In the period of three years, a total of 645 ExPEC strains were studied. 522 of the samples were urine, 54, 28, 21, 6, 12, 3 and 1 of them were wound, blood, sputum, aspirate, sterile body fluid, vaginal and urosepsis, respectively (Fig. 1). In 156 (24%) of the 645 clinical samples, at least 1 virulence gene-positive was found. One hundred fourteen (18%) of these samples had only CNF (cnf1, cnf2, cnf3 and cnf1-cnf3 genes were identified in 78, 12, 20 and 6 of the samples, respectively) (Fig. 5), thirty-six (5,4%) had only CDT (cdt1, cdt2, cdt3, cdt4 and cdt1-cdt4 genes were identified in 20, 4, 4, 4 and 4 of the samples, respectively) (Fig. 6), and six (0,9%) had both toxins-factors) (Fig. 7) (Table 3).
According to the clinical origins of 645 ExPEC isolates, the distribution of virulence factors was found to be 3% in the ExPEC strains of the cdt gene family isolated from the urinary system and 15% in the non-urinary system isolates. While the rate of Cnf gene family was 20% in the urinary system, the ratio was 3% for the cdt gene family (Table 3).
Table 3
Distribution of Toxin Genes in 645 ExPEC strains
VFs (%)
|
All isolates
(n = 645)
n (%)
|
Urinary system
n = 522 (80)
|
Non-urinary system
|
Total
n = 123 (20)
|
Wound (n = 54)
|
Blood
(n = 28)
|
Sputum
(n = 21)
|
Aspirte
(n = 6)
|
Sterile body fluid
(n = 12)
|
Urosepsis
(n = 3)
|
Vagen
(n = 1)
|
cnf1
|
78(12)
|
68 (13)
|
10 (8)
|
3 (5)
|
3 (11)
|
3 (14)
|
1 (16)
|
-
|
-
|
-
|
cnf2
|
12 (2)
|
10 (2)
|
2 (1,6)
|
1 (2)
|
1 (4)
|
-
|
-
|
-
|
-
|
-
|
cnf3
|
20 (3)
|
16 (3)
|
4 (3,2)
|
2 (4)
|
1 (4)
|
1 (5)
|
-
|
-
|
-
|
-
|
cnf1-cnf3
|
6 (0,9)
|
5 (1)
|
1 (0,8)
|
-
|
1 (4)
|
-
|
-
|
-
|
-
|
-
|
Total cnfs
|
116 (18)
|
97 (20)
|
17(13)
|
|
|
|
|
|
|
|
cdt1
|
20 (3)
|
12 (2)
|
8 (7)
|
-
|
1 (4)
|
2 (10)
|
2 (32)
|
2 (16)
|
1 (33)
|
-
|
cdt2
|
4 (0,6)
|
2 (0,3)
|
2 (1,7)
|
1 (2)
|
1 (4)
|
-
|
-
|
-
|
-
|
-
|
cdt3
|
4 (0,6)
|
2 (0,3)
|
2 (1,7)
|
-
|
-
|
1 (5)
|
-
|
-
|
-
|
-
|
cdt4
|
4 (0,6)
|
2 (0,3)
|
2 (1,7)
|
1 (2)
|
-
|
-
|
1 (16)
|
-
|
-
|
-
|
cdt1-cdt4
|
4 (0,6)
|
1 (0,2)
|
3 (3,2)
|
2 (4)
|
1 (4)
|
-
|
1 (16)
|
-
|
-
|
-
|
Total cdts
|
36 (5,4)
|
19 (3)
|
18 (15)
|
|
|
|
|
|
|
|
cnf1-cdt1
|
4 (0,9)
|
3 (0,8)
|
1 (1,7)
|
1 (2)
|
-
|
-
|
-
|
-
|
-
|
-
|
In 156 E. coli isolates, at least one of whose virulence genes defined for ExPEC were positive and therefore can be identified as ExPEC (cnf1, cnf2, cnf3-cdt1, cdt2, cdt3, cdt4), the highest prevalence was cnf1 among the virulence genes with a rate of 50% + 7.6% in binary combinations. This was followed by cdt1 with a rate of 12.8% + 2.5% in binary combinations (Table 4).
Table 4
Distribution of Toxin Genes in CDT and CNF positive 156 ExPEC strains
Virulence factor
|
cnf1
|
cnf2
|
cnf3
|
cnf1-cnf3
|
cdt1
|
cdt2
|
cdt3
|
cdt4
|
cdt1-cdt4
|
cnf1-cdt1
|
Total
(n = 156)
|
78
|
12
|
20
|
6
|
20
|
4
|
4
|
4
|
4
|
4
|
n/total
|
78/156
|
12/156
|
20/156
|
6/156
|
20/156
|
4/156
|
4/156
|
4/156
|
4/156
|
4/156
|
%
|
50
|
7,6
|
12,8
|
3,8
|
12,8
|
2,5
|
2,5
|
2,5
|
2,5
|
2,5
|
In the distribution of virulence genes / gene combinations detected in these strains via the clinical materials from which the strains were isolated, it was observed that 121 (78%) of 156 strains, where at least 1 virulence gene was positive, were associated with the urinary system, and the remaining 35 (22%) were isolated from extra-intestinal urinary infection foci. The most common of the virulence genotypes in 121 clinical materials originating from the urinary system was the CNF gene family with a rate of 78%. The CDT gene family was 17%. In CNF-CDT positive isolates, it was 2%. (Table 5).
While 68 of 78 cnf1 positive strains originated from urinary system, 10 of them were isolated from other extraintestinal infection foci. Similar rates were observed in cnf2 and cnf3. However, among the positive genes of cdt1, cdt2, cdt3, cdt4, only cdt1 proved superiority in the urinary system with a rate of 10%, while cdt2, cdt3 and cdt4 were found in equal percentages. The cdtB gene family proved its superiority, especially in aspirated, sputum, peritoneal fluid, urosepsis and CSF material. The strains with at least 2 gene regions were found to be predominant in strains isolated from non-urinary tract infection. (Table 5).
Table 5
Distribution of factor-toxin genes of 156 ExPEC isolates by clinical origin / The virulence factor distributions in the urinary and non-urinary tract foci in CDT and CNF positive ExPEC strains
n(%)
Urinary system n = 121 (78)
|
|
n(%)
Non-urinary system n = 35 (22)
|
Isolates
VFs
|
Urine
(n = 121)
|
Wound
(n = 11)
|
Blood
(n = 9)
|
Sputum (n = 7)
|
Aspirate (n = 5)
|
CSF
(n = 1)
|
Peritoneal fluid (n = 1)
|
Urosepsis (n = 1)
|
cnf1
|
68 (56)
|
3 (27)
|
3 (33)
|
3 (43)
|
1 (20)
|
-
|
-
|
-
|
cnf2
|
10 (8)
|
1 (9)
|
1 (11)
|
-
|
-
|
-
|
-
|
-
|
cnf3
|
16 (13)
|
2 (18)
|
1 (11)
|
1 (14)
|
-
|
-
|
-
|
-
|
cnf1-cnf3
|
5 (4)
|
-
|
1 (11)
|
-
|
-
|
-
|
-
|
-
|
cdt1
|
12 (10)
|
-
|
1 (11)
|
2 (29)
|
2 (40)
|
1
|
1
|
1
|
cdt2
|
2 (2)
|
1 (9)
|
1 (11)
|
-
|
-
|
-
|
-
|
-
|
cdt3
|
2 (2)
|
-
|
-
|
1 (14)
|
1 (20)
|
-
|
-
|
-
|
cdt4
|
2 (2)
|
1 (9)
|
-
|
-
|
1 (20)
|
-
|
-
|
-
|
cdt1-cdt4
|
1 (1)
|
2 (18)
|
1 (11)
|
-
|
-
|
-
|
-
|
-
|
cnf1-cdt1
|
3 (2)
|
1 (9)
|
-
|
-
|
-
|
-
|
-
|
-
|
Of the 156 ExPEC strains with at least one virulence gene, group b2 was the mostly associated with virulence with a rate of 60.8%. This was followed by group D at a rate of 30.7%. The rates of groups A and b1 were 5.12% and 3.2%, respectively (Table 6).
It was observed that cnf1 was found to be significantly more frequent in the phylogroup b2 urinary tract strains than the phylogroup b2 non-urinary tract strains. While the same situation was valid for group D as well, the opposite was the case in group b1, and it was more common in non-urinary system isolates (Table 6).
Table 6
Distribution of 156 ExPEC isolates virulence genes by phylogenetic group and clinical origin
Virulence genes
|
No. (%) of virulance isolates (n = 156)
|
No (%) of isolates
|
Urinary/Others
(n = 121)/(n = 35)
|
Group A n = 8 (5,1)
|
Group b1 n = 5 (3,2)
|
Group b2 n = 95 (60)
|
Group D n = 48 (30)
|
Urinary/Others
|
Urinary/Others
|
Urinary/Others
|
Urinary/Others
|
cnf1
|
68 (56) 10 (29)
|
0 0
|
0 3 (60)
|
37 (39) 4 (4)
|
30 (62) 4 (8)
|
cnf2
|
10 (8) 2 (6)
|
3 (37) 0
|
0 0
|
7 (7) 2 (2)
|
0 0
|
cnf3
|
16 (13) 4 (11)
|
2 (25) 2 (25)
|
0 0
|
12 (13) 2 (2)
|
2 (4) 0
|
cdt1
|
12 (10) 8 (23)
|
0 1 (13)
|
0 0
|
12 (13) 7 (7)
|
0 0
|
cdt2
|
2 (2) 2 (6)
|
0 0
|
0 0
|
0 0
|
2 (4) 2 (4)
|
cdt3
|
2 (2) 2 (6)
|
0 0
|
0 0
|
0 0
|
2 (4) 2 (4)
|
cdt4
|
2 (2) 2 (6)
|
0 0
|
0 2 (40)
|
2 (2) 0
|
0 0
|
cnf1-cnf3
|
5 (4) 1 (3)
|
0 0
|
0 0
|
5 (5) 1
|
0 0
|
cdt1-cdt4
|
1 (1) 3 (9)
|
0 0
|
0 0
|
1 (5) 3 (3)
|
0 0
|
cnf1-cdt1
|
2 (2) 2 (6)
|
0 0
|
0 0
|
0 0
|
2 (4) 2 (4)
|
With the XbaI-PFGE method, 156 strains determined to be ExPEC based on 80% similarity were collected in 106 clusters. It was revealed that four of the test strains (Z23, Z25, Z36, Z38) had 5 members, 8 of them (A, E, G, Z60, Z64, Z80, Z83 and Z88) had three members and 15 of them had two members (C, F, I, M, O, R, Z2, Z3, Z5, Z6, Z24, Z56, Z58, Z73 and Z76). In response to this, 81 strains were distributed into single-member clusters (Fig. 8).
The strains collected in a single-member cluster and in the same subset were determined to have different virulence factors. ExPEC isolates isolated from blood and urine clinical materials from the patients with urosepsis were found to be distributed in different clusters and had different virulence factors (for EXPEC strains, while cdt1 was positive in the blood sample (Z), it was negative in the urine sample in terms of any virulence factors (Z10)) using the PFGE method (Fig. 9).