# Characterization of Integrons and Antimicrobial Resistance inEscherichia coli Sequence Type 131 Isolates

Authors: Jiangqing Huang; Fangjun Lan; Yanfang Lu; Bin Li
Journal: Canadian Journal of Infectious Diseases and Medical Microbiology
 (2020)

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## Abstract

Background. Escherichia coli sequence type 131 (ST131) is an important multidrug-resistant extraintestinal pathogen, which can cause many kinds of infections. Integrons may play a crucial role in the dissemination of antibiotic resistance genes. The purpose of this study was to characterize the prevelance of integrons among E. coli ST131 strains in China. Methods. Eighty-three E. coli ST131 isolates were used in this study. The antibiotic susceptibility test was performed by the disk diffusion method. The presence and characterization of class 1, 2, and 3 integrons, as well as promotor of gene cassettes and other antimicrobial resistance genes, were detected by PCR and DNA sequencing. Transfer of integrons was carried out using a broth culture mating method. Clonal relatedness of E. coli ST131 isolates was analyzed by PFGE. Results. Overall, 26.5% (22/83) of the E. coli ST131 isolates carried class 1 integrons. Class 2 and 3 integrons were not found in this study. Two types of gene cassette arrays were demonstrated in this study and were as follows: dfrA17-aadA5 and aac(6′)-Ib-cr-cmlA5. Only one type of Pc promoter variant was detected among 22 integron-positive isolates (PcW). In vivo transfer of integron was successful for 9 of integron-positive E. coli ST131 isolates harboring resistance gene cassettes. Results of PFGE demonstrated that the integron-positive E. coli ST131 isolates were grouped into 12 different PFGE clusters. Conclusions. Our study showed a low prevalence of integrons was detected in E. coli ST131. Continued surveillance of this mobile genetic element should be performed to study the evolution of antibiotic resistance among E. coli ST131.


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## Body

## 1. Introduction

Integrons are genetic elements found in bacteria, which can capture and express antimicrobial resistance gene cassettes [1]. Integrons are strongly associated with the dissemination of antimicrobial genes among different genera of bacteria [2]. Several types of integron classes have been found nowadays in bacteria [3]. Integrons are often located on transmissible plasmids or transposons, which can facilitate the transferability of antimicrobial genes among bacteria [4].Escherichia coli sequence type 131 (E. coli ST131) is an important multidrug-resistant extraintestinal pathogen. It is firstly identified in 2008 and now disseminated globally [5]. E. coli ST131 is becoming the predominant extraintestinal pathogenic E. coli lineage and can cause many clinical infections, such as sepsis and urinary tract infections [6]. E. coli ST131 are usually reported to produce extended spectrum β-lactamases and show resistance to fluoroquinones [7,8]. The emergence of such pathogen has made a significant contribution to the rising prevalence of antibiotic resistance [9].Integrons, especially class 1 and 2 ones, have a worldwide distribution inEnterobacteriaceae [10, 11]. This has led to the speculation that integrons probably involve in the dissemination of antimicrobial resistance genes in E. coli ST131. Thus, in this study, we determined the distribution of various integrons among E. coli ST131 collected at a university hospital in southern China.


## 2. Materials and Methods

### 2.1. Bacterial Isolates

A total of 83E. coli ST131 were used in this study, which were collected at a Chinese university hospital (Fujian Medical University Union Hospital, Fuzhou, Fujian province, China) between August 2014 and August 2015. These strains were isolated and reported in our previous study [8].


### 2.2. Antimicrobial Susceptibility Testing

The antimicrobial sensitivity was tested by the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI, 2016) guidelines [12]. The antibiotics used for the susceptibility tests were cefotaxime (CTX), ceftazidime (CAZ), cefepime (FEP), ciprofloxacin (CIP), levofloxacin (LEV), amikacin (AMK), imipenem (IMP), piperacillin-tazobactam (TZP), aztreonam (ATM), trimethoprim-sulfamethoxazole (SXT), and ertapenem (ETP). E. coli ATCC 25922 was used for quality control.


### 2.3. Detection of Integrons, Their Pc Promoters, and Associated Resistance Genes

The presence of class 1, 2, and 3 integrons was analyzed by PCR inE. coli ST131 as described previously [13]. The characterization of the variable region of integrons was detected by PCR and DNA sequencing. In those isolates that were positive for intI1, the Pc promotor region was detected as described previously [14].The presence of genes associated with ampicillin (blaTEM, blaSHV, and blaOXA-1), tetracycline (tetA-tetE), streptomycin (aadA), sulphonamide (sul1, sul2, and sul3), kanamycin (aphA1 and aphA2), chloramphenicol resistance (cmlA and floR), and plasmid-borne quinolone resistance genes (qnrA, qnrB, and qnrS) was also analyzed by PCR [13, 15].


### 2.4. Conjugation Experiments and Plasmids Detection

A conjugation experiment was performed in broth culture withE. coli J53 Azr as the recipient [16]. Conjugation experiments were carried out with integron-positive strains harboring resistance gene cassettes. Transconjugants were selected on MacConkey agar plates containing streptomycin at 50 mg/liter and sodium azide at 100 mg/liter for selection. The grown isolates were selected and identified by the Vitek system.


### 2.5. Pulsed-Field Gel Electrophoresis Analysis (PFGE)

The integron-positiveE. coli ST131 isolates were subjected to molecular typing using XbaI digestion by pulsed-field gel electrophoresis, which clonally evaluates isolates. A PFGE dendrogram was constructed with BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) according to the unweighted pair group method based on Dice coefficients. Isolates with a Dice similarity index ≥70% were considered to belong to the same PFGE cluster [17].


### 2.6. Statistical Analysis

SPSS22.0 statistical software was used for statistical analysis, and the chi-square test or Fisher’s exact test (two tailed) or Mann–Whitney test was performed for data comparison. Onlyp<0.05 was considered statistically significant.


## 2.1. Bacterial Isolates

A total of 83E. coli ST131 were used in this study, which were collected at a Chinese university hospital (Fujian Medical University Union Hospital, Fuzhou, Fujian province, China) between August 2014 and August 2015. These strains were isolated and reported in our previous study [8].


## 2.2. Antimicrobial Susceptibility Testing

The antimicrobial sensitivity was tested by the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI, 2016) guidelines [12]. The antibiotics used for the susceptibility tests were cefotaxime (CTX), ceftazidime (CAZ), cefepime (FEP), ciprofloxacin (CIP), levofloxacin (LEV), amikacin (AMK), imipenem (IMP), piperacillin-tazobactam (TZP), aztreonam (ATM), trimethoprim-sulfamethoxazole (SXT), and ertapenem (ETP). E. coli ATCC 25922 was used for quality control.


## 2.3. Detection of Integrons, Their Pc Promoters, and Associated Resistance Genes

The presence of class 1, 2, and 3 integrons was analyzed by PCR inE. coli ST131 as described previously [13]. The characterization of the variable region of integrons was detected by PCR and DNA sequencing. In those isolates that were positive for intI1, the Pc promotor region was detected as described previously [14].The presence of genes associated with ampicillin (blaTEM, blaSHV, and blaOXA-1), tetracycline (tetA-tetE), streptomycin (aadA), sulphonamide (sul1, sul2, and sul3), kanamycin (aphA1 and aphA2), chloramphenicol resistance (cmlA and floR), and plasmid-borne quinolone resistance genes (qnrA, qnrB, and qnrS) was also analyzed by PCR [13, 15].


## 2.4. Conjugation Experiments and Plasmids Detection

A conjugation experiment was performed in broth culture withE. coli J53 Azr as the recipient [16]. Conjugation experiments were carried out with integron-positive strains harboring resistance gene cassettes. Transconjugants were selected on MacConkey agar plates containing streptomycin at 50 mg/liter and sodium azide at 100 mg/liter for selection. The grown isolates were selected and identified by the Vitek system.


## 2.5. Pulsed-Field Gel Electrophoresis Analysis (PFGE)

The integron-positiveE. coli ST131 isolates were subjected to molecular typing using XbaI digestion by pulsed-field gel electrophoresis, which clonally evaluates isolates. A PFGE dendrogram was constructed with BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) according to the unweighted pair group method based on Dice coefficients. Isolates with a Dice similarity index ≥70% were considered to belong to the same PFGE cluster [17].


## 2.6. Statistical Analysis

SPSS22.0 statistical software was used for statistical analysis, and the chi-square test or Fisher’s exact test (two tailed) or Mann–Whitney test was performed for data comparison. Onlyp<0.05 was considered statistically significant.


## 3. Results

### 3.1. Integrons, Pc Promoters, and Associated Resistance Genes

Of the 83E. coli ST131 isolates, 22 (27%) carried class 1 integrons. Class 2 and class 3 integrons were not detected in the study. The clinical characteristics of E. coli ST131 are showed in Table 1. The result of serotype revealed the integron-positive E. coli ST131 belonged to two types, and the most prevalence type was O25b (77.3%, 17/22). Results of phylogenetic analysis showed that 21/22 (95.5%) of isolates belonged to group B2 and 1/22 (4.5%) were D group. Several types of fragments ranging in size from 0.15 kb to 3.0 kb were identified (Table 2). Sixteen isolates carried a single integron with a same gene cassette (dfrA17-aadA5). Two gene cassette arrangements were detected in 19 (86.4%) of integron-positive isolates. In addition, only one isolate carried the aac(6′)-Ib-cr-cmlA5 gene cassette array with the largest size (3.0 kb). In this study, only one Pc variant (PcW) was found in 22 integron-positive E. coli ST131, as shown in Table 1.Table 1
Phenotypic and genotypic characteristics of 22 integron-positiveE. coli ST131 isolates.
Isolates
Ward
Source
Age
Sex
Genetic material in isolate with integron
Serotype
Phylogenetic group
Resistance proflies
Conjugation experiments
Promoter
Gene cassette(s)
EC327
Urinary surgery
Urine
48
F
PcW
−1
O25b
B2
SXT
EC431
Emergency surgery
Drain
78
F
PcW
−
O16
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXTCTX, CIP, LEV, SXT
EC455
Colorectal surgery
Drain
62
F
PcW
dfrA17-aadA5
O16
B2
+2
EC485
Urinary surgery
Urine
60
M
PcW
aac(6′)-Ib-cr-cmlA5
O16
B2
−
EC541
Neurosurgery
Blood
69
M
PcW
dfrA17-aadA5
O25b
B2
CTX, SXT
EC549
Neurosurgery
Urine
39
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, AMK, ATM, SXT
EC568
Hepatobiliary surgery
Blood
75
M
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, AMK, ATM, SXT
EC570
Urinary surgery
Urine
51
F
PcW
dfrA17-aadA5
O25b
B2
CTX, FEP, CIP, LEV, AMK, ATM, SXT
EC578
Urinary surgery
Urine
68
M
PcW
dfrA17-aadA5
O16
D
CTX, FEP, CIP, LEV, SXT
+
EC668
Oncology
Urine
59
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CIP, LEV, SXT
+
EC686
Cardiology
Urine
78
M
PcW
dfrA17-aadA5
O16
B2
CIP, LEV, SXT
+
EC771
Outpatient
Blood
61
F
PcW
-
O25b
B2
SXT
EC786
Nephrology
Blood
64
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXT
+
EC792
Colorectal surgery
Secretion
52
M
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXT
EC913
Gastrointestinal surgery
Urine
52
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CIP, LEV, SXT
+
EC934
Hematology
Urine
54
F
PcW
−
O25b
B2
SXT
EC935
Emergency surgery
Secretion
55
F
PcW
−
O25b
B2
SXT
EC978
Hematology
Blood
43
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXT
+
EC1044
Hematology
Urine
64
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXT
+
EC1065
Urinary surgery
Urine
56
M
PcW
dfrA17-aadA5
O25b
B2
SXT
EC1085
Oncology
Ascitic fluid
64
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXT
+
EC1151
Outpatient
Blood
82
M
PcW
dfrA17-aadA5
O25b
B2
CTX, FEP, CIP, LEV, ATM, SXT
F, female; M, male; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CIP, ciprofloxacin; LEV, levofloxacin; AK, amikacin; IPM, imipenem; TZP, piperacillin-tazobartan; ATM, aztreonam; SXT, trimethoprim-sulfamethoxazole; ETP, ertapenem,1−: not detected, 2+: successfully transferred.Table 2
Size, number, and gene cassetes amplified from the variable region of integron inE.coli ST131.
Approximate length (kb)
Isolate (n)
Gene cassette (s)
0.151
2
—
1.6
4
dfrA17-aadA5
1.6 + 0.72
12
dfrA17-aadA5
3.0
1
aac (6′)-Ib-cr-cmlA5
Total
19
1The 0.15 kb amplicon was found to be a integron variable region without any gene cassette present but contained partial sequences of 5′ and 3′ conserved segments of integrons. 2The 0.7 kb amplicon was found to be a integron variable region without any gene cassette present.Table3 shows the antimicrobial resistance genes detected in our E. coli ST131 isolates. The blaTEM gene was predominant in integron-positve E. coli ST131 isolates, and the blaOXA-1 gene was in 9 isolates. The blaSHV gene was not found in this study. Regarding tetracycline resistance, tetA + tetC was more common in those strains with integron than those strains without integron. The following genes were detected among 56 SXT-resistant isolates (integron-positive/integron-negative isolates): sul1 (15/7), sul2 (2/5), and sul1 + sul2 (3/20). Among the 16 ciprofloxacin-resistant integron-positive isolates, the qnrB gene was detected in 2 isolates. Among the 22 integron-positive E. coli ST131 isolates, the clmA gene and clmA + floR gene were found in two different isolates. The aadA gene was observed in two of 22 integron-positive isolates, and none of these isolates carried aphA1 and aphA2 gene which conferred kanamycin resistance.Table 3
Genes of resistance detected amongE.coli ST131 strains with and without integrons.
Antibiotics
Integron-positive strains (n = 22)
Integron-negative strains (n = 61)
No. of resistance isolates
Genes detected (no. of strains)
No. of resistance isolates
Genes detected (no. of strains)
Ampicillin
—∗
bla
TEM (11)
—
bla
TEM (23)
bla
OXA-1 (9)
bla
OXA-1 (1)
Tetracycline
—
tetA (1)
—
tetA (20)
tetC (3)
tetB (2)
tetA + tetB (6)
tetC (8)
tetA + tetC(10)
TetA + tetC (10)
tetB + tetC (1)
Sulphonamide
21
sul1 (15)
35
sul1 (7)
sul2 (3)
sul2 (5)
sul1 + sul2 (3)
sul1 + sul2 (20)
Ciprofloxaci
16
qnrB (2)
43
qnrS (1)
Streptomycin
—
aadA (2)
—
ND
Kanamycin
—
ND
—
aphA1 (1)
aphA1 + aphA2 (1)
Chloramphenicol
—
cmlA (1)
—
floR (2)
clmA + floR (1)
∗Not detected; ND:no gene detected


### 3.2. Antimicrobial Susceptibility Testing

E. coli ST131 showed high resistance to CTX (71.1%), CIP (69.9%), LEV (69.9%), and SXT (67.5%). However, resistance rates were low to ATM (36.1%), FEP (31.3%), CAZ (26.5%), AK (8.4%), IPM (3.6%), and TZP (1.2%). None of the isolates were resistant to ETP. The results of the antimicrobial susceptibility test were shown in Table 4. Compared with integron-negative E.coli ST131, integron-positive ones were shown to have a higher resistance to SXT and FEP (p<0.05, respectively). Multidrug resistance (defined as resistance to six or more antibiotics) rates of integron-positive and -negative E.coli ST131 isolates were 45.5% and 18.0%, respectively.Table 4
Association between antibiotic profile and integron inE. coli ST131.
Antibiotic
Antibiotic susceptibility
p value
All isolates (n = 83)
Integron-positive isolates (n = 22)
Integron-negative isolates (n = 61)
S (%)
I (%)
R (%)
S (%)
I (%)
R (%)
S (%)
I (%)
R (%)
CTX
25.3
3.6
71.1
31.8
0.0
68.2
23.0
4.9
72.1
0.726
CAZ
49.4
24.1
26.5
54.5
9.1
36.4
47.5
29.5
23.0
0.222
FEP
55.4
13.3
31.3
50.0
0.0
50.0
57.4
18.0
24.6
0.028
CIP
28.9
1.2
69.9
27.3
4.5
68.2
29.5
0.0
70.5
0.840
LEV
30.1
0.0
69.9
31.8
0.0
68.2
29.5
0.0
70.5
0.840
AK
79.5
12.1
8.4
68.2
18.2
13.6
83.6
9.8
6.6
0.564
IPM
96.4
0.0
3.6
100.0
0.0
0.0
95.1
0.0
4.9
0.562
TZP
95.2
3.6
1.2
90.9
9.1
0.0
96.8
1.6
1.6
1.000
ATM
37.4
26.5
36.1
50.0
4.5
45.5
32.8
34.4
32.8
0.289
SXT
32.5
0.0
67.5
4.5
0.0
95.5
42.6
0.0
57.4
0.003
ETP
98.8
1.2
0.0
100.0
0.0
0.0
98.4
1.6
0.0
—
CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CIP, ciprofloxacin; LEV, levofloxacin; AK, amikacin; IPM, imipenem; TZP, piperacillin—tazobartan; ATM, aztreonam; SXT, trimethoprim-sulfamethoxazole; ETP, ertapenem.


### 3.3. Conjugation Experiments

In this study, transfer of integrons was successful in 9 of 17 integron-positiveE. coli ST131 isolates harboring resistance gene cassettes (Table 1). The presence of integrons in the transconjugants was confirmed by PCR.


### 3.4. Bacterial Clonal Relatedness

PFGE analysis of 22 integron-positiveE. coli ST131 isolates revealed that these isolates were divided into 12 clusters (named A-L, in Figure 1) using 70% similarity cutoff value. Two clusters (A and G) were frequent, containing 4 isolates, respectively.Figure 1
Dendrogram generated by bionumerics software that demonstrates the relationship of 22 integron-positiveE. coli ST131 isolates at 70% similarity level, after digestion with XbaI. Strain designation, sample source, and O serotype are shown. The analysis of the bands generated was performed using the Dice coefficient and unweighted pair group method.PFGE analysis of the 83E. coli ST131 isolates (integron-positive and -negative isolates) demonstrated these subtypes were grouped into 22 different PFGE clusters (named 1–22, in Supplementary Data) using 70% similarity cutoff value. The integron-positive isolates were scattered in 10 groups. And 10/22 (45.5%) of integron-positive isolates were clustered into the same clonal group (3).


## 3.1. Integrons, Pc Promoters, and Associated Resistance Genes

Of the 83E. coli ST131 isolates, 22 (27%) carried class 1 integrons. Class 2 and class 3 integrons were not detected in the study. The clinical characteristics of E. coli ST131 are showed in Table 1. The result of serotype revealed the integron-positive E. coli ST131 belonged to two types, and the most prevalence type was O25b (77.3%, 17/22). Results of phylogenetic analysis showed that 21/22 (95.5%) of isolates belonged to group B2 and 1/22 (4.5%) were D group. Several types of fragments ranging in size from 0.15 kb to 3.0 kb were identified (Table 2). Sixteen isolates carried a single integron with a same gene cassette (dfrA17-aadA5). Two gene cassette arrangements were detected in 19 (86.4%) of integron-positive isolates. In addition, only one isolate carried the aac(6′)-Ib-cr-cmlA5 gene cassette array with the largest size (3.0 kb). In this study, only one Pc variant (PcW) was found in 22 integron-positive E. coli ST131, as shown in Table 1.Table 1
Phenotypic and genotypic characteristics of 22 integron-positiveE. coli ST131 isolates.
Isolates
Ward
Source
Age
Sex
Genetic material in isolate with integron
Serotype
Phylogenetic group
Resistance proflies
Conjugation experiments
Promoter
Gene cassette(s)
EC327
Urinary surgery
Urine
48
F
PcW
−1
O25b
B2
SXT
EC431
Emergency surgery
Drain
78
F
PcW
−
O16
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXTCTX, CIP, LEV, SXT
EC455
Colorectal surgery
Drain
62
F
PcW
dfrA17-aadA5
O16
B2
+2
EC485
Urinary surgery
Urine
60
M
PcW
aac(6′)-Ib-cr-cmlA5
O16
B2
−
EC541
Neurosurgery
Blood
69
M
PcW
dfrA17-aadA5
O25b
B2
CTX, SXT
EC549
Neurosurgery
Urine
39
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, AMK, ATM, SXT
EC568
Hepatobiliary surgery
Blood
75
M
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, AMK, ATM, SXT
EC570
Urinary surgery
Urine
51
F
PcW
dfrA17-aadA5
O25b
B2
CTX, FEP, CIP, LEV, AMK, ATM, SXT
EC578
Urinary surgery
Urine
68
M
PcW
dfrA17-aadA5
O16
D
CTX, FEP, CIP, LEV, SXT
+
EC668
Oncology
Urine
59
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CIP, LEV, SXT
+
EC686
Cardiology
Urine
78
M
PcW
dfrA17-aadA5
O16
B2
CIP, LEV, SXT
+
EC771
Outpatient
Blood
61
F
PcW
-
O25b
B2
SXT
EC786
Nephrology
Blood
64
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXT
+
EC792
Colorectal surgery
Secretion
52
M
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXT
EC913
Gastrointestinal surgery
Urine
52
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CIP, LEV, SXT
+
EC934
Hematology
Urine
54
F
PcW
−
O25b
B2
SXT
EC935
Emergency surgery
Secretion
55
F
PcW
−
O25b
B2
SXT
EC978
Hematology
Blood
43
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXT
+
EC1044
Hematology
Urine
64
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXT
+
EC1065
Urinary surgery
Urine
56
M
PcW
dfrA17-aadA5
O25b
B2
SXT
EC1085
Oncology
Ascitic fluid
64
F
PcW
dfrA17-aadA5
O25b
B2
CTX, CAZ, FEP, CIP, LEV, ATM, SXT
+
EC1151
Outpatient
Blood
82
M
PcW
dfrA17-aadA5
O25b
B2
CTX, FEP, CIP, LEV, ATM, SXT
F, female; M, male; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CIP, ciprofloxacin; LEV, levofloxacin; AK, amikacin; IPM, imipenem; TZP, piperacillin-tazobartan; ATM, aztreonam; SXT, trimethoprim-sulfamethoxazole; ETP, ertapenem,1−: not detected, 2+: successfully transferred.Table 2
Size, number, and gene cassetes amplified from the variable region of integron inE.coli ST131.
Approximate length (kb)
Isolate (n)
Gene cassette (s)
0.151
2
—
1.6
4
dfrA17-aadA5
1.6 + 0.72
12
dfrA17-aadA5
3.0
1
aac (6′)-Ib-cr-cmlA5
Total
19
1The 0.15 kb amplicon was found to be a integron variable region without any gene cassette present but contained partial sequences of 5′ and 3′ conserved segments of integrons. 2The 0.7 kb amplicon was found to be a integron variable region without any gene cassette present.Table3 shows the antimicrobial resistance genes detected in our E. coli ST131 isolates. The blaTEM gene was predominant in integron-positve E. coli ST131 isolates, and the blaOXA-1 gene was in 9 isolates. The blaSHV gene was not found in this study. Regarding tetracycline resistance, tetA + tetC was more common in those strains with integron than those strains without integron. The following genes were detected among 56 SXT-resistant isolates (integron-positive/integron-negative isolates): sul1 (15/7), sul2 (2/5), and sul1 + sul2 (3/20). Among the 16 ciprofloxacin-resistant integron-positive isolates, the qnrB gene was detected in 2 isolates. Among the 22 integron-positive E. coli ST131 isolates, the clmA gene and clmA + floR gene were found in two different isolates. The aadA gene was observed in two of 22 integron-positive isolates, and none of these isolates carried aphA1 and aphA2 gene which conferred kanamycin resistance.Table 3
Genes of resistance detected amongE.coli ST131 strains with and without integrons.
Antibiotics
Integron-positive strains (n = 22)
Integron-negative strains (n = 61)
No. of resistance isolates
Genes detected (no. of strains)
No. of resistance isolates
Genes detected (no. of strains)
Ampicillin
—∗
bla
TEM (11)
—
bla
TEM (23)
bla
OXA-1 (9)
bla
OXA-1 (1)
Tetracycline
—
tetA (1)
—
tetA (20)
tetC (3)
tetB (2)
tetA + tetB (6)
tetC (8)
tetA + tetC(10)
TetA + tetC (10)
tetB + tetC (1)
Sulphonamide
21
sul1 (15)
35
sul1 (7)
sul2 (3)
sul2 (5)
sul1 + sul2 (3)
sul1 + sul2 (20)
Ciprofloxaci
16
qnrB (2)
43
qnrS (1)
Streptomycin
—
aadA (2)
—
ND
Kanamycin
—
ND
—
aphA1 (1)
aphA1 + aphA2 (1)
Chloramphenicol
—
cmlA (1)
—
floR (2)
clmA + floR (1)
∗Not detected; ND:no gene detected


## 3.2. Antimicrobial Susceptibility Testing

E. coli ST131 showed high resistance to CTX (71.1%), CIP (69.9%), LEV (69.9%), and SXT (67.5%). However, resistance rates were low to ATM (36.1%), FEP (31.3%), CAZ (26.5%), AK (8.4%), IPM (3.6%), and TZP (1.2%). None of the isolates were resistant to ETP. The results of the antimicrobial susceptibility test were shown in Table 4. Compared with integron-negative E.coli ST131, integron-positive ones were shown to have a higher resistance to SXT and FEP (p<0.05, respectively). Multidrug resistance (defined as resistance to six or more antibiotics) rates of integron-positive and -negative E.coli ST131 isolates were 45.5% and 18.0%, respectively.Table 4
Association between antibiotic profile and integron inE. coli ST131.
Antibiotic
Antibiotic susceptibility
p value
All isolates (n = 83)
Integron-positive isolates (n = 22)
Integron-negative isolates (n = 61)
S (%)
I (%)
R (%)
S (%)
I (%)
R (%)
S (%)
I (%)
R (%)
CTX
25.3
3.6
71.1
31.8
0.0
68.2
23.0
4.9
72.1
0.726
CAZ
49.4
24.1
26.5
54.5
9.1
36.4
47.5
29.5
23.0
0.222
FEP
55.4
13.3
31.3
50.0
0.0
50.0
57.4
18.0
24.6
0.028
CIP
28.9
1.2
69.9
27.3
4.5
68.2
29.5
0.0
70.5
0.840
LEV
30.1
0.0
69.9
31.8
0.0
68.2
29.5
0.0
70.5
0.840
AK
79.5
12.1
8.4
68.2
18.2
13.6
83.6
9.8
6.6
0.564
IPM
96.4
0.0
3.6
100.0
0.0
0.0
95.1
0.0
4.9
0.562
TZP
95.2
3.6
1.2
90.9
9.1
0.0
96.8
1.6
1.6
1.000
ATM
37.4
26.5
36.1
50.0
4.5
45.5
32.8
34.4
32.8
0.289
SXT
32.5
0.0
67.5
4.5
0.0
95.5
42.6
0.0
57.4
0.003
ETP
98.8
1.2
0.0
100.0
0.0
0.0
98.4
1.6
0.0
—
CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CIP, ciprofloxacin; LEV, levofloxacin; AK, amikacin; IPM, imipenem; TZP, piperacillin—tazobartan; ATM, aztreonam; SXT, trimethoprim-sulfamethoxazole; ETP, ertapenem.


## 3.3. Conjugation Experiments

In this study, transfer of integrons was successful in 9 of 17 integron-positiveE. coli ST131 isolates harboring resistance gene cassettes (Table 1). The presence of integrons in the transconjugants was confirmed by PCR.


## 3.4. Bacterial Clonal Relatedness

PFGE analysis of 22 integron-positiveE. coli ST131 isolates revealed that these isolates were divided into 12 clusters (named A-L, in Figure 1) using 70% similarity cutoff value. Two clusters (A and G) were frequent, containing 4 isolates, respectively.Figure 1
Dendrogram generated by bionumerics software that demonstrates the relationship of 22 integron-positiveE. coli ST131 isolates at 70% similarity level, after digestion with XbaI. Strain designation, sample source, and O serotype are shown. The analysis of the bands generated was performed using the Dice coefficient and unweighted pair group method.PFGE analysis of the 83E. coli ST131 isolates (integron-positive and -negative isolates) demonstrated these subtypes were grouped into 22 different PFGE clusters (named 1–22, in Supplementary Data) using 70% similarity cutoff value. The integron-positive isolates were scattered in 10 groups. And 10/22 (45.5%) of integron-positive isolates were clustered into the same clonal group (3).


## 4. Discussion

This study demonstrated the occurrence of integrons amongE. coli ST131 clinical isolates. The results showed that the prevalence of integrons was low, and some were located on transferable plasmids. The prevalence of the integron (26.5%) in this study was similar to that in our previous report (26.7%) [18]; however, it is lower than that found in clinical Enterobacteriaceae isolates in China (59.9%) [19]. The result suggested that the prevalence of integron was lower in E. coli ST131 as compared with other clinical isolates.E. coli ST131 isolates showed high antimicrobial resistance to SXT, CIP, LEV, and CTX. In China, sulfonamides (such as SXT), β-lactams (such as CTX), and quinolones (such as CIP) are often used to cure E. coli infections; therefore, the high resistance of E. coli ST131 may be due to the frequent and inappropriate use of these antimicrobials [20–22].Integrons are known to be correlated with multidrug resistance (MDR) [23, 24]. Multiple antibiotic resistance rates of integron-positive and -negative strains were 45.5% and 13.3%, respectively. This survey demonstrated MDR are widely distributed in E. coli ST131 (Table 1).As previous studies reported, many antibiotic resistance gene cassettes in integrons probably play an important role in the development of antibiotics resistance [2, 25]. In this study, integrons were significantly associated with SXT. Two different gene cassettes were detected (Table 2), which correlated with resistance to a variety of aminoglycosides (aac and aad), chloramphenicol (cmlA), and trimethoprim (dfrA17), and the dfrA17-aadA5 (84.2%) gene cassette dominated in integron-positive isolates in this study, which may contributed to resistance to SXT. The gene cassettes identified were not novel in this study, and it was also the most prevalent gene cassette in E. coli in other previous studies [26, 27]. According to the database in GenBank, a novel cassette array, aac(6′)-Ib-cr-cmlA5, was first reported in E. coli, suggesting the ability of integrons to capture and integrate resistance gene and to form novel cassettes that mediate resistance and multidrug resistance of E. coli ST131 clinical isolates. However, the aac(6′)-Ib-cr-cmlA5-carrying isolate was found to be susceptible (or intermediate) to all antimicrobials (Table 1) which indicated that there may exist some unknown mechanism leading to this phenomenon.We also explored the presence of antimicrobial resistance genes that played a significant role in antimicrobial resistance (Table3). The blaOXA-1 gene was found in 9 of 22 integron-positive isolates. This gene was often found as gene cassettes while not detected in variable region in this study [28, 29]. The fact could be explained by the limitation of PCR or loss of 5′ and 3′ conserved segments of integrons. Regarding tet gene, the tetA gene was the most common one in integron-positive isolates, supporting the previous reports highlighting that the tetA gene was located on the same conjugative plasmid with integron [30]. In this study, the higher percentage of resistance to STX might be attributed to the higher prevalence of sul gene among those integron-positive isolates, and this result corroborates a study reported previously that sul1 gene is usually encoded by class 1 integrons and is part of the 3′ conserved segment [31].Additionally, similar to another previous study, PcW was the most frequent common promoter in clinicalE. coli isolates in this study, a relatively weak promoter [32]. However, compared with another previous finding in other bacteria, only one promoter was detected in this study that was a strange phenomenon [33, 34]. As discussed before, the prevalence of the promoter in E. coli ST131 and whether this is a feature of E. coli ST131 strains should be further investigated. Furthermore, the weaker Pc promoter showed the more efficient excision activity, which promotes integration of exogenous gene cassettes, and these suggested that PcW-carrying E. coli ST131 isolates have a strong capability to excise and capture gene cassettes, that might promote the spread of antimicrobial resistance [35].In this study, 9 (52.9%)E. coli ST131 isolates transferred integron and resistance gene cassettes to E. coli J53 Azr by conjugation. Many previous studies found that most of the resistance determination and integron in E. coli were encoded in a transferable plasmid, which might be horizontally transferred to the isolates among E. coli ST131 or different bacteria species by conjugation, potentially resulting in multiple drug resistance of bacteria [36, 37].The result of PFGE demonstrated that integron-positive isolates were highly diverse in this study and there was no dominant clone (Figure1). Interestingly, nearly half of integron-positive isoaltes belonged to the same group when we performed PFGE analysis on all isolates (Supplementary Data). Future study will be carried out to investigate this phenomenon.


## 5. Conclusions

In conclusion, a low prevalence of integrons was detected inE. coli ST131 clinical isolates. Continued surveillance of this mobile genetic element should be performed to study the evolution of antibiotic resistance among E. coli ST131.



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*Source: 3826186-2020-02-24.xml*