Antimicrobial resistance patterns of isolates
Among 300 urine samples included in this study, 100 E. coli isolates were detected. The demographic information and distribution of E. coli isolates are shown in Table 1. The isolates showed resistance patterns to the following antibiotics: AMP (92%; n=92), CTX (85%; n=85) CZA (74%; n=74), SXT (63%; n=63), ATM (54%; n=54), NAL (51%; n=51), CIP (49%; n=49), SAM (38%; n=38), amoxicillin-clavulanic acid (31%; n=31), TZP (26%; n=26), NFT (21%; n=21), AMK (20%; n=20), gentamicin (18%; n=18), FO (18%; n=18), ETP (12%; n=12), IPM (12%; n=12), and Mpm (%11; n=11). A total of 36/100 (36%) E. coli isolates were phenotypically ESBL producers. In ESBL-producing isolates, 14/36 (38.8%) and 12/36 (33.3%) were temocillin and carbapenem resistant, respectively, and 11/36 (30.5%) were CP-E.coli, with a MIC≥4 µg/ml against Mpm. In addition, 9/36 (25%) and 6/36 (16.6%) of the isolates were resistant to CAZ/AVI (MIC≥128 µg/ml) and colistin (MIC>4 µg/ml), respectively. The E. coli isolates indicated multiple resistance to antibiotics cephalosporins, sulfonamides, and fluoroquinolones, and 48/100 (48%) of the isolates were classified as MDR. Prior antibiotic consumption and hospitalization were significant risk factors for the isolation of ESBL E. coli isolates (p=0.01 and p=0.02). Noticeably, there was no association between age, gender, different wards and underlying diseases with EP- E. coli isolation (p>0.05).
Table 1. Epidemiological characteristics of ESBL and non-ESBL-producing E. coli
P
|
Non-ESBL
No. (%)
(N=64)
|
ESBL No. (%)
(N=36)
|
Total
(N=100)
|
Characteristics
|
0.907
|
51.5
(n=33)
48.5
(n=31)
|
52.8 (n=19)
47.2 (n=17 )
|
52
48
|
Gender
Male
Female
|
0.315
|
20.3
(n=13)
34.3
(n=22)
45.3
(n=29)
|
13.8
(n=5)
25.0
(n=9)
6.1
(n=22)
|
18
31
51
|
Age
<40
40-60
>60
|
0.933
|
15.6
(n=10)
40.6
(n=26)
26.5
(n=17)
12.5
(n=8)
4.6
(n=3)
|
22.2
(n=8)
38.8
(n=14)
25.0
(n=9)
11.11
(n=4)
2.7
(n=1)
|
18
40
26
12
4
|
Clinical distribution
ICU
Nephrology
Hematology
Emergency
Other
|
0.022
|
54.6
(n=35)
45.3
(n=29)
|
77.7 (n=28)
22.3
(n=8)
|
63
37
|
Healthcare occupation
Positive
Negative
|
0.01
|
45.3
(n=29)
54.6
(n=35)
|
88.8 (n=32)
11.1
(n=4)
|
61
39
|
Prior antibiotic use
Positive
Negative
|
0.04
|
17.2
(n=12)
|
38.8 (n=14)
|
26
|
Biofilm formation
|
0.937
|
26.5
(n=17)
18.7
(n=12)
12.5
(n=8)
10.9
(n=7)
15.6
(n=5)
6.25
(n=4)
17.1
(n=11)
|
27.7
(n=10)
22.2
(n=8)
13.8
(n=5)
13.8
(n=5)
8.3
(n=3)
0.0
(n=0)
13.8
(n=5)
|
27
20
13
12
8
4
16
|
Underlying disease
Diabetes
Cancer
Kidney disease
Liver disease
Hart disease
Other
Non Underlying disease
|
Prevalence of ESBL and carbapenemase genes
Among the 36 ESBL-producing E. coli isolates surveyed, 29 (80.5%), 19 (52.7%), and 17 (47.2%) harbored blaCTX-M, blaTEM, and blaSHV genes, respectively. All 36 (100%) isolates carried the blaCTXM gene and was also ESBL producers. Among 11 carbapenemase-producing E. coli isolates, 54.5% (n=6) and 18% (n=2) harbored blaNDM and blaOXA-48 genes, respectively, and 27.2% (n=3) of the isolates harbored both blaNDM and blaOXA-48 genes. None of the isolates carried blaKPC, blaIMP, and blaVIM genes.
Analysis of isolates by PFGE
PFGE analysis of 36 EP- E. coli demonstrated 34 pulsotypes classified into 32 singletons with unique patterns, as well as 2 clusters, each consists of two strains numbered from P1 to P34. Distribution of pulsotypes in different hospital wards and different dates is shown in Figure 1.
Results of biofilm formation
Totally 26% (26/100) isolates were biofilm producer. As showed in table 1, Biofilm formation has significant association with EP- E. coli isolates (p=0.04). In EP- E. coli 14 isolates were biofilm producers which 28.5% (n=4/14) was strong biofilm producers, while 57.1% (n=8/14) and 14.2 % (n=2/14) were moderate and weak biofilm producers, respectively.
Recognition of phylogroups and serogroups
The majority of the EP- E. coli isolates belonged to the phylogenetic groups B2 (52.7%; n=19/36) and D (33.3%; n=12/36), followed by groups B1 (8.3%; n=3/36), A (2.7%; n=1/36), and F (2.7%; n=1/36). Also, serogroup O1 was detected in 36.1% (n=13/36) of the isolates and then in serogroups O25 (22.2%; n=8/36), O75 (13.8%; n= 5/36), O18 (8.3; n=3/36), O15 (5.5; n=2/36), O4 (2.7%; n=1/36), and O16 (2.7%; n=1/36). The serogroup of 8.3% (n=3/36) of the isolates could not be detected.
Identification of virulence factor genes
Among the EP- E. coli, 97.2% (n=35), 86.1% (n=31), 83.3% (n=30), 80.5% (n=29), 33.3% (n=12), 30.5% (n=11), and 27.7% (n=10) carried fimH, iutA, fyuA, inh, traT, papП, and csgA genes, respectively. In addition, 30.5% (n=11), 27.7% (n=10), 25% (n=9), 19.4% (n=7), 16.6% (n=6), 13.8% (n=5), 13.8% (n=5), and 8.33% (n=3) carried fimA, ompT, usp, sfa/foc, hly, cnf-1, afa, and iroN, respectively.
Detection of ST131 clone
A total of 19.4% (n=7/36) isolates in EP- E. coli isolates were identified as ST131. All the isolates (100%; n=7/7) were detected as O25b-ST131 clone, which 57.1% (n=4/7) were carbapenemase-producing isolates containing blaCTX-M, blaNDM and blaOXA-48 genes (Table 2). As shown in Table 2, most of the carbapenemase-producing E. coli isolates were serogroup O1, and three of these isolates containing blaNDM and blaoxa-48 genes belonged to O25 (ST131 clone).
Table 2. Characteristics of Carbapenemase-producing E.coli isolates
ESBL
|
Mpm
MIC (µg/mL)
|
CTX
MIC (µg/mL)
|
CAZ/AVI MIC
(µg/mL)
|
Colistin
MIC
(µg/mL)
|
Temocillin sensitivity
|
Resistance genes
|
Phyl/Sero
|
Sequence typing
|
Yes
|
64
|
32
|
128
|
0.5
|
R
|
CX-M/OXA-48/NDM
|
B2/O25
|
ST131
|
Yes
|
16
|
32
|
128
|
16
|
R
|
CTX-M/NDM
|
B1/O1
|
Non-ST131
|
Yes
|
8
|
32
|
128
|
32
|
R
|
CTX-M/NDM
|
B2/O1
|
Non-ST131
|
Yes
|
8
|
32
|
128
|
0.5
|
S
|
CTX-M/NDM
|
B2/O25
|
ST131
|
Yes
|
8
|
32
|
128
|
1
|
R
|
CX-M/OXA-48/NDM
|
B2/O25
|
ST131
|
Yes
|
128
|
32
|
128
|
16
|
R
|
CTX-M/NDM
|
B2/O1
|
Non-ST131
|
Yes
|
4
|
32
|
0.5
|
0.5
|
R
|
CTX-M/OXA-48
|
B2/O1
|
Non-ST131
|
Yes
|
128
|
32
|
128
|
0.5
|
R
|
CX-M/OXA-48/NDM
|
B2/O25
|
ST131
|
Yes
|
4
|
32
|
0.5
|
1
|
S
|
CTX-M/OXA-48
|
B1/O1
|
Non-ST131
|
Yes
|
4
|
32
|
128
|
32
|
R
|
CTX-M /NDM
|
B2/O1
|
Non ST131
|
Yes
|
4
|
32
|
128
|
0.5
|
R
|
CTX-M/NDM
|
B2/O16
|
Non-ST131
|