐γ‐H2AX positive cells in tissues with high (>50%)
abundance of Proteobacteria (Figure 3D).
3.3 | Effect of E. coli on NF‐κB activity and DNA
damage in prostate epithelial cells
Our gross histological analysis showed that tissues with a higher level
of inflammation have more COX2 expression (Figure 1B) in both
stromal and luminal cells. The COX2 expression and NF‐κB activation
were co‐ordinately involved in different cancer cells.32‐35 Our mi-
crobial culture and genome analysis data demonstrated E. coli as one
of the most commonly BPH‐tissue associated bacteria. Therefore, we
wanted to investigate the effect of E. coli isolated from BPH tissues
on the NF‐κB activity of prostate epithelial cells. For this purpose,
one clinical isolate identified as E. coli (based on culture and 16S
rRNA sequencing data) was cultured, heat‐killed and treated to
RWPE‐1‐NF‐κB‐Luc cells and DU145‐NF‐κB‐Luc at different MOI
(details mentioned in materials and methods section). The luciferase
intensity measured after 6 hours of treatment showed significantly
higher NF‐κB activity in a dose‐dependent manner (Figure 4A). There
is significantly (P < .005, the Student t test) higher NF‐κB activity in
the cells treated with heat killed E. coli at a MOI of 100 and 200
compared with the untreated cells (Figure 4A).
Different pathological bacteria and/or their components are known
to induce damage to host cellular DNA.15‐17 Frequent DNA damage and
its repair put cells under a risk to acquire genetic mutations. To check
whether the clinical isolates collected in our study has any DNA dama-
ging effect on prostate epithelial cells, we cultured human normal im-
mortalized prostate epithelial RWPE‐1 cells in the presence of the
aforementioned E. coli bacteria. The results (Figure 4B) clearly exhibits
induction of double‐stranded breaks in most of the (90%, P < .005, the
Student t test) etoposide treated cells compared with the untreated cells.
The RWPE‐1 cells treated with bacteria (MOI 200) for 4 hours also
showed significantly (P < .05, the Student t test) higher percentage (56%)
of γ‐H2AX positive cells compared with the uninfected cells. This data
suggests that bacterial infection of prostate might induce DNA damage in
normal prostate epithelial cells.
In human and animal prostate tissue, multiple exogenous factors
could induce inflammation and could induce DNA damage in pro-
static cells. Immunohistochemistry for γ‐H2AX in BPH prostate tis-
sues used in this study clearly showed the presence of DNA damage
(Figure 4C). The γ‐H2AX positive cells were sporadically present in
the sections. The observed differential p‐γ‐H2A.X staining pattern of
6 | JAIN ET AL.
JAIN ET AL. | 7

F I G U R E 2 Bacterial culture report of BPH tissue samples. A, The pie chart indicates the percentage of different bacteria found in BPH tissues
from the microbial culture of tissues collected through transurethral resection of prostate (TURP). N = 36. B, Dot plot showing inflammation score
of individual BPH tissues and their associated groups of microbes (i). Dot plot showing inflammation score of BPH tissues, either positive or
negative result for any bacterial growth (ii). N = 36. BPH, benign prostate hyperplasia [Color figure can be viewed at wileyonlinelibrary.com]

prostatic cells corroborates previous reports.18,19 The extent of
p‐γ‐H2AX staining (as DNA damage readout) was also correlated to
the level of inflammation in different BPH tissues (Figure 4C).
4 | D I S C U S SI O N
Advancement in the high throughput sequencing techniques revealed
the presence of microbes in most of the organs of our body and in
disease conditions their abundance and composition get altered.3,9,10
External factors like geographical location, lifestyle, and food habit
also contribute to the microbiota composition of healthy and dis-
eased organs of our body. Prostatitis or inflammation of the prostate
is one of the main reasons for the urologic problem in men by the age
of 50. Multiple factors, including bacterial infection, are believed to
induce and/or promote prostatitis and BPH.36 Among different
methods to identify and characterize microorganisms, 16S rRNA‐
based PCR assays are more sensitive. To best of our knowledge, the

F I G U R E 1 Extent of inflammation in benign prostate hyperplasia (BPH) tissues. A, Representative hematoxylin and eosin (H&E) stained images
of BPH tissues showing the different extent of inflammation and basement membrane damage (yellow arrowhead). Scale bar = 50 µm.
Mild, moderate and severe represents the level of inflammation (based on scoring, as mentioned in materials and methods). B, Micrograph of
immunohistochemical staining for cyclooxygenase‐2 (COX2) showing the different level of COX2 in BPH tissues with varied inflammation. Scale
bar = 50 µm. Quantification bar graph showing the extent of COX2 staining in the form of % area per field. N = 5, **P < .005, ***P < .001. Mild, moderate
and severe represents the level of inflammation (based on scoring, as mentioned in Section 2) [Color figure can be viewed at wileyonlinelibrary.com]
8 | JAIN ET AL.

F I G U R E 3 Prostate tissue microbiota from 20 BPH patients identified by 16S ribosomal ribonucleic acid (rRNA) sequencing. A, The relative
abundances of bacterial phylum identified in different tissue samples were visualized by bar plot. Each bar represents a subject sample and each
colored box represents a bacterial phylum. The height of a colored box represents the relative abundance of that phylum within the sample.
N = 20. B, Dot plot showing the % abundance of different bacteria from our next‐generation sequencing (NGS) analysis. The statistical analysis
(one way analysis of variance [ANOVA]) was carried out to compare the significance of the difference between the abundance of Proteobacteria
and other groups. ***P < .001. N = 20. C, Dot plot showing the inflammation score of BPH tissues do not have a significant difference if the
tissues were categorized into tissues with high abundance Proteobacteria (>50%) and low Proteobacteria (<50%). D, Dot plot showing significantly
more % p‐γ‐H2A.X positive cells in tissues with a higher abundance Proteobacteria (>50%) than the tissues with low abundance of Proteobacteria
(<50%). **P < .005. BPH, benign prostate hyperplasia [Color figure can be viewed at wileyonlinelibrary.com]

current study is the first report that has used NGS techniques to
profile the microbiota of BPH tissues.
In the current study, to avoid possibilities of postsurgical infec-
tion, all the patients were treated with antibiotics before TURP. Most
of the patients were treated with ceftriaxone + tazobactam, amikacin,
and nitrofurantoin antibiotic. Always, the microbial study raises
doubts about the probable contamination of samples during collec-
tion and processing procedure. Hence, throughout the procedure,
extreme measures were taken at multiple steps to prevent con-
tamination. Because of presurgery antibiotic treatment, there was
uncertainty in getting any viable bacteria from the prostate tissues.
However, the results show that 20 out of 36 patients that were
culture positive for bacteria. This observation supports that the
bacteria isolated through culture were not due to the contamination
that might have happened during tissue isolation and/or handling. In
our study, identification of single bacterial isolates corroborates the
previous reports.37 Identification of Proteobacteria, Firmicutes, and
Actinobacteria as the most common type of phylum in our study also
matches with the previous report.8 Culture of only one type of
bacteria might be due to multiple reasons like selective culture
conditions in the laboratory might have allowed the growth of only
one type of bacteria and/or pretreatment of patients with antibiotic
might have led to the survival of only one type of bacteria, which is
resistant to that particular therapy. Moreover, the pre‐surgical an-
tibiotic treatment might have also effect in reshaping the resident
prostate microbiota, and its consequence on our microbiota profiling
results cannot be ruled out. It is well known that use of TURP can
burn the tissues which may limit the usage of tissue samples in the
downstream experimental procedures. While collecting tissue frag-
ments, we grossly evaluated those tissues and have tried to collect
fragments without any visible damage. During the histo‐pathological
evaluation of all the samples, we did not observe any microscopic
JAIN ET AL. | 9
10 | JAIN ET AL.

tissue damage that might have incurred due to tissue charring.
Despite all these measures, the possible effect of TURP procedure on
our findings can't be completely ruled out.
In the present study, microbiota analysis through bacterial DNA
sequencing showed the presence of multiple bacteria in all the tissues.
The inability of this technique to exactly estimate the live bacteria that
were present in each tissue is a known limitation of this technique.
However, isolation of live bacteria through microbial culture and their
genome identification through DNA sequencing indicates the presence
of certain viable bacteria in those tissues. In future, sequencing of
other variable regions of 16S rRNA gene along with V3 might enable
us identifying bacteria at the species level. The pathological pro-
liferation of both prostatic epithelial cells and fibroblast cells leads to
BPH. Presence of live bacteria and bacterial components like en-
dotoxin might elicit inflammation and activate multiple signaling
events in prostate epithelial and stromal cells. High level of COX2
expression in both stroma and luminal cells indicates the inflammatory
effects on both the cell types. Inflammatory factors like bacterial in-
fection might have both a direct and indirect effect on prostatic epi-
thelial cells. Careful observation of COX2 stained sections showed
that almost all the sites with inflammatory lesions were with COX2
positive stromal cells. However, only in certain cases, the luminal
epithelial cells were strongly positive for COX2 expression. Particu-
larly, the glands with loss of basement membrane integrity were po-
sitive for COX2 expression which might be due to the fact that loss of
basement membrane integrity (Figure 1B; severe inflammation) has
allowed the inflammatory mediators to reach the epithelial cells and
activated the intracellular signaling cascades. In the beginning, we had
an intention of correlating the level of inflammation with the presence
of specific bacteria. The technical limitation arose as the pieces of
tissue used for microbial culture experiment, histopathological analy-
sis, and DNA isolation are different. Further, the level of inflammation
varied from region to region within the same tissue. So, the level of
inflammation could be more reliably correlated with the molecular
events like COX2 expression, DNA damage and basement membrane
damage from the histopathological analysis. Inflammation inducing
property of bacteria depends both on its abundance and type. In our
microbiota analysis, we got phylogenetic information up to genus,
which drastically confined our ability to correlate the presence of any
specific bacteria with the level of inflammation.
At the molecular level, activation of NF‐κB signaling is a major
signaling molecule involved in COX2 overexpression and is known to
have an effect on prostatic cancer cells survival and proliferation.24,38
Moreover, constitutive activation of the NF‐κB pathway is known to
promote PC progression.21 Activation of NF‐κB signaling in normal
immortalized RWPE‐1 prostate cells through PKCϵ is known to
confer a growth advantage to these cells.20 Hence, activation of
NF‐κB signaling in RWPE‐1 cells by clinical isolates underscores the
implication of bacterial infection on prostate epithelial cells survival
and growth. At the same time, bacterial infection and/or chronic in-
flammation induced by these organisms could lead to the transfor-
mation of normal epithelial cells by inducing genetic alteration.
Although there is a clear demarcation between BPH, prostatitis, and
PC,11,12 inflammation is the common phenomena among all the above
disease pathogenesis. It cannot be denied completely the possibility that
the initiation of BPH mediated inflammation may develop chronic pros-
tatitis and may further develop PC.2,10,14,36 In this current study, the
inflammation score used represents the level of inflammation specifically
in BPH tissue which corroborates earlier reports showing BPH‐
associated inflammation.2,27 Though BPH by itself is not a causative
factor for PC, BPH‐associated inflammation and further development of
prostatitis might promote PC initiation and progression.39 The prostatic
epithelial cells if exposed to exogenous and/or endogenous DNA dama-
ging agents, unrepaired DNA damage might result in the transformation
of normal cells. Recent studies have shown the effect of different bacteria
in inducing DNA damage response in eukaryotic cells.16 Certain geno-
toxins produced by bacteria could directly induce DNA damage.17 Pre-
vious studies have already reported E. coli induced DNA damage in vivo
and its effect in triggering genomic instability in mammalian cells.15
Indirect recruitment and activation of immune cells as a host response to
infection would generate a high level of reactive oxygen species, which
are also potent DNA damaging agents.40 Our study has provided evi-
dence that shows the presence of different microbes in BPH tissues. We
observed an association of abundance of Proteobacteria with the level of
inflammation and the level of DNA damage (% of p‐γ‐H2AX positive cells)
(Figure 3).

F I G U R E 4 Role of E. coli on inducing nuclear factor‐κB (NF‐κB) activity and DNA damage in prostate epithelial cells. A, RWPE‐1‐NF‐κB‐Luc
or DU145‐NF‐κB‐Luc (2 × 103) cells were cocultured with different multiplicity of infection (MOI) of E. coli for 6 hours. Bioluminescence was
measured after adding luciferin to all the wells. The result showed that E. coli significantly increased NF‐κB activity in treated cells. The changes
in the luciferase activity were expressed as % of control and results represent the mean ± SD from three independent experiments. *P < .05,
**P < .005, and ***P < .001. N = 3. Lipopolysaccharide (LPS) 10 ng/mL for RWPE‐1‐NF‐κB‐Luc and 10 μg/mL for DU145‐NF‐κB‐Luc cells were
used as a positive control. B, DNA damage ability of clinically isolated E. coli was checked by incubating them with RWPE‐1 cells at an MOI of
200 for 4 hours and then stained for phosphorylated γ‐H2A.X protein. Etoposide, an inducer of DNA double‐stranded breaks, was used as a
positive control. Scale bar = 50 µm. Bar graph showing quantification; 1.74‐fold increases in the number of γ‐H2A.X‐positive cells in infected
RWPE‐1 cells (MOI = 1:200) as compared with uninfected cells. *P < .05, **P < .005, and ***P < .001. N = 5. Etoposide = 100 ng/mL. C, Micrograph
images of phosphorylated γ‐H2A.X positive cells in human BPH tissue with mild, moderate and severe inflammation. The field inside the yellow
dotted rectangle is magnified below the corresponding micrograph images to show the p‐γ‐H2A.X positive cells (yellow arrowhead pointing
positively stained cell). Scale bar = 50 µm. Bar graph represents the quantification expressed as the percentage of positive cells relative to the
total number of cells. *P < .05, **P < .005, and ***P < .001. N = 5. Mild, moderate and severe represents the level of inflammation (based on
scoring, as mentioned in Section 2). DAPI, 4′,6‐diamidino‐2‐phenylindole [Color figure can be viewed at wileyonlinelibrary.com]
JAIN ET AL.
The data showed (Figure 4B) demonstrates the DNA damaging
effect of a clinical isolate (E. coli) on a normal immortalized prostate cell
line (RWPE‐1) in vitro. Although we have not checked the expression
status of different genotoxins in this clinical isolate, induction of DNA
damage in RWPE‐1 cells indicates the direct effect of these bacteria on
these cells. At the same time, though we have not investigated the
effect of bacteria on the viability of RWPE‐1 cells, we believe that DNA
damage response activated in these cells and cells that will fail to repair
the damage may undergo cell death.41 The p‐γ‐H2AX positive cells
detected in BPH tissues indicate the presence of damaged DNA in these
tissues. It also corroborates a previous study that showed increased
oxidative DNA damage in BPH tissues.14 In the future, animal models
that would induce bacterial infection specifically in prostate might help
in understanding the actual contribution of bacterial infection in indu-
cing DNA damage in prostatic cells and its long‐term consequences.7,42
A C K N O W L E D GM E N T S
The authors thank Mr Madan Mohan Mallick and Mr Sushanta Kumar
Swain for their efficient technical support. The authors acknowledge
the director, Institute of Life sciences for supporting the project. The
project is funded by intramural support provided by Institute of Life
Sciences, Bhubaneswar. SBS thanks the Department of Biotechnology,
Govt. of India for the financial support (BT/ILS/Flagship/2019).
CO NFLICT OF I NTERE STS
The authors declare that there are no conflict of interests.
E TH ICS S TA T EM E NT
All experiments involving patient samples were conducted with prior
approval from the Institutional Human Ethical Committee, Institute
of Life Sciences, Bhubaneswar, India and Ispat General Hospital
(IGH), Rourkela, India. All the patients were informed, agreed to
participate and provided the consent to participate.
D A T A A V A I L A B I LI TY S TA T E ME N T
All the data are available in the main text and in the supplementary
material. The data generated, used and/or analyzed during the current
study are available from the corresponding author on reasonable request.
OR CID
Shantibhusan Senapati http://orcid.org/0000-0001-7108-8255
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SUPPO RTING IN F ORMATION
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Jain S, Samal AG, Das B, et al.
Escherichia coli, a common constituent of benign prostate
hyperplasia‐associated microbiota induces inflammation and
DNA damage in prostate epithelial cells. The Prostate.
2020;1–12. https://doi.org/10.1002/pros.24063