Isolation and Characterization of Carbapenem-Resistant Escherichia coli Carrying blaNDM and mcr-1 from Recurrent Urinary Tract Infection Patient

Objective The emergence of carbapenem-resistant E. coli (CRECO), leading to few antibacterial drugs available for CRECO infection. In this study, we report three carbapenem-resistant Escherichia coli (E. coli) isolates coproducing blaNDM and mcr-1 from patients with recurrent urinary tract infection (RUTI). Carbapenem-resistant E. coli strains, E55, E84, and E85, were isolated from the urine sample of RUTI patients. Methods Antimicrobial susceptibility testing (AST) was conducted with VITEK-2 compact system and Kirby–Bauer (K-B) disk diffusion method. The ESBL test was detected by the disk diffusion method. The EDTA-modified carbapenem inactivation method (eCIM) and modified carbapenem inactivation method (mCIM) were performed for screening the carbapenemase. Multilocus sequence typing (MLST) was performed for molecular typing of the strains. The resistance genes were detected by PCR. Results The three isolates were all susceptible to tigecycline and nitrofurantoin. The blaNDM-1, blaCMY-6, blaTEM-1 and blaCTX-M-1, mcr-1, and porin loss expression of outer membrane protein F (OmpF) were detected in E55, which was assigned to ST2. The E84 and E85 were identified as ST471 carrying blaNDM-5, blaCTX-M55, and blaTEM-1 and the quinsolone-resistant genes aac(6′)-Ib-cr and mcr-1. Conclusion To our knowledge, our study is the first to report carbapenem-resistant E. coli strains carrying blaNDM and mcr-1 from urine of the recurrent urinary tract infection patients. These E. coli strains carrying blaNDM and mcr-1 should be closely monitored.


Introduction
Escherichia coli (E.coli) is an important pathogen in the clinical infectious disease, especially in the urinary tract infections (UTIs) [1].Te emergence of carbapenemresistant E. coli (CRECO) leads to few therapeutic options available.Carbapenem antibiotics play an important role in treatment of these multiple drug-resistant (MDR) bacterial infections and have been widely used in clinical practices [2].Carbapenem-resistant Enterobacteriaceae (CRE) remains a global public health threat, like tuberculosis (TB) [3].New Delhi metallo-β-lactamase (NDM) is an important carbapenemase that confers resistance to almost all β-lactams.Polymyxins are considered among the last therapeutic options to therapy the serious infections caused by CRECO [4].However, the frst report about mobile colistin resistance 1 (mcr-1) was published in 2016, detected from ECO and Klebsiella pneumoniae isolates recovered from animals and patients in China, which is responsible for colistin resistance [5].Since then, more and more literatures were published to report the detection of the mcr-1 gene from more sites from animals and humans in Europe, Canada, Vietnam, Hong Kong, and Taiwan [6][7][8][9][10].But there are few reports of mcr s detected from clinical patient samples, especially the urine of patients with recurrent urinary tract infection (RUTI).Here, we report three isolated E. coli strains, which coproduce mcr-1 and bla NDMs from patients with RUTI.

Bacterial Isolation and Identifcation
. Tree E. coli strains, E55, E84, and E85, were isolated from the urine sample of RUTI patients.Te three isolates were identifed by the VITEK-2 compact system (bioMérieux, France) and 16S rRNA sequencing.

Antimicrobial Susceptibility Testing.
In vitro antimicrobial susceptibility testing (AST) was conducted using VITEK-2 compact system (bioMérieux, France) and Kirby-Bauer (K-B) disk difusion method (flter paper from Oxoid), using E. coli (ATCC 25922) as the control.Te results were interpreted following the Clinical and Laboratory Standards Institute (CLSI), and colistin and tigecycline were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (https://www.eucast.org/).

Phenotypic Detection of ESBL and Carbapenemase.
Recommended by the CLSI [11], ESBL production was confrmed by combined disk approach, and phenotypic screening for preliminarily determining whether the strains produced metallo-carbapenemase was performed in accordance with the modifed carbapenem inactivation method (mCIM) and EDTA-modifed carbapenem inactivation method (eCIM).

DNA Extraction.
Colonies of the clinical E. coli strains were transferred to a microcentrifuge tube with sterile distilled water.Te samples were boiled to prepare the DNA templates for polymerase chain reaction (PCR).

Antimicrobial Susceptibility
Testing.Te three strains showed multidrug-resistant phenotypes.E55 was resistant to most of the antibiotics, excluding tigecycline and nitrofurantoin.E84 was only susceptible to amikacin, doxycycline, tigecycline, trimethoprim-sulfamethoxazole, gentamicin, minocycline, and nitrofurantoin.E85 was only susceptible to trimethoprim-sulfamethoxazole, gentamicin, nitrofurantoin, tigecycline, and amikacin.Te results of the antibiotics tested are presented in Table 1.

Characterization of Carbapenem-Resistant E. coli.
ESBL tests of the three strains were all negative.mCIM tests of the three strains were all positive, then eCIM tests were done, and the results were all positive too (Table 2).
To determine the mechanism of colistin resistance and carbapenem resistance, we initially investigated the presence of resistance genes.We identifed that there were the betalactam resistance genes bla NDM-1 , bla CMY-6 , bla TEM-1 and bla CTX-M-1 , and mcr-1, accompanied by the loss of OmpF in E55, and the MLST typing of E55 belongs to ST2.E84 and E85 were identifed as ST471 carrying bla NDM-5 , bla CTX-M55 , and bla TEM-1 , aac(6′)-Ib-cr, and mcr-1.Te results of antimicrobial resistance genes and MLST are presented in Table 2.

Discussion
UTI is an infammation of the urinary tract caused by bacteria.It is one of the most common bacterial infection diseases [1].In a survey of 289 female patients with recurrent urinary tract infections from 2006 to 2014, it was found that 71% of persistent and 47% of recurrent urinary tract infections were caused by E. coli [12].Gordon and Jones reported that the isolation rate of E. coli in UTI was as high as 47% in North America, Europe, and Latin America from a retrospective investigation of SENTRY program in 2000 [13].Some other studies had shown that among the pathogenic bacteria of urinary tract infection detected from 2016 to 2017, E. coli ranked no. 1, accounting for 28.85%.Te data reported by other domestic literatures were basically consistent [14,15].In this study, we found three carbapenemresistant E. coli isolates coproducing bla NDM and mcr-1 from patients with recurrent urinary tract infection (RUTI).

2
Canadian Journal of Infectious Diseases and Medical Microbiology   Te three strains were all MDR isolates.Compared with E84 and E85, E55 showed more resistance to antibiotics; it was only susceptible to tigecycline and nitrofurantoin.While nitrofurantoin showed susceptibility to the three strains, it suggested that nitrofurantoin might be a treatment option for treatment of RUTI caused by colistin and carbapenemresistant E. coli.
In our study, the three E. coli isolates showed multidrug resistance not only because of bla NDM and mcr-1 gene but also due to other resistance mechanisms, for example, the bla CMY-6 , bla TEM-1 , and bla CTX-M-1 , which code the AmpC and ESBLs in E55; the bla CTX-M55 and bla TEM-1 and aac(6′)-Ib-cr+ which code ESBLs and the quinolone resistance genes in E84 and E85.But in E55, quinolone-resistant genes and aminoglycoside-resistant genes were not detected, and the isolate showed resistance to the two antibiotics, maybe because of the deletion of the OmpF gene.Most of the membrane porins that β-lactam antibiotics can pass through are mainly OmpF and OmpC, which are characterized by a signifcant reduction in the permeability of negatively charged substances, while a positive charge can promote solute passage.It is suggested that the loss of porin is related to the resistance of bacteria to β-lactam antibiotics.It has been found that the loss of pore protein contributes more to carbapenem resistance than ESBLs or carbapenemases' presence.Because the drugs targeting porin loss are limited, related drugs targeting the mechanism must be developed to address the carbapenem resistance issue of pathogenic bacteria [16].
In addition, our results showed that E55 coproduced mcr-1 and bla NDM1 , while E84 and E85 coproduced mcr-1 and bla NDM5 .Te bacteria carried the New Delhi metalloβ-lactamase gene (bla NDM−1 ) which was frst identifed in 2009 [17], and then E. coli carried mcr-1 which was frst identifed in 2016 [5].Because the mcr-1 and bla NDM genes can disseminate widely around the world and across species [5], E. coli with mcr-1 or bla NDM can also be detected gradually from some patients, but the related reports are rare.MCR-1 and NDM-5 coproducing isolate was frst reported from a duck sample [18], but soon reported from urine [19][20][21][22], blood [21], ascites [21], and abdominal drainage [22].And soon more literatures had reported the coexistence of mcr-1 and bla NDM-5 genes in clinic origin and animal origin.Han et al. represented the frst report of a wild-derived E. coli strain harboring mcr-1 and bla NDM-5 genes simultaneously [23].So far, strains coproducing NDM-1 and MCR-1 had been reported from blood and feces [24][25][26].However, there is no report from urine.As we know, this is the frst report of carbapenem-resistant E. coli strain carrying bla NDMs and mcr-1 from urine of the RUTI patients.
In this study, the ESBL tests of the three carbapenemresistant E. coli strains were negative, but bla TEM-1 and bla CTX-M-1 were detected in E55, bla TEM-1 and bla CTX-M-55 which were detected in E84 and E85.Te reason is the production of carbapenemase by bacteria which cannot be inhibited by any of the β-lactamase inhibitors.So, under the action of carbapenemase and other resistance mechanisms, the antibiotics with enzyme inhibitors, for example, ticarcillin/clavulanic acid, piperacillin-tazobactam, ampicillin/sulbactam, and cefperazone-sulbactam, also showed resistance.
Te results of mCIM and eCIM are consistent with gene detection in the study.Te specifcity and sensitivity of mCIM and eCIM can reach 100%.It is simple, cost-efective, criteria clear and can be available easily in any laboratory.Te mCIM and eCIM were recommended by CLSI in 2017.Te mCIM and eCIM have become a useful tool in microbiology laboratories, but its limitation is timeconsuming [27].
To this study, there were some limitations.First, in our study, we detected ompA, ompC, and ompF genes deletion but did not determine the expression level of omps using RT-PCR or western blot.Second, pulsed feld gel electrophoresis analysis of S1 nuclease-digested DNA (S1-PFGE), followed by southern blotting should be conducted to identify the location of mcr-1 and bla NDMs -carrying plasmids.Finally, the genetic characteristics of the mcr-1-and bla NDMs -harboring plasmids also should be analyzed.

Conclusion
As we know, this is the frst report of carbapenem-resistant E. coli strain carrying bla NDMs and mcr-1 from urine of the RUTI patients.Te coexistence of mcr-1 and carbapenemase genes in E. coli may weaken the efectiveness of therapy and pose a potential threat to public health.Constant surveillance of polymyxin-and carbapenem-resistant organisms is imperative in order to prevent the spread of mobile antibiotic resistance mechanisms.
Infectious Diseases and Medical Microbiology

Table 2 :
Characterization of carbapenem-resistant Escherichia coli carrying bla