In total, 100 isolates, of 385 urine specimens from UTI patients, were identified as UPEC and of which, 62% and 38% were related to females and males, respectively. The age range of patients in this study was between 4 months and 78 years. The highest prevalence of the disease in all isolates was observed in the age range of 0-5 years (29 samples), followed by 18 patients in the range age of 30-39 years in women and 17 patients in the range age of 20-29 years in men (Fig. 1). There was a statistically significant relationship between the frequency of bacteria and gender (P = 0.006).
Antibiotic resistance profiles
Among the UPEC isolates tested in this study, resistance rates were as follow: Piperacillin (92%), Ticarcillin (91%), Amoxicillin/Clavulanic acid (88%), Trimethoprim-sulfamethoxazole and Doxycycline (87%), Cefixime (65%), Aztreonam (59%), Ceftriaxone (58%), Ceftazidime (56%), Nitrofurantoin (45%), Cefoxitin (44%), Norfloxacin and Gentamicin (28%), Ciprofloxacin (27%), Amikacin (11%), and Imipenem (5%). Also, 94% of the isolates were resistant to at least three classes of antibiotics (multidrug-resistant, MDR). Statistical results showed that the overall antibiotic resistance rate in male patients was higher than in female patients (P <0.038), and there was a significant relationship between antibiotic resistance and patient age (Fig. 2). The rate of antibiotic resistance has increased in elderly patients (P <0.05).
Prevalence of virulence genes
The results showed that 96% of the isolates had at least one variant of the genes encoding virulence factors (Fig. 3). The highest and lowest prevalence were related to the fimH gene and the afa gene, respectively. The results also showed a high prevalence of virulence genes among the isolates. The distribution of virulence genes in this study were fimH (96%), aer (47%), papC (36%), cnf1 (16%), hly (15%), and afa (8%) respectively. According to the results, the most frequent gene, fimH, was found in 33% of isolates; the most two frequent genes, fimH and aer, which were identified in 13% of isolates; the most three frequent genes were fimH, aer, and papC, which were identified in 16% of the isolates; the most four frequent genes were fimH, aer, papC, and cnf1, which were identified in 5% of isolates; and finally, the most five frequent genes were fimH, aer, papC, cnf1, and hly genes which were identified in 4% of isolates. A total of six genes together was not detected in any of the isolates.
Prevalence of phylogenetic groups
The classification of the phylogenetic groups of E. coli strains was done by the triplex PCR technique using two genes, ChuA and YjaA, and a DNA fragment of TSPE4.C2 (Fig. 3). The highest prevalence was related to phylogenetic group B2, and there was no observation of phylogenetic group B1 between the studied isolates. In this study, 55% of isolates belonged to group B2, 32% to group D, and 13% to group A.
Relationship between the phylogenetic group and patient profile
Among phylogenetic groups, group B2 had the highest prevalence in female (59.6%) and male (50%) patients. The phylogenetic group D was more prevalent in females (33.8%) than males (28.9%). In contrast, group A had the highest prevalence in men (21%) than women (6.4%). The highest and lowest frequencies of phylogenetic groups were in the range of 0-5 years and 50-78 years, respectively (Fig. 4). No significant difference between the distribution of phylogenetic groups in isolates of male and female patients was shown by statistical analysis. Comparing of the prevalence of phylotypes in both sexes showed that phylotype A is more common in isolates of male patients than females (P = 0.047).
Virulence gene patterns
All the studied strains exhibited 17 virulence gene patterns, referred to as Ec. Ec2 was characterized only by the fimH gene's presence and was the most noted pattern found in 33 isolates. Four isolates belonged to the Ec1 pattern that didn’t have any virulence genes (Table 2). The B2 group had the highest frequency (15 patterns) among the obtained patterns. Group D and A were present in 9 and 7 patterns, respectively.
Table 2 Virulence gene patterns identified among the studied isolates
pattern
|
fimH
|
aer
|
papC
|
cnf1
|
hlyA
|
afa
|
No. of Strains
|
Phylogenetic groups
|
Ec1
|
-
|
-
|
-
|
-
|
-
|
-
|
4
|
B2, D, A
|
Ec2
|
+
|
-
|
-
|
-
|
-
|
-
|
33
|
B2, D, A
|
Ec3
|
+
|
+
|
-
|
-
|
-
|
-
|
13
|
B2, D
|
Ec4
|
+
|
+
|
+
|
-
|
-
|
-
|
16
|
B2
|
Ec5
|
+
|
+
|
+
|
+
|
-
|
-
|
5
|
B2, D, A
|
Ec6
|
+
|
+
|
+
|
+
|
+
|
-
|
4
|
B2, D, A
|
Ec7
|
+
|
+
|
-
|
-
|
-
|
+
|
6
|
B2, D
|
Ec8
|
+
|
-
|
-
|
-
|
+
|
-
|
1
|
B2, A
|
Ec9
|
+
|
-
|
-
|
+
|
+
|
-
|
3
|
B2
|
Ec10
|
+
|
-
|
+
|
-
|
-
|
-
|
3
|
B2, A
|
Ec11
|
+
|
-
|
-
|
-
|
-
|
+
|
2
|
D
|
Ec12
|
+
|
+
|
+
|
-
|
+
|
-
|
2
|
B2, D
|
Ec13
|
+
|
+
|
-
|
+
|
+
|
-
|
2
|
B2
|
Ec14
|
+
|
-
|
-
|
+
|
-
|
-
|
1
|
B2
|
Ec15
|
-
|
+
|
+
|
+
|
-
|
-
|
1
|
A
|
Ec16
|
+
|
-
|
+
|
-
|
+
|
-
|
3
|
B2, D
|
Ec17
|
+
|
-
|
+
|
+
|
-
|
-
|
1
|
B2
|
Total
|
96
|
47
|
36
|
16
|
15
|
8
|
100
|
|
Distribution of antibiotic resistance among phylogenetic groups
The highest rates of resistance among phylogenetic groups were group B2 (56.47%), followed by group A (54.32%) and group D (42.18%). Of the 16 antibiotics studied, the most prevalent antibiotic resistance was observed in phylogenetic group B2 with nine antibiotics, including Amoxicillin/Clavulanic acid, Trimethoprim, Sulfamethoxazole, Doxycycline, Cefixime, Ceftriaxone, Ceftazidime, Cefoxitin, Norfloxacin, and Gentamicin. Phylogenetic group A showed the highest resistance rate to 5 antibiotics of Aztreonam, Nitrofurantoin, Ciprofloxacin, Amikacin, and Imipenem. Phylogenetic group D showed the highest resistance rate to 2 antibiotics of Piperacillin and Ticarcillin. The distribution of antibiotic resistance among phylogenetic groups is shown in Table 3. Significant differences were seen between phylogenetic groups and resistance to all anti-infection agents studied, except for Piperacillin, Amikacin, and Ciprofloxacin (P <0.05).
In the present study, the rate of multidrug resistance among the isolates ranged from 3 to 8 classes of antibiotics of which, 94% of isolates showed MDR resistance to at least three classes of antibiotics. The frequency of samples among the classes of antibiotics showed that most isolates belong to five classes (31.91%), four classes (25.53%), and six classes (22.34%), respectively. The highest prevalence of MDR strains was observed in phylogenetic group B2. The MDR distribution among phylogenetic groups ranks between groups B2 (56.3%), D (30.8%), and A (12.7%), respectively.
Table 3 Distribution of antibiotic resistance among phylogenetic groups
Drugs
|
Total Resistance
|
B2 (55)
|
D (32)
|
A (13%)
|
Piperacillin
|
92%
|
50(90.9%)
|
32(100%)
|
10(76.92%)
|
Ticarcillin
|
91%
|
50(90.9%)
|
31(96.87%)
|
10(76.92%)
|
Amoxicillin clavulanic acid
|
88%
|
49(89.09%)
|
28(87.5%)
|
11(84.61%)
|
Trimethoprim sulfamethoxazole
|
87%
|
50(90.9%)
|
27(84.37%)
|
10(76.92%)
|
Doxycycline
|
87%
|
49(89.09%)
|
27(84.37%)
|
11(84.61%)
|
Cefixime
|
65%
|
40(72.72%)
|
18(56.25%)
|
7(53.84%)
|
Aztreonam
|
59%
|
34(61.81%)
|
16(50%)
|
9(69.23%)
|
Ceftriaxone
|
58%
|
34(61.81%)
|
18(56.25%)
|
6(46.15%)
|
Ceftazidime
|
56%
|
31(56.36%)
|
18(56.25%)
|
7(53.84%)
|
Nitrofurantoin
|
45%
|
21(38.18%)
|
15(46.87%)
|
9(69.23%)
|
Cefoxitin
|
44%
|
29(52.72%)
|
10(31.35%)
|
5(38.46%)
|
Norfloxacin
|
28%
|
17(30.90%)
|
7(21.87%)
|
4(30.76%)
|
Gentamicin
|
28%
|
17(30.90%)
|
8(25%)
|
3(23.07%)
|
Ciprofloxacin
|
27%
|
17(30.90%)
|
5(15.62%)
|
5(38.46%)
|
Amikacin
|
11%
|
8(14.54%)
|
0(0%)
|
3(23.07%)
|
Imipenem
|
5%
|
1(1.81%)
|
1(3.12%)
|
3(23.07%)
|
Total
|
100%
|
497(56.47%)
|
261(42.18%)
|
113(54.32%)
|
Prevalence of virulence genes between the phylogenetic groups
Of the 219 virulence genes observed among the strains studied, the highest frequency was seen in phylogenetic group B2 (58.9%), and the lowest gene prevalence was observed in group D (31.96%) and group A (9.25%), respectively. Exceptionally, the afa gene had the highest prevalence in groups D and B2, A, respectively. Table 4 shows the prevalence rates of each gene between phylogenetic groups.
Table 4 Prevalence of virulence genes among phylogenetic groups B2, D and A
Phylogenetic groups
|
fimH (96)
|
aer (47)
|
papC (36)
|
cnf-1 (17)
|
hlyA (15)
|
afa (8)
|
Total (219)
|
B2 (55)
|
53(55.2%)
|
29(61.7%)
|
23(63.88%)
|
11(68.75%)
|
10(66.66%)
|
3(37.5%)
|
129(58.9%)
|
D (32)
|
32(33.33%)
|
15(31.91%)
|
10(27.77%)
|
4(25%)
|
5(33.33%)
|
4(50%)
|
70(31.965)
|
A (13)
|
11(11.45%)
|
3(6.38%)
|
3(8.335)
|
2(12.5%)
|
0
|
1(12.5%)
|
20(9.25%)
|