Background
Infectious microorganisms are often thought to be associated with human carcinogenesis. For example,
Helicobacter pylori, a Gram-negative bacterium chronically residing in the human stomach, is referred to as a definite carcinogen of gastric cancer [
1‐
3]. Human hepatitis B and C viruses are also established as causative agents of hepatocellular carcinoma [
4,
5]. Similar association is also established between some human papilloma viruses and carcinoma of the cervix uteri [
6].
Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths in the world [
7], and its prevention and early detection are among the most urgent needs regarding public health. Human colon is colonized by more than 10
14 bacteria which can be classified into at least 1000 species, making up what is called the microbiota, and it would be natural to assume that some of these bacteria are associated with the pathogenesis of human CRC. Recently, several studies have indicated that certain strains of
Escherichia coli (
E. coli) possessing a gene cluster named the
pks island might have a causative role in human CRC development [
8,
9].
E. coli, a Gram-negative, facultative anaerobic rod, a member of the family
Enterobacteriaceae, is found widely in the gastrointestinal tract of many mammals, including almost all humans. This species is further divided into phylogenetic groups A, B1, B2, and D. Most strains belonging to group B2 are known for their extraintestinal pathogenic nature causing urinary tract infection, sepsis, and newborn meningitis in humans, which are called extraintestinal pathogenic
E. coli (ExPEC), whereas strains in groups A and D are mostly nonpathogenic commensals or pathogens which mainly cause intestinal disorders presenting as diarrhea [
10,
11]. The
pks island, made up of approximately 54,000 base pairs, consists of genes coding three nonribosomal peptide megasynthases (NRPS), three polyketide megasynthases (PKS), and two hybrid NRPS/PKS megasynthases [
12], and is thought to produce a peptide–polyketide genotoxin named colibactin. This pathogenic island is found mostly in phylogenetic group B2
E. coli [
9,
13,
14], although there are also a much smaller number of
pks-positive strains in group B1
E. coli and other bacterial species in the family
Enterobacteriaceae [
15].
Previous in vitro experiments showed that
pks-positive
E. coli induces DNA double-strand breaks and transient G2-M cell cycle arrest in its host mammalian cells, whereas
pks-negative
E. coli does not [
13,
16]. Those infected host cells can survive after incomplete DNA repair, resulting in higher mutation rates, presumably leading to tumorigenesis. They also showed that
pks-positive
E. coli loses this genotoxic property by knocking out the
pks island, while
pks-negative
E. coli acquires genotoxicity by introducing a gene construct containing the
pks island [
13,
16]. In vivo studies used colitis-susceptible interleukin-10-deficient mice under germ-free condition, and when they were associated with either
pks-positive
E. coli or
pks-negative
E. coli, together with azoxymethane, the group of mice receiving
pks-positive
E. coli showed significantly greater increase in colon tumor occurrence and invasiveness compared to the other group, while the severity of colitis was similar between the two groups [
8]. When host cells are infected with greater number of
pks-positive
E. coli, they show senescence-associated secretory phenotype, by which the cells secrete growth factors such as hepatocyte growth factor, stimulating non-infected neighboring cells to proliferate, also possibly causing tumor formation [
17].
Concerning humans, two studies from Europe showed that
pks-positive
E. coli are more prevalent in colon tissue specimens derived from CRC patients compared to those from non-CRC controls. Arthur et al. studied on tissue specimens from 21 CRC patients and 24 controls and found
pks-positive
E. coli from 67 and 21% of each category, respectively [
8]. Likewise, Buc et al. studied on tissue specimens from 38 CRC patients and 31 diverticulosis patients and found
pks-positive
E. coli from 55 and 19%, respectively [
9]. Thus, the
pks island may act as a tumor promoter for CRC, and could be used as a predictive biomarker for CRC development.
In the present study, we sought to clarify the difference in pks prevalence between CRC patients and non-CRC controls in the Japanese population, by using non-invasive endoscopic sample collection technique.
Methods
Patients
Patients who received total colonoscopy at the University of Tokyo Hospital from January 2014 through May 2015 were asked for participation, and written consent was obtained from every patient who agreed to participate. Clinical characteristics and history of previous and present endoscopic findings of each patient were recorded. Patients were classified into three disease categories: CRC, adenoma, and control; regardless of whether the disease was diagnosed by previous or present session of colonoscopy. In all CRC patients, endoscopic diagnosis was confirmed by pathological examination of previous or concomitant biopsy samples. k-ras gene status of the tumor was determined by direct sequencing of tumor DNA extracted from paraffin-embedded tumor tissues.
This study was approved by the ethics committee of the University of Tokyo (approval number 10329).
Ten milliliters of residual suspension in the colonic lumen after lavage was collected during colonoscopy of every patient, using a syringe connected to the endoscope. The samples were then transiently stored and transported at 4 °C. Solid component within each suspension sample, which contained bacterial cells, was isolated by centrifugation and was washed with phosphate buffer saline for three times, in order to remove possible water-soluble distractors such as polyethylene glycol contained in oral bowel preparation solution. Then total bacterial DNA was extracted using QIAamp DNA Stool Mini Kit (QIAGEN), and was stored at −30 °C until use.
Biopsy samples were washed with phosphate buffer saline in order to remove mucus and mucus-associated bacteria. DNA was extracted from the tissue and co-existing mucosa-associated bacteria using QIAamp DNA Mini Kit (QIAGEN), and was stored at −30 °C until use.
PCR analysis
Both conventional qualitative PCR and real-time quantitative PCR were used to evaluate fecal DNA samples. In order to detect
pks-positive bacterial DNA, primers were used to amplify a 283 bp sequence in
clbB gene of
E. coli, a part of the
pks island, with the forward primer 5′-GCGCATCCTCAAGAGTAAATA-3′ and the reverse primer 5′-GCGCTCTATGCTCATCAACC-3′ [
8]. Primers to amplify a 147 bp sequence in
E. coli-specific gene
uidA with the forward primer 5′-TGGTAATTACCGACGAAAACGGC-3′ and the reverse primer 5′-ACGCGTGGTTACAGTCTTGCG-3′, and those to amplify a 144 bp universal bacterial sequence in 16S rRNA gene with the forward primer 5′-GGTGAATACGTTCCCGG-3′ and the reverse primer 5′-TACGGCTACCTTGTTACGACTT-3′, were also used in order to detect total
E. coli DNA and total bacterial DNA, respectively [
18,
19].
Twenty microliters of reaction mixture for conventional qualitative PCR consisted of 1 μL of template DNA solution, each 1 μL of 10 μmol/L forward and reverse primer solutions, deoxynucleotide mixture, the DNA polymerase AmpliTaq Gold (Applied Biosystems), and buffer according to the manufacturer’s instructions. The PCR conditions were 7 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C, and 7 min at 72 °C.
Patients were considered pks-positive when their fecal bacterial DNA tested pks-positive after conventional qualitative PCR, and for each disease category, pks prevalence was defined as the proportion of the number of pks-positive patients to that of total patients in that category.
We have isolated several
E. coli strains from patients. One of these isolates was positive for
clbB gene,
uidA gene, and 16S rRNA gene by conventional qualitative PCR and DNA sequencing. This isolate was also positive for
clbQ and
clbA genes, which are located near the 5′ and 3′ terminals of the
pks island, respectively, thus suggesting this isolate contained the entire sequence of the
pks island [
14]. Primers to amplify a 308 bp sequence in
clbQ gene with the forward primer 5′-GCACGATCGGACAGGTTAAT-3′ and the reverse primer 5′-TAGTCTCGGAGGGATCATGG-3′, and those to amplify a 342 bp sequence in
clbA gene with the forward primer 5′-AAGCCGTATCCTGCTCAAAA-3′ and the reverse primer 5′-GCTTCTTTGAGCGTCCACAT-3′, were used. We used this
E. coli isolate as the positive control for the
pks island. As the negative control for the
pks island and the positive control for
uidA gene and 16S rRNA gene, str. K-12 substr. MG1655 was used.
Twenty microliters of reaction mixture for real-time quantitative PCR consisted of 2 μL of template DNA solution, each 0.6 μL of 10 μmol/L forward and reverse primer solutions, and the FastStart Universal SYBR Green Master (ROX) mix (Roche) which contains the DNA polymerase. The PCR conditions were 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 60 s at 60 °C, followed by an appropriate dissociation phase.
For each fecal bacterial DNA sample, relative concentration of pks-positive bacterial DNA was defined as the proportion of calculated concentration of pks-positive DNA given by real-time quantitative PCR to that of total bacterial DNA.
Statistical analysis
Continuous variables were compared using the Mann–Whitney U test, and categorical variables were compared using the χ2 test. All tests were two-sided, and a p value <0.05 was considered significant.
Discussion
Our present study is the first to investigate the difference in pks prevalence between CRC patients and non-CRC controls in the Japanese population. It is also unique in that it used colonic lavage samples derived during colonoscopy, and the samples were collected and stored in a uniform method performed by a limited number of investigators with good understanding of the study, which might be advantageous compared to asking patients to collect their stool for themselves. Using colonic lavage samples is less invasive compared to biopsy, which also makes this method easy to practice in clinical settings.
This study showed that the prevalence of
pks-positive
E. coli was not significantly higher in CRC patients compared to controls. Based on the findings in the previous study [
8], we had assumed that
pks prevalence in CRC and control patients would be 67 and 21%, respectively. According to this assumption and a two-sided α level of 0.05, 35 CRC patients and 26 controls in the present study give the statistical power of 92%, which makes it likely that the result in this study is a true negative.
Using colonic lavage samples,
pks prevalence in CRC patients was 43%. This value is lower compared to those of CRC patients in two previous studies from Europe using colon tissue specimens, which are 67 and 55%, respectively (Table
4) [
8,
9]. On the other hand,
pks prevalence in our non-CRC controls (46%) appears higher than their counterparts in those studies (21 and 19%, respectively). This discrepancy in
pks prevalence might simply reflect the difference between mucosa-associated bacteria and those floating in the gut lumen, but in two studies from the United States using stool instead of tissue specimens showed
pks prevalence of 32 and 20% [
14,
20], which are not very different from those using tissue specimens. We also evaluated
pks prevalence using colonic lavage samples and concurrent biopsy samples from 13 patients, which indicated good correlation. Another possible explanation to this discrepancy is the microbial alteration by colonic lavage procedure before colonoscopy. However, several previous reports show such changes rarely overcome interpersonal differences in bacterial profile at phylum or genus level [
21,
22].
Table 4
Comparison of pks prevalence among previous reports and the present study
| Tissue | 45 | UK | CRC 67% Control 21% (p < 0.001) |
| Tissue | 69 | France | CRC 55% Diverticulosis 19% (p = 0.0024) |
| Stool (rectal swab) | 69 | USA | CRC NA Control 32% |
| Stool | 41 | USA (Puerto Rico) | CRC NA Control 20% |
Present study | Colonic lavage | 61†
| Japan | CRC 43% Control 46% (p = 0.798) |
We should also note that healthy Japanese population seems to show relatively high prevalence of phylogenetic group B2 strains among their stool
E. coli compared to their Western counterparts. In one report from Tokyo, Japan, group B2 strain accounted for 44% of all
E. coli isolates [
23]. On the other hand, this value was 16, 32, 33, and 37% in one report from France [
24], 29% in another from the same country [
24], and 48% in one from the United States [
25]. Taking into account that most
pks-positive bacteria belong to phylogenetic group B2
E. coli, this can lead to high prevalence of
pks-positive bacteria among healthy Japanese people, including non-CRC controls in this study.
Post-hoc subgroup analysis on CRC patients in this study showed no significant association between clinical parameters and pks prevalence. However, it might be worth noting that pks-positive E. coli was detected only in k-ras mutant CRC patients, although the difference did not reach to statistical significance due to small sample size. It might imply a possibility that pks-positive E. coli can induce k-ras gene mutation through its genotoxic property.
Authors’ contributions
TS collected, analyzed, and interpreted the patient data regarding colonic lavage samples, biopsy samples, and endoscopic findings, and was a major contributor in writing the manuscript. All authors participated in collecting patient data. All authors read and approved the final manuscript.