Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation

Table 1 List of primers used for PCR assays

Primer name	Sequence (5’-3’)	Region specific for	Ref.
afa-f	CGGCTTTTCTGCTGAACTGGCAGGC	afaC	[49]
afa-r	CCGTCAGCCCCCACGGCAGACC
afa1	GCTGGGCAGCAAACTGATAACTCTC	afaBC
afa2	CATCAAGCTGTTTGTTCGTCCGCCG
afaE-f1	TTAGACCGTACTGTTGTGTTACCCCC
afaE1-r	CATCGCCCGTCGCAGAGCCCAT	afaE1
afaE2-r	GTTTCCCAGTAGACTGGAATGAAGC	afaE2
afaE3-r	CCCTATTGTTGTCGCTGATCAGGAAG	afaE3
daaE-r	CGGCTAGTCATATATAGATTTGTCGC	daaE
afaE5-f	TCAACTCACCCAGTAGCCCCAG	afaE5
afaE5-r	AGGAAGTGGTAGCACCGGTACG
afaE7-f	GCTAAATCAACTGTTGATGTT	afaE7
afaE7-r	GGACAATCCAAATGGCGAATTA
afaE8-f	CTAACTTGCCATGCTGTGACAGTA	afaE8
afaE8-r	TTATCCCCTGCGTAGTTGTGAATC
aggR1	CTAATTGTACAATCGATGTA	aggR	[50]
aggR2	CTGAAGTAATTCTTGAA
pksORF9-10.1KJ	ATTCGATAGCGTCACCCAAC	clbK-clbJ	[51]
pksORF9-10.2KJ	TAAGCGTCTGGAATGCAGTG
CNF-1s	GGGGGAAGTACAGAAGAATTA	cnf1	[51]
CNF-1as	TTGCCGTCCACTCTCACCAGT
CNF-2s	TATCATACGGCAGGAGGAAGCACC	cnf2
CNF-2as	GTCACAATAGACAATAATTTTCCG
CNF3-3D	TAACGTAATTAGCAAAGA	cnf3
CNF-3as	GTCTTCATTACTTACAGT
CDT-s1	GAAAGTAAATGGAATATAAATGTCCG	cdtB-II, cdtB-III, cdtB-V	[51]
CDT-as1	AAATCACCAAGAATCATCCAGTTA
CDT-IIas2	TTTGTGTTGCCGCCGCTGGTGAAA	cdtB-II
CDT-IIIas2	TTTGTGTCGGTGCAGCAGGGAAAA
CDT-s2	GAAAATAAATGGAACACACATGTCCG	cdtB-I, cdtB-IV
CDT-as2	AAATCTCCTGCAATCATCCAGTTA
CDT-Is	CAATAGTCGCCCACAGGA	cdtB-I
CDT-Ias	ATAATCAAGAACACCACCAC
CDT-IVs	CCTGATGGTTCAGGAGGCTGGTTC	cdtB-IV
CDT-IVas	TTGCTCCAGAATCTATACCT
P105	GTCAACGAACATTAGATTAT	cdtC-V
c2767r	ATGGTCATGCTTTGTTATAT
cif-int-s	AACAGATGGCAACAGACTGG	cif	[51]
cif-int-as	AGTCAATGCTTTATGCGTCAT
clbQ-F	TTGTATAGTTACACAACTATTTC
clbQ-R	CCTGTTAGCTTTCGTTC		This study
MIclbQaadA7-F	CATTAAATCATCAAATTAAACGAATTCTATTACACAACAAGGAGTGGGACGCACTGGCATTTAATAACGCGTC
MIClbQaadA7-R	GATGATGGAACAGCCATATCTATTGCTCCTTGTATAGTTACACAACTATTTTTAATCACTTTACTTTTATC

¹The afa-f and afa-r primers detect all afa-positive strains, irrespective of the afaE subtype and the binding properties of the adhesins (Afa/Dr+ and Afa/Dr-). Specific detection of strains encoding Afa/Dr+ adhesins was performed using afa-1 and afa-2 primers;

²Primers used with CDT-s1 primer.

Table 2 Histological grading of intestinal inflammation

Symptoms	Characteristics
Infiltration of inflammatory cells
0	Rare inflammatory cells in the lamina propria
1	Increased numbers of inflammatory cells, including neutrophils in the lamina propria
2	Confluence of inflammatory cells extending into the submucosa
3	Transmural extension of the inflammatory cell infiltrate
Infiltration of epithelium by polynuclear cells
0	No infiltration
1	Surface
2	Inside the crypt
3	Cryptic abscess
Severity of epithelial damage
0	Absence of mucosal damage
1	Lymphoepithelial lesions
2	Mucosal erosion/ulceration
3	Extensive mucosal damage and extension through deeper structures of the bowel wall
Surface of epithelial damage
0	Normal
1	Focal
2	Wide

Table 3 List of primers used for RT qPCR assays

Primer name	Sequence (5’-3’)	Region specific for	Ref.
mmu-26s-FW	TGTCATTCGGAACATTGTAG	S26	This study
mmu-26s-RV	GGCTTTGGTGGAGGTC
mmu-PCNA-FW	CCACATTGGAGATGCTGTTG	PCNA
mmu-PCNA-RV	CAGTGGAGTGGCTTTTGTGA

Table 4 Distribution of Escherichia coli strains producing various cyclomodulins according to phylogroups and specimen origins n (%)

Phylogoups		E. coli strains exhibiting cyclomodulin-encoding genes
		pks	cnf	cdt	cif
Colon cancer-associated E. coli strains (n = 88)1	A (n = 20)	0 (0)	0 (0)	0 (0)	2 (2)
	B1 (n = 14)	0 (0)	1 (1)	1 (1)	1 (1)
	B2 (n = 38)	23 (26)	16 (18)	4 (11)	0 (0)
	D (n = 16)	0 (0)	1 (1)	1 (1)	0 (0)
Diverticulosis-associated E. coli strains (n = 46)2	A (n = 17)	0 (0)	0 (0)	0 (0)	0 (0)
	B1 (n = 3)	0 (0)	0 (0)	0 (0)	0 (0)
	B2 (n = 15)	6 (13)	4 (9)	0 (0)	0 (0)
	D (n = 11)	0 (0)	0 (0)	0 (0)	0 (0)

¹Isolated from 48 colon cancer patients;

²Isolated from 33 patients with diverticulosis. E. coli: Escherichia coli.

Table 5 Phylogroup distribution of cyclomodulin-positive Escherichia coli strains according to specimen origins n (%)

	Phylogroups
	A	B1	B2	D
Cyclomodulin-positive E. coli strains isolated from colon cancer patients	2 (6)	2 (6)	26 (84)	1 (3)
Cyclomodulin-positive E. coli strains isolated from patients with diverticulosis	0 (0)	0 (0)	6 (100)	0 (0)
Cyclomodulin-positive E. coli strains	2 (5)	2 (5)	32 (86)	1 (3)

E. coli: Escherichia coli.

Table 6 Hemolysin expression and presence of cyclomodulin- and adhesin-encoding genes in B2 phylogroup Escherichia coli strains

E. coli strains	Haemolytic phenotype1	Cyclomodulin-encoding genes				Adhesin-encoding genes
		pks	cnf	cdtB	cif	afa	dra	aagR
Colon cancer
1C12	+	+	cnf1	-	-	-	-	-
1D2	-	+	-	-	-	-	-	-
1F8	-	-	-	-	-	-	-	-
2D5	-	-	-	-	-	-	-	-
2F8	+	+	cnf1	cdtB-IV	-	-	-	-
2G2	+	-	-	-	-	-	-	-
4A9	-	-	-	-	-	-	-	-
6A8	+	+	cnf1	-	-	-	-	-
6G8	+	+	cnf1	-	-	-	-	-
6G10	-	-	-	cdtB-IV	-	-	-	-
6G11	-	-	-	-	-	-	-	-
7G1	-	-	-	-	-	-	-	-
7G2	+	+	cnf1	-	-	-	-	-
8A9	+	+	-	-	-	-	-	-
8A10	-	-	-	-	-	-	-	-
8F1	+	-	cnf1	-	-	-	-	-
8G8	-	-	-	-	-	-	-	-
9G5	+	+	cnf1	-	-	+	-	-
10D12	-	+	-	-	-	-	-	-
10E9	+	+	cnf1	-	-	-	-	-
11F1	-	-	-	-	-	-	-	-
11G5	-	+	-	-	-	-	-	-
12B1	+	+	cnf1	-	-	-	-	-
13H2	+	+	cnf1	-	-	-	-	-
14H4	-	+	-	-	-	+	+ (afaE5)	-
15D1	+	-	-	-	-	-	-	-
15D3	+	+	cnf1	-	-	-	-	-
16C1	-	+	-	-	-	-	-	-
17G3	+	-	cnf1	-	-	-	-	-
18C3	+	+	cnf1	-	-	-	-	-
18C5	-	-	-	-	-	-	-	-
18H5	-	+	-	-	-	-	-	-
19D12	+	+	cnf1	-	-	-	-	-
19G1	-	+	-	-	-	-	-	-
19H2	+	+	cnf1	cdtB-IV	-	-	-	-
20B6	+	+	cnf1	-	-	-	-	-
20C3	-	+	-	cdtB-I	-	-	-	-
20D5	-	-	-	-	-	-	-	-
Diverticulosis
1D5	-	-	-	-	-	-	-	-
4D5	+	+	cnf1	-	-	-	-	-
9D7	-	-	-	-	-	-	-	-
9F1	-	-	-	-	-	-	-	-
9F4	-	-	-	-	-	-	-	-
11D9	+	+	cnf1	-	-	-	-	-
12H1	-	+	-	-	-	-	-	-
13D1	+	+	cnf1	-	-	-	-	-
15C1	-	-	-	-	-	-	-	-
16A4	-	+	-	-	-	-	-	-
16A8	-	-	-	-	-	-	-	-
17C1	-	-	-	-	-	-	-	-
17F2	-	-	-	-	-	-	-	-
17E1	-	-	-	-	-	-	-	-
18E6	+	+	cnf1	-	-	-	-	-

¹Alpha-hemolysin expression was analysed after over-night growth of bacteria on agar plates containing sheep blood. cnf: Cytotoxic necrotizing factor; cdt: Cytolethal distending toxins; cif: Cycle-inhibiting factor; afa: Afimbrial adhesion. E. coli: Escherichia coli.