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BMC Microbiology

, 18:125 | Cite as

Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli isolated from the rivers and lakes in Northwest China

  • Haixia Liu
  • Hongchao Zhou
  • Qinfan Li
  • Qian Peng
  • Qian Zhao
  • Jin Wang
  • Xiaoqiang Liu
Open Access
Research article
Part of the following topical collections:
  1. Applied microbiology

Abstract

Background

Extended-spectrum β-lactamases (ESBLs)-producing Escherichia coli (E. coli) isolates in environment water become progressively a potential threat to public health, while the detailed information about the ESBL-producing E. coli isolates in the rivers and lakes in Northwest China is scarce. In the present study, it was aimed to characterize the ESBL-producing E. coli isolated from the surface waters in Northwest China.

Results

A total of 2686 E. coli isolates were obtained from eleven rivers and lakes in Northwest China to screen for ESBL producers. Seventy-six (2.8%) isolates were classified as ESBL producers, and phylogenic groups D and A accounted for 59.2% of the ESBL producers. CTX-Ms were the predominant ESBLs genotype, and they were represented by seven blaCTX-M subtypes. blaCTX-M-14 was the most prevalent specific CTX-M gene, followed by blaCTX-M-9, blaCTX-M-123, blaCTX-M-15, blaCTX-M-27, blaCTX-M-1 and blaCTX-M-65. Moreover, 54 of the 76 ESBL producers carried at least one plasmid-mediated quinolone resistance (PMQR) gene, and aac(6′)-Ib-cr was predominant. The overall occurrence of virulence factors ranged from 1.3% (eae) to 48.7% (traT). Thirty-seven sequence types (STs) were confirmed among the 76 ESBL producers, and the predominant was ST10, which was represented by 10 isolates; importantly, clone B2-ST131, associated with severe infections in humans and animals, was detected three times.

Conclusion

The prevalence of ESBL-producing E. coli from the rivers and lakes in Northwest China was low (2.8%), and the extraintestinal pathogenic E. coli (ExPEC) pathotype was the most commonly detected on the basis of the virulence factor profiles. 76.3% of ESBL producers harbored more than one β-lactamase gene, and blaCTX-M-14 was the predominant genotype. Notably, one ST131 isolate from Gaogan Canal simultaneously harbored blaCTX-M-9, blaCTX-M-15, blaCTX-M-123, blaKPC-2, blaNDM-1, blaOXA-2 as well as the PMQR genes qnrA, qnrS and aac(6′)-Ib-cr.

Keywords

Escherichia coli Surface water Antibiotic resistance β-Lactamase PMQR 

Abbreviations

EAEC

Enteroaggregative E. coli

EIEC

Enteroinvasive E. coli

EPEC

Enteropathogenic E. coli

ESBL

Extended-spectrum β-lactamase

ETEC

Enterotoxigenic E. coli

ExPEC

Extraintestinal pathogenic E. coli

PMQR

Plasmid-mediated quinolone resistance

STEC

Shiga toxin-producing E. coli

UPEC

Uropathogenic E. coli

Background

The use of a wide variety of antimicrobials in human medicine, veterinary clinics, livestock industries and aquaculture has resulted in the emergence and spread of antibiotic-resistant bacteria in different environments, particularly in many developing countries [1, 2]. It becomes evident that the resistance genes can be introduced into the natural bacterial community as the antibiotic-resistant bacteria in humans and animals entered the water bodies [3]. Hence, it is necessary to clarify the potential threat associated with the occurrence of antibiotic-resistant bacteria in water environments in order to further evaluate public health risk and prevent waterborne infections. As one of the most typical indicator bacterium of fecal contamination in the environments, Escherichia coli (E. coli) can easily acquire resistance to antibiotics consumption in humans and animals [4]. Generally, pathogenic E. coli isolates were categorized into several pathotypes based on the clinical symptoms of the patients and the distinct virulence traits of the bacteria. Therefore, E. coli isolates are characterized by their virulence properties and mechanisms of pathogenicity into the enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC) as well as extraintestinal pathogenic E. coli (ExPEC) [5, 6]. STEC isolates are defined as E. coli isolates expressing either stx1 or stx2; EPEC isolates are defined as eae-harboring diarrheagenic E. coli isolates that do not possess the stx gene; ETEC isolates are characterized by estA and eltB; isolates carrying aggR and ipaH are referred to as EAEC and EIEC, respectively [7]. Lastly, ExPEC isolates are associated with fyuA, iutA, afa, papA, focG, sfaS, kpsMII, hlyD and traT. Thus, the pathotypes of the uncharacterized isolates can be inferred from their virulence properties.

Since the extended-spectrum β-lactamases (ESBLs) was firstly reported in 1979 [8], the prevalence of ESBL-producing bacteria have been frequently detected worldwide from clinical isolates due to the increasing use of β-lactam antibiotics and carbapenems; the latter are usually used as the last resort for most serious bacterial infections. Moreover, some ESBL-producing isolates have been recovered from surface waters, where contamination from unmetabolized antibiotics may exert a selective pressure on bacteria, resulting in the emergence and spread of antibiotic-resistant isolates, especially the multidrug-resistant (MDR) isolates during their migration in water resources [3]. Relatedly, plasmid-mediated quinolone resistance (PMQR) determinants also pose a serious threat to public health, and some PMQR genes are considered to be associated with the ESBLs encoding genes [9]. The spread of E. coli co-expressing quinolone resistance along with ESBLs into rivers and lakes is worrisome and contributes to the growing concerns about resistant E. coli and their potential hazards to the environment.

Until now, little data are available on the ESBL-producing E. coli isolates in the surface waters in Northwest China. Thus, the current study was designed to gain insight into the prevalence of ESBL-producing E. coli isolates obtained throughout March 2015 to November 2016 from the rivers and lakes in Shaanxi province, and to further analyze the molecular characteristics of the ESBL producers.

Methods

Collection of isolates

Between March 2015 and November 2016, a total of 2686 E. coli isolates were obtained from eleven water bodies located in Shaanxi province, Northwest China, including Hei River (n = 177), Ying Lake (n = 194), Xianyang Lake (n = 196), Qishui River (n = 264), East Lake of Fengxiang county (n = 154), Wei River (n = 343), Ba River (n = 256), Shichuan River (n = 294), Xiaowei River (n = 265), Qixing River (n = 276) and Gaogan Canal of Yangling (n = 267) (Fig. 1). Among these water bodies, Hei River functioned as a public water supply source, while the others were scenic spots or functioned as floodways of the cities and countryside. All sampling sites were sampled once or multiple times, and all samples were collected in sterile 500-ml polyethylene bottles without preservatives and transported at 4 °C to the Veterinary Pharmacology Laboratory in Northwest A&F University, where primary isolation of E. coli was performed. Briefly, multiple volumes of untreated water were membrane filtered directly through 0.45-μm pore size filters, and the filters were placed on MacConkey agar plates (Solarbio Science & Technology, Co., Ltd., Beijing, China) at 37 °C for the identification of E. coli isolates. All 2686 putative E. coli colonies on MacConkey agar were restreaked onto Eosin Methylene Blue agar (Solarbio Science & Technology, Co., Ltd., Beijing, China), and then the suspicious colonies of E. coli were further identified with standard biochemical tests. Finally, the confirmed isolates as E. coli were stored at − 80 °C in Tryptic Soy broth (Solarbio Science & Technology, Co., Ltd., Beijing, China) containing 30% glycerol until use.
Fig. 1

The map of sample locations

Antimicrobial susceptibility testing

The broth microdilution procedure recommended by Clinical Laboratories Standards Institute (CLSI) [10] was performed to determine the antimicrobial susceptibility of all E. coli isolates against 16 antimicrobials representing six antimicrobial classes: β-lactams, including penicillins (ampicillin, amoxicillin-clavulanic acid and ticarcillin-clavulanic acid), the first-generation cephalosporins (cephalothin), the third-generation cephalosporins (cefotaxime, ceftazidime and ceftriaxone), cephamycins (cefoxitin), and carbapenems (meropenem); tetracyclines (tetracycline); amphenicols (thiamphenicol); quinolones (nalidixic acid and ciprofloxacin); aminoglycosides (gentamicin and amikacin); sulfonamides (sulfamethoxazole-trimethoprim). The control strain for susceptibility testing was E. coli ATCC 25922.

Moreover, ESBL production among the E. coli isolates resistant to the third-generation cephalosporins was detected phenotypically by the double disk synergy test with disks supplemented with cefotaxime and ceftazidime alone or coupled with clavulanic acid [10]. Initial screening analyses indicated that 2.8% (n = 76) E. coli isolates were phenotypic ESBL-positive isolates, and these isolates were used for further analysis.

Phylogenetic typing and determination of virulence factors

Total DNA was isolated from the ESBL producers by using the boiling method. Phylogenetic grouping was determined for the ESBL-producing isolates according to the novel quadruplex PCR method [11]. Meanwhile, seven virulence factor genes known to be characteristic of intestinal pathogenic E. coli (IPEC), including aggR for EAEC, stx1 and stx2 for STEC; eae for EPEC, estA and eltB for ETEC, EIEC-specific gene ipaH; as well as seven markers of virulence associated with uropathogenic E. coli (UPEC), including traT, fyuA, papC, chuA, afa/dra, iutA and PAI [12], were performed by PCR.

Characterization of β-lactamase and PMQR genes

PCR detection and gene identification were performed for β-lactamase genes (TEM, SHV, CTX-Ms), plasmid-mediated AmpC β-lactamase (CMY-2) and carbapenemase genes (class A, KPC-2; class B, NDM-1; class D, OXA) in ESBL-producing E. coli. blaCTX-M group-specific primers for CTX-M-1, CTX-M-2, CTX-M-8 and CTX-M-9 were used to detect of blaCTX-M genes. The PCR products were purified and sequenced by Sangon Biotech (Shanghai, China), and then the β-lactamase genes were identified using the β-lactamase database (http://www.lahey.org/studies/webt.asp) after all the sequences were analyzed online using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Moreover, all the 76 ESBL-producing E. coli isolates were screened by PCR for PMQR genes (qnrA, qnrB, qnrD, qnrS, aac(6′)-Ib-cr, oqxAB and qepA) as described previously [13, 14].

Conjugation experiments

Potential horizontal transferability of β-lactamase and PMQR genes from 15 randomly selected ESBL-producing E. coli isolates (at least one isolate per sampling site) was assessed by conjugation studies (broth mating method) using E. coli J53 AZr as the recipient [15]. The Mueller-Hinton agar supplemented with 150 μg/ml sodium azide and 2 μg/ml cefotaxime were used to select the transconjugants, which were subsequently analyzed by PCR to determine the transferability of β-lactamase and PMQR genes. In addition, the resistance patterns of the recipient and all transconjugants were analyzed.

Multilocus sequence typing (MLST) determination

Internal fragments of seven conserved housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) of each ESBL-producing E. coli isolate were amplified by PCR. A detailed scheme of the MLST procedure, including the primers, PCR conditions, allelic type and sequence type assignment methods, is available at MLST database website (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli).

Statistical analysis

Pearson′s Chi-squared test was used for statistical analysis, and the statistical significance level was established at P < 0.05.

Results

Antimicrobial susceptibility

Among the 2686 E. coli isolates collected, 76 (2.8%) isolates were identified as the ESBL-producing isolates, which were unevenly distributed in 11 sampling sites at levels ranging from 1.1 to 6.4%. Moreover, 64 of the 76 (84.2%) isolates expressed the MDR phenotype. The 76 ESBL-producing isolates showed high resistance to tetracycline (97.3%), followed by ticarcillin-clavulanic acid (90.8%), cephalothin (89.5%), nalidixic acid (81.6%), cefotaxime (77.6%), ciprofloxacin (69.7%), sulfamethoxazole-trimethoprim (69.7%), thiamphenicol (63.2%), and cefoxitin (57.6%), whereas they exhibited high susceptibility to meropenem (96.1%).

Phylogenetic groups and the virulence genes distribution

Phylogenetic analysis showed that the 76 ESBL-producing isolates were composed of phylogenetic groups D (n = 24), A (n = 21), B2 (n = 15), B1 (n = 10), C (n = 4), and E (n = 2). Overall, 78.9% (60/76) of ESBL-producing isolates harbored as least one virulence factor, and the prevalence of individual virulence genes ranged from 1.3% (eae) to 52.6% (traT). estA and aggR were detected in ten and two isolates, respectively, while stx1, stx2 and ipaH were not detected. The virulence genes associated with UPEC isolates were detected throughout the sources, whereas the virulence genes associated with STEC and EIEC isolates were not detected.

Distribution of β-lactamase and PMQR genes

As shown in Table 1, blaSHV, blaTEM and blaCTX-M were detected in 36.8% (n = 28), 43.4% (n = 33) and 76.3% (n = 58) of ESBL producers, respectively, and 58 of the 76 isolates possessed more than one β-lactamase gene. It is interesting that the number of the β-lactamase genes in an E. coli isolate was positively correlated the prevalence of the ESBL producer in each sampling site. For the blaCTX-M positive isolates, blaCTX-M-14 (n = 35) was the predominant genotype, followed by blaCTX-M-9 (n = 17), blaCTX-M-123 (n = 15), blaCTX-M-15 (n = 7), blaCTX-M-27 (n = 4), blaCTX-M-1 (n = 3) and blaCTX-M-65 (n = 3). On the other hand, blaOXA-2, blaKPC-2, blaCMY-2 and blaNDM-1 were detected in five, four, two and one isolate, respectively. It is noteworthy that 80% (4/5) of blaOXA-2 positive isolates were isolated from Gaogan Canal. Among the 33 TEM-positive isolates, two were blaTEM-3 and the rest were non-ESBL gene blaTEM-1. The blaSHV genes were represented by blaSHV-2 (n = 7) and blaSHV-12 (n = 21), and it is interesting to note that ESBL gene blaSHV-12 and non-ESBL gene blaTEM-1 simultaneously appeared in 20 isolates. Furthermore, 54 of 76 (71.1%) ESBL-producing isolates harbored at least one PMQR gene, which was co-located in the ESBL producers with β-lactamase genes. Aac(6′)-Ib-cr (n = 46) was the most dominant PMQR gene, followed by the qnr genes (n = 34). Moreover, one isolate harbored the qepA gene, while the oqxAB gene was not detected in any isolate.
Table 1

ESBL-producing E. coli isolates from rivers and lakes in the Northwest China

Sampling sites

Isolates No.

PG

Antimicrobial resistance profiles

β-lactamase genes

PMQR genes

Virulence genes

MLST

Hei River

HH1609014

B1

AMP AMC TIM CEP CTX CEX FOX TEC GEM AMK SXT

CTX-M-14

 

fyuA, traT

ST155

HH1510025

B2

AMP CEP TPH GEM SXT

TEM-1, SHV-12

qnrB

 

ST1587

Ying Lake

YH1507022

A

AMP AMC TIM CEP CTX CAZ CEX FOX TEC TPH NAC CIP SXT

TEM-1, CTX-M-14

aac(6′)-Ib-cr

traT, papC, chuA

ST617

YH1606032

A

AMP AMC TIM CEP CTX CAZ CEX FOX TEC NAC CIP GEM AMK SXT

CTX-M-14

aac(6′)-Ib-cr

afa/dra, PAI

ST44

YH1607018

D

AMP AMC TIM CEP CAZ TEC TPH GEM AMK

TEM-1, SHV-12

 

traT, chuA

ST2148

Xianyang Lake

XY1608045

B2

AMP AMC TIM CEP CTX CAZ TEC TPH NAC CIP GEM AMK SXT

CTX-M-14

aac(6′)-Ib-cr

traT, iutA

ST602

XY1605044

D

AMP AMC TIM CEP CAZ CEX FOX TEC

CTX-M-14

  

ST393

XY1605033

D

AMP AMC TIM CEP CEX TEC NAC CIP SXT

TEM-1, SHV-12

qnrB, aac(6′)-Ib-cr

traT, chuA

ST393

XY1507042

E

AMP AMC CTX CAZ TPH NAC CIP

TEM-1, SHV-12

 

fyuA, traT

ST1301

Qishui River

QS1608021

A

AMP AMC TIM CEP CTX CEX FOX TEC NAC CIP SXT

TEM-1, CTX-M-1

aac(6′)-Ib-cr

estA

ST10

QS1607026

A

AMP AMC TIM CEP CTX CAZ TEC NAC CIP GEM SXT

CTX-M-9

 

traT, chuA

ST4429

QS1608034

B2

AMP AMC TIM CEP CEX FOX TEC TPH NAC CIP SXT

CTX-M-9

qnrB, qnrS

traT, chuA

ST331

QS1610030

C

AMP AMC TIM CEP CEX TEC TPH NAC CIP

TEM-1, SHV-12

qnrB, aac(6′)-Ib-cr

estA

ST23

East Lake

EH1507029

A

AMP AMC CTX CAZ TEC NAC CIP SXT

CTX-M-1

qnrB, aac(6′)-Ib-cr

 

ST10

EH1607033

A

AMP AMC TIM CTX CAZ CEX FOX TEC SXT

CTX-M-14

aac(6′)-Ib-cr

traT, chuA, papC,

ST10

EH1607014

A

AMP AMC TIM CEP CTX CAZ CEX FOX TEC TPH NAC CIP GEM SXT

TEM-1, CTX-M-14

aac(6′)-Ib-cr

traT, PAI

ST167

EH1608016

A

AMP AMC TIM CEP CAZ TEC NAC CIP

SHV-12

aac(6′)-Ib-cr

 

ST167

Wei River

WH1606023

A

AMP AMC CEP CEX FOX TEC TPH

TEM-1, SHV-12

aac(6′)-Ib-cr

fyuA

ST10

WH1508055

B1

AMP AMC TIM CTX CAZ TEC NAC CIP GEM AMK SXT

CTX-M-27

aac(6′)-Ib-cr

traT, afa/dra

ST58

WH1606078

D

AMP AMC TIM CEP CEX FOX TEC TPH NAC CIP GEM AMK SXT

CTX-M-14

qnrS

traT, papC, afa/dra

ST609

WH1510002

D

AMP AMC TIM CEP CTX CAZ CEX

CTX-M-9

qnrB, aac(6′)-Ib-cr

fyuA, traT, papC

ST38

WH1607120

E

AMP AMC TIM CEP CTX CAZ TEC NAC CIP GEM AMK SXT

CTX-M-14

aac(6′)-Ib-cr

traT

ST1301

Ba River

BA1605012

A

AMP AMC TIM CEP TEC TPH NAC CIP SXT

TEM-1, SHV-12

aac(6′)-Ib-cr

 

ST44

BA1605022

B1

AMP AMC TIM CEP CTX CEX TEC NAC CIP SXT

CTX-M-9, CTX-M-14

qnrS, aac(6′)-Ib-cr

traT, afa/dra

ST155

BA1508024

D

AMP AMC TIM CEP CAZ TEC NAC

TEM-1, SHV-12

  

ST4068

BA1510031

D

AMP AMC TIM CEP CTX CAZ CEX FOX TEC TPH SXT

CTX-M-9, CTX-M-14

 

traT, papC, sfaS

ST2003

BA1509025

D

AMP AMC TIM CTX CAZ CEX TEC TPH NAC CIP GEM AMK

CTX-M-14, CTX-M-15

qnrB, aac(6′)-Ib-cr

fyuA, traT, papC

ST69

BA1509015

D

AMP AMC TIM CTX CEX FOX TEC TPH

CTX-M-14, CTX-M-15

qnrS, aac(6′)-Ib-cr

fyuA, iutA

ST405

Shichuan River

SC1608022

A

AMP AMC TIM CEP CTX CAZ TEC SXT

SHV-12, CTX-M-123

 

traT, chuA

ST93

SC1506012

A

AMP AMC TIM CEP CTX CAZ CEX FOX TEC TPH NAC SXT

CTX-M-14

  

ST746

SC1507014

A

AMP AMC TIM CEP CTX CAZ CEX FOX TEC NAC CIP SXT

TEM-3, CTX-M-123

aac(6′)-Ib-cr

traT, papC, hlyD

ST2376

SC1604029

B1

AMP AMC TIM CEX FOX TEC TPH NAC CIP GEM AMK

TEM-1, SHV-12

qnrS, aac(6′)-Ib-cr

 

ST155

SC1607063

B2

AMP AMC TIM CEP CTX CEX FOX MEM TEC TPH NAC CIP GEM AMK SXT

CTX-M-15, CTX-M-123

qnrS, aac(6′)-Ib-cr

fyuA, traT, papC

ST131

SC1608102

B2

AMP AMC TIM CEP CTX CAZ CEX TIC TPH NAC

TEM-1, SHV-2

 

fyuA

ST95

SC1610005

D

AMP AMC TIM CEP CTX CEX FOX TEC NAC CIP GEM AMK SXT

TEM-1, SHV-12, CTX-M-15

qnrS, aac(6′)-Ib-cr

estA

ST38

SC1609081

D

AMP AMC TIM CEP CTX CEX TEC NAC CIP GEM SXT

TEM-1, CTX-M-14

qnrB, aac(6′)-Ib-cr

estA

ST405

Xiaowei River

XW1608112

A

AMP AMC TIM CEP CTX CEX FOX TEC TPH NAC CIP GEM AMK SXT

TEM-1, SHV-12

qnrB, aac(6′)-Ib-cr

iutA, afa/dra

ST10

XW1608047

A

AMP AMC TIM CEP CTX CAZ CEX FOX TEC NAC SXT

TEM-1, SHV-12, CTX-M-14

  

ST44

XW1609034

B1

AMP AMC TIM CEP CTX CAZ CEX TEC TPH NAC CIP SXT

CTX-M-14, CTX-M-65

qnrS

fyuA, PAI

ST75

XW1608023

B2

AMP AMC TIM CEP CTX CEX TEC TPH NAC

TEM-1, SHV-2

 

traT, chuA

ST95

XW1607012

B2

AMP AMC TIM CEP CTX CAZ CEX FOX TEC TPH NAC CIP SXT

CTX-M-9, CTX-M-14, CTX-M-123

aac(6′)-Ib-cr

fyuA, traT, iutA

ST12

XW1607055

B2

AMP AMC TIM CEP CTX CAZ FOX TEC TPH NAC CIP SXT

CTX-M-14

  

ST2855

XW1609057

D

AMP AMC TIM CEP CTX CAZ TEC TPH SXT

TEM-1, SHV-12

  

ST5164

XW1608026

D

AMP AMC TIM CEP CTX CAZ CEX FOX TEC NAC CIP GEM AMK SXT

CTX-M-1

aac(6′)-Ib-cr

traT, hlyD

ST3880

XW1607034

D

AMP AMC TIM CEP CTX CAZ CEX FOX TEC NAC CIP SXT

CTX-M-14, CTX-M-123

aac(6′)-Ib-cr

fyuA, traT

ST38

XW1609038

D

AMP AMC TIM CEP CTX CEX FOX MEM TEC NAC CIP GEM AMK SXT

CTX-M-15

qnrB, aac(6′)-Ib-cr

traT, iutA, papC

ST69

XW1608041

D

AMP AMC CEP CTX CEX TEC TPH NAC CIP SXT

TEM-1, SHV-2, CTX-M-14

 

traT, chuA

ST609

Qixing River

QX1608021

A

AMP AMC TIM CEP CTX CAZ TEC TPH NAC SXT

TEM-1, SHV-12

  

ST10

QX1608013

A

AMP AMC TIM CEP CEX TEC TPH SXT

TEM-1, SHV-12

aac(6′)-Ib-cr

traT, papC, PAI

ST10

QX1509072

A

AMP AMC TIM CEP CTX CAZ FOX TEC NAC GEM AMK SXT

TEM-1, SHV-2

qnrS, aac(6′)-Ib-cr

estA

ST10

QX1608015

A

AMP AMC TIM CEP CTX CEX FOX TEC TPH NAC CIP GEM AMK SXT

CTX-M-9, CTX-M-27

qnrS

traT, papC

ST3902

QX1605083

B1

AMP AMC TIM CEP CTX CAZ CEX FOX TEC TPH NAC CIP GEM AMK SXT

TEM-1, CTX-M-9, KPC-2

 

fyuA, papC, traT

ST3160

QX1608005

B1

AMP AMC TIM CEP CTX CAZ CEX TEC NAC CIP SXT GEM SXT

CTX-M-14

qnrB

estA

ST75

QX1507055

B2

AMP AMC TIM CEP CTX CAZ CEX FOX TEC TPH NAC CIP GEM SXT

CTX-M-27

qnrS

aggR

ST1304

QX1508112

B2

AMP AMC TIM CEP CTX CAZ FOX TEC TPH NAC CIP GEM AMK SXT

TEM-1, SHV-12, CTX-M-9, OXA-2

qnrB, qnrS, aac(6′)-Ib-cr

traT, iutA, PAI

ST12

QX1608059

B2

AMP AMC CEP CAZ CEX FOX TEC TPH NAC CIP GEM AMK

CTX-M-14, CTX-M-123

 

estA

ST2077

QX1510043

C

AMP AMC TIM CEP CTX CAZ CEX FOX TEC NAC CIP GEM SXT

CTX-M-9, CTX-M-14

aac(6′)-Ib-cr

traT, papC

ST23

QX1608046

D

AMP AMC TIM CEP CTX CAZ CEX FOX TEC TPH NAC CIP SXT

CTX-M-14, CTX-M-123, CTX-M-65

 

afa/dra, hlyD

ST3880

QX1604103

D

AMP AMC TIM CEP CEX FOX TEC TPH NAC CIP GEM AMK SXT

TEM-1, SHV-2, CTX-M-14, CTX-M-123

qnrB, aac(6′)-Ib-cr

fyuA, iutA, PAI

ST609

QX1609108

D

AMP AMC TIM CEP CTX CAZ CEX FOX MEM TEC TPH NAC CIP SXT

TEM-3, CTX-M-14

 

fyuA, afa/dra

ST2148

Gaogan Canal

GG1505017

A

AMP AMC TIM CEP CTX CEX TEC NAC CIP GEM AMK SXT

TEM-1, SHV-2, CTX-M-14

aac(6′)-Ib-cr

 

ST10

GG1509025

A

AMP AMC TIM CEP CEX TEC TPH GEM AMK SXT

TEM-1, SHV-12, CTX-M-65

aac(6′)-Ib-cr

estA

ST10

GG1508074

B1

AMP AMC TIM CEP CTX CAZ CEX MEM TEC TPH NAC CIP SXT

TEM-1, SHV-12, CTX-M-9

aac(6′)-Ib-cr

eae

ST58

GG1609024

B1

AMP AMC TIM CEP CTX CAZ CEX MEM TEC TPH NAC CIP SXT

CTX-M-9, CTX-M-123

qnrB

traT, papC, afa/dra

ST155

GG1609158

B1

AMP AMC CEP CEX FOX TEC TPH N GEM SXT

TEM-1, SHV-12

  

ST1049

GG1609019

B2

AMP AMC TIM CEP CTX CAZ FOX TEC TPH SXT

CTX-M-9, CTX-M-14

 

iutA, afa/dra

ST3252

GG1609022

B2

AMP AMC TIM CEP CTX CAZ CEX FOX TEC NAC CIP

CTX-M-9, CTX-M-14

qnrA, aac(6′)-Ib-cr

fyuA, traT, iutA, PAI

ST12

GG1609068

B2

AMP AMC CEP CTX CEX FOX TEC NAC CIP SXT

CTX-M-14, KPC-2, OXA-2

qnrB, qnrS, aac(6′)-Ib-cr

fyuA, papC, traT, iutA

ST131

GG1610109

B2

AMP AMC TIM CEP CTX CAZ CEX FOX MEM TEC TPH NAC CIP GEM AMK SXT

CTX-M-9, CTX-M-15, CTX-M-123,

KPC-2, NDM-1, OXA-2

qepA, qnrS, aac(6′)-Ib-cr

fyuA, papC, traT, chuA, iutA

ST131

GG1609086

C

AMP AMC TIM CEP CTX CAZ FOX TEC TPH NAC CIP SXT

CTX-M-9, CTX-M-14, CTX-M-123

qnrS, aac(6′)-Ib-cr

traT, afa/dra, papC, PAI

ST410

GG1607066

C

AMP AMC TIM CEP CTX CAZ CEX TEC TPH NAC CIP GEM SXT

TEM-1, SHV-12, CTX-M-123

qnrB, aac(6′)-Ib-cr

traT, chuA

ST88

GG1609121

D

AMP AMC TIM CEP CTX CAZ TEC TPH NAC CIP GEM AMK SXT

CTX-M-15, CTX-M-123

aac(6′)-Ib-cr

estA

ST38

GG1609016

D

AMP AMC TIM CEP CTX CAZ CEX FOX TEC TPH NAC SXT

CTX-M-14, CMY-2

aac(6′)-Ib-cr

fyuA, traT

ST69

GG1604028

D

AMP AMC TIM CTX CEX FOX TEC NAC CIP GEM AMK

CTX-M-14, CTX-M-123

aac(6′)-Ib-cr

aggR

ST69

GG1506027

D

AMP AMC TIM CEP CTX CAZ CEX FOX TEC TPH NAC CIP GEM AMK SXT

CTX-M-9, CTX-M-123, KPC-2, OXA-2

qnrB, aac(6′)-Ib-cr

fyuA, traT, chuA, iutA

ST405

GG1608063

D

AMP AMC TIM CEP CTX CAZ CEX FOX MEM TEC TPH NAC CIP SXT

CTX-M-14, CTX-M-27, CMY-2, OXA-2

qnrS, aac(6′)-Ib-cr

fyuA, traT, chuA, iutA, PAI

ST405

AMP ampicillin, AMC amoxicillin-clavulanic acid, TIM ticarcillin-clavulanic acid, CEP cephalothin, CTX cefotaxime, CAZ ceftazidime, CEX ceftriaxone, FOX cefoxitin, MEM meropenem, TEC tetracycline, TPH thiamphenicol, NAC nalidixic acid, CIP ciprofloxacin, GEN gentamicin, AMK amikacin, SXT sulfamethoxazole-trimethoprim

Conjugation experiments

Ten out of fifteen ESBL producers were horizontally transferred to recipient strain E. coli J53 AZr. PCR demonstrated the presence of β-lactamase and PMQR genes in transconjugants (Table 2). Antimicrobial susceptibility patterns revealed that all transconjugants kept the similar antibiotic resistance profiles to ampicillin, amoxicillin-clavulanic acid, ticarcillin-clavulanic acid, cefotaxime, ceftazidime, ceftriaxone and cefoxitin compared with the donors, and all transconjugants exhibited at least 8-fold increase in MICs compared with the recipient. The ciprofloxacin MICs for eight transconjugants harboring PMQRs ranged from 0.125 to 1 μg/ml, representing an increase of 2-fold to 16-fold compared with the recipient (Table 2). However, the transconjugants were still susceptible to meropenem, tetracycline, ciprofloxacin, gentamicin, thiamphenicol and sulfamethoxazole-trimethoprim.
Table 2

Antimicrobial susceptibility profiles of ESBL-producing E. coli isolates used in the conjugation experiments

Isolates

MIC (μg/ml) of antimicrobials

Presence of

AMP

AMC

TIM

CTX

CAZ

CEX

FOX

MEM

TEC

TPH

CIP

GEN

SXT

β-lactamase genes

PMQR genes

Donors

 HH1609014

256

32

32

32

4

32

8

0.03

32

2

0.5

32

128

CTX-M-14

 

 XY1608045

512

32

16

32

64

4

2

0.125

64

32

64

32

32

CTX-M-14

aac(6′)-Ib-cr

 EH1607014

256

32

32

64

64

128

16

0.063

32

64

128

16

64

TEM-1, CTX-M-14

aac(6′)-Ib-cr

 WH1510002

256

32

32

32

128

64

1

0.063

0.25

1

2

4

16

CTX-M-9

qnrB, aac(6′)-Ib-cr

 BA1605022

512

64

16

64

8

64

2

0.063

64

0.25

32

2

64

CTX-M-9, CTX-M-14

qnrS, aac(6′)-Ib-cr

 QX1604103

256

64

32

4

256

128

32

0.03

128

128

128

64

256

TEM-1, SHV-2, CTX-M-14, CTX-M-123

qnrB, aac(6′)-Ib-cr

 SC1610005

512

64

32

32

8

64

32

0.03

32

2

16

32

128

TEM-1, SHV-12, CTX-M-15

qnrS, aac(6′)-Ib-cr

 XW1609038

256

32

32

32

4

64

16

4

0.25

0.5

64

128

64

CTX-M-15

qnrB, aac(6′)-Ib-cr

 GG1509025

256

64

16

4

2

32

2

0.125

128

64

2

64

64

TEM-1, SHV-12, CTX-M-65

aac(6′)-Ib-cr

 GG1610109

512

64

32

128

64

128

32

16

128

128

128

128

128

CTX-M-9, CTX-M-15, CTX-M-123, KPC-2, NDM-1, OXA-2

qepA, qnrS, aac(6′)-Ib-cr

Recipient J53AZr

4

1

1

0.125

0.063

0.063

0.125

0.03

0.25

0.125

0.063

0.25

0.25

  

Transformants

 Trans-HH1609014

128

16

16

16

1

8

8

0.03

0.5

0.25

0.125

0.25

0.5

CTX-M-14

 

 Trans-XY1608045

256

32

16

16

32

0.5

0.5

0.063

0.25

0.125

0.125

0.125

0.25

CTX-M-14

aac(6′)-Ib-cr

 Trans-EH1607014

256

16

16

32

32

64

8

0.03

0.125

0.125

0.5

0.125

1

CTX-M-14

aac(6′)-Ib-cr

 Trans-WH1510002

128

32

16

32

32

64

0.5

0.03

0.063

0.063

0.063

0.063

0.25

CTX-M-9

aac(6′)-Ib-cr

 Trans-BA1605022

128

32

32

64

1

16

0.5

0.03

0.5

0.063

0.125

0.03

0.5

CTX-M-9, CTX-M-14

qnrS, aac(6′)-Ib-cr

 Trans-QX1604103

128

32

32

1

64

64

16

0.125

0.125

0.125

0.5

0.125

2

TEM-1, CTX-M-14

aac(6′)-Ib-cr

 Trans-SC1610005

128

16

16

16

1

16

4

0.03

0.5

0.25

0.5

0.5

1

TEM-1, SHV-12, CTX-M-15

qnrS, aac(6′)-Ib-cr

 Trans-XW1609038

128

16

16

16

1

32

16

0.063

0.063

0.063

0.125

0.25

0.5

CTX-M-15

qnrB, aac(6′)-Ib-cr

 Trans-GG1509025

256

32

16

1

0.5

32

1

0.063

0.125

0.125

0.125

0.125

0.25

SHV-12, CTX-M-65

aac(6′)-Ib-cr

 Trans-GG1610109

256

32

32

32

32

64

16

0.03

0.25

0.063

1

0.25

0.5

CTX-M-15, CTX-M-123, KPC-2, NDM-1

qepA, qnrS, aac(6′)-Ib-cr

MLST determination

The diversity and phylogenetic relationships of the ESBL-producing E. coli isolates were evaluated by MLST. MEGA 6.0 software was used to construct the phylogenetic tree for 76 ESBL-producing E. coli isolates using the maximum likelihood approach with on the basis of the Tamura-Nei model and seven concatenated housekeeping gene sequences (Fig. 2). The 76 ESBL producers belonged to 37 STs (Fig.1 and Table 1). Among of them, 19 STs were represented by more than two isolates, and the other 18 STs represented a single isolate each. ST10 (n = 10) was more prevalent compared with other STs (P < 0.001). It is difficult to infer a significant correlation between the water bodies and the STs because of the limited number of ESBL producers. Nevertheless, we found that some ESBL producers from different water bodies shared the same STs, and some STs, e.g., ST10, ST38, ST69, ST405, identified in this study were also found among the E. coli isolates from dogs in Shannxi province. Three ST131 isolates were from Shichuan River and Gaogan Canal, which flowed through several cities and villages. Furthermore, the ST131 isolate from Shichuan River simultaneously harbored blaCTX-M-15 and blaCTX-M-123; one ST131 isolate from Gaogan Canal harbored blaCTX-M-9, blaCTX-M-15, blaCTX-M-123, blaKPC-2, blaNDM-1, blaOXA-2 as well as PMQR genes qnrA, qnrS and aac(6′)-Ib-cr, while another ST131 isolate from Gaogan Canal harbored blaCTX-M-14, blaKPC-2, blaOXA-2 as well as qnrB, qnrS and aac(6′)-Ib-cr.
Fig. 2

Phylogenetic tree showing the relationship of 76 ESBL-producing E. coli isolates. The dendrogram was constructed by using the nucleotide sequences of the seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) of 76 ESBL-producing E. coli isolates from rivers and lakes in Northwest China with the maximum likelihood method. Sampling sites, phylogenetic group (PG), sequence type (ST) and ST clonal complex (STcc) were displayed the right of the dendrogram

Discussion

The spread of ESBL-producing E. coli isolates in the environment, especially in water is worrisome both in developing and developed countries as they pose potential risks to public health [16, 17, 18]. Rivers and lakes are usually considered to be of special importance as a reservoir of resistance genes because they can collect the surface waters containing contaminants from different origins, e.g., municipal wastewater, agricultural activities, or the sewage from the hospitals and livestock, which include abundant antibiotic-resistant bacteria. In this study, 2686 E. coli isolates were collected from 11 water bodies between March 2015 and November 2016, with 90.9% (10/11) of sampling sites located in Guanzhong region, an economically developed and densely populated area in Shaanxi province. Generally, the prevalence rate of ESBL producers was 2.8%, which was much lower than the prevalence of ESBL producers among the E. coli isolated from dogs (24.2%), retail meat (22.3%) and pigs (9.6%, unpublished data from our group) in Shaanxi province [19, 20]. Meanwhile, the frequency of ESBL-producing E. coli varied significantly at different sampling sites, and it was more frequently isolated in the Gaogan Canal (6.4%), Qixing River (4.3%) and Xiaowei River (4.2%) compared with Hei River (1.1%) (P < 0.01). It is noteworthy that a blaNDM-1-producing ST131 clone, and four of the five blaOXA-2-producing isolates were isolated from Gaogan Canal. There is a high probability that the Gaogan Canal, Qixing River and Xiaowei River were contaminated by the wastewater from the hospitals, pharmaceutical manufactures or livestock farms, which are located in or adjacent to cities or rural villages. However, ESBL-producing E. coli isolates were seldom detected in the Hei River, Ying Lake and Qishui River, which belong to public water supply source or scenic spots. The results indicate that there is a positive linear relationship between the occurrence of ESBL producers and discharge of wasterwater, such as the sewage of the hospitals and the livestock farms.

It is of particular concern that the majority (84.2%) of 76 ESBL-producing isolates included in this study expressed the MDR phenotype and showed high resistance rates to amoxicillin-clavulanic acid (98.7%), tetracycline (97.3%) and ticarcillin-clavulanic acid (90.8%). Moreover, it is worrisome that most ESBL producers were commonly located on conjugative plasmids that also harbor genes conferring cross-resistance to non-β-lactam antibiotics [21]. Traditionally, phylogroups A and B1 contain commensal isolates, while groups B2 and D are considered to be opportunistic ExPEC isolates. The 76 ESBL-producing E. coli isolates surveyed belonged mainly to phylogroups D and A (59.2%), followed by group B2 (19.7%). Normally, virulence factors are ideal targets for determining the pathogenic potential of a given E. coli isolate. Most of our ESBL-producing isolates (65.8%) possessed UPEC-related virulence factors, followed by estA, which is associated with the ETEC. Our results generally agree with a previous study that found ExPEC as the main pathotype in E. coli isolates from other water sources [6]. However, our findings tend to strongly disagree with the previous finding of significantly higher prevalence of ETEC isolates in surface waters of developing countries [22, 23], which may be due to the large differences in the sampling environments. It has been shown that ExPEC isolates can exist as commensals in the guts of healthy animals and humans, where they may gain or lose virulence genes through genetic exchange [6]. Moreover, UPEC isolates, the primary ExPEC associated with urinary tract infections, are also an important source of ESBLs entering the water system [24].

In recent years, CTX-M subtypes of the CTX-M-1 and CTX-M-9 groups have become the most prevalent ESBL-encoding genes among the E. coli from clinical and aquatic environments [4]. In the present study, CTX-Ms were represented by seven blaCTX-M subtypes that mostly expressed blaCTX-M-14. Two recent studies in our laboratory revealed that the predominant blaCTX-M subtypes in the ESBL-producing E. coli isolated from dogs and pigs, respectively, in the Guanzhong region of Shaanxi province [20, 25]. blaCTX-M-15 and blaCTX-M-14 were also prevalence in humans in Asia [26]. We identified three isolates that harbored blaCTX-M-65, which has not been reported before in Northwest China, although it has been frequently reported in other places in China [27, 28, 29]. All 76 ESBL-producing isolates were assigned to 37 STs, with ST10 as the most predominant. In contrast to the genetic characteristics of the ESBL-producing E. coli isolates from other sources, all the ESBL producers were much more diverse compared to the isolates from pigs and dogs in Shaanxi province. The emergence of clone ST131 represents a major challenge to public health worldwide since it was first discovered in human clinical samples. Subsequently, it has disseminated to various animal species and environments [4]. Our study indicated that three (3.9%, 3/76) ST131 isolates were detected in Shichuan River and Gaogan Canal, of which two ST131 isolates harbored blaCTX-M-15 and one harbored blaCTX-M-14, blaKPC-2 and blaOXA-2. The previous study suggested that the worldwide pandemic B2-ST131 E. coli isolates harboring blaCTX-M-27-producing have been closely associated with underlying severe infections in human and animal medicine [30]. We also detected four blaCTX-M-27-producing E. coli isolates, although these were not of the ST131 clone. Hence, further studies will need to be performed to explore these isolates, while at the same time, appropriate measures urgently need to be enforced to alleviate the stress posed by antibiotic resistance in the environments.

We found that almost all blaSHV-12 genes mainly co-existed with non-ESBL gene blaTEM-1 but not the other β-lactamase genes (Table 1). With respect to PMQR genes, their prevalence among E. coli isolates from humans and animals has been described frequently. However, there are few reports on the presence of PMQR genes in the ESBL-producing E. coli in water bodies. Our surface water E. coli isolates yielded one or more PMQR genes in 71.1% of the ESBL-producing isolates tested, with aac(6′)-Ib-cr as the most prevalent (63.2%), which was similar with a previous study in our laboratory that showed aac(6′)-Ib-cr as the most prevalent PMQR gene in extended-spectrum cephalosporin-resistant E. coli isolates from dogs in Shaanxi [20]. However, a previous study in Heilongjiang province showed that the oqxAB gene was the most dominant in the ESBL-producing E. coli from piglets [31]. All the PMQR genes co-localized with blaCTX-M in our E. coli isolates. The emergence of PMQRs indicates that quinolone resistance can also be acquired through horizontal gene transfer, and PMQR genes qnr and aac-(6′)-Ib-cr were co-transferred with β-lactamase genes, which were confirmed by the conjugation experiments in the present study. Notably in this study, one ST131 isolate from Gaogan Canal simultaneously harbored blaCTX-M-9, blaCTX-M-15, blaCTX-M-123, blaKPC-2, blaNDM-1, blaOXA-2 as well as the PMQR genes qnrA, qnrS and aac(6′)-Ib-cr. To our knowledge, this is the first description of the coexistence of so many resistance genes in one E. coli isolate from water. Hence, more studies should be carried out in the future in order to judge if these genes are located on the same plasmid.

Conclusion

In conclusion, the prevalence of ESBL-producing E. coli from the rivers and lakes in Northwest China was 2.8%, and the ExPEC pathotype was the most frequently detected depending on the virulence factor profiles. 76.3% of ESBL producers harbored more than one β-lactamase gene, and blaCTX-M-14 was the predominant genotype; the most dominant PMQR gene was aac(6′)-Ib-cr. The ESBL producers showed a high degree of overlaps in terms of resistance phenotypes, β-lactamases, PMQR genes and other genetic characteristics. The most prevalent sequence type was ST10, and three ST131 clones were detected.

Notes

Acknowledgements

The authors are thankful to Haohao Feng, Jinglong Ye, Runan Zuo and Yuyang Miao for their assistance in sample collection.

Funding

This study was supported by the Key Research and Development Project of Shaanxi Province (No. 2018NY-109, No. 2018NY-005), and the Agricultural Science and Technology Promotion Project of Yangling Demonstration Zone (No. TS-2016-12). The funding bodies are play role in provide research funding of the study. They have no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials

All the data supporting our findings is contained within the manuscript.

Authors′ contributions

XL and HL conceived and guided the experiments. HL also drafted the manuscript. HZ, QZ and QP participated in the identification of the isolates, and performed the antimicrobial susceptibility assays. QZ, JW and QL participated in the conjugation experiments, and contributed to the manuscrip revision. HL, XL, QZ and QL performed the molecular studies, and analyzed the experimental data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

In this study, informed consent was not necessary because the isolates included in the study were obtained from surface waters. Ethics approval and consent to participate.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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© The Author(s). 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Haixia Liu
    • 1
  • Hongchao Zhou
    • 2
  • Qinfan Li
    • 2
  • Qian Peng
    • 2
  • Qian Zhao
    • 1
  • Jin Wang
    • 1
  • Xiaoqiang Liu
    • 2
  1. 1.Department of Aquaculture, College of Animal Science and TechnologyNorthwest A&F UniversityYanglingChina
  2. 2.Department of Basic Veterinary, College of Veterinary MedicineNorthwest A&F UniversityYanglingChina

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