Introduction
============

Diarrheal diseases are the second leading cause of death in children under 5 years of age ([@B28]). Each year, diarrhea kills ∼525,000 children worldwide ([@B41]). Enterotoxigenic *Escherichia coli* (ETEC) is an extremely important causative agent of diarrhea in the developing world, due to the prevalence of unhygienic conditions that include the unavailability of clean water and poor sanitation. It is the most frequently encountered bacterial agent causing diarrhea in both children and adults, as well as the most common cause of traveler's diarrhea in these areas ([@B40]; [@B4]). ETECs are defined by their ability to produce one or both of two types of enterotoxins \[heat-stable (ST) and heat-labile (LT) ([@B34]; [@B29])\], besides which, they may also produce several colonization factors (CFs) ([@B34]) and carry chromosomal determinants associated with ETEC virulence ([@B8]).

Antimicrobial resistance (AMR) has now been recognized as a significant global threat to human and animal health ([@B42]). Due to the widespread and indiscriminate use of antibiotics in countries where diarrhea is endemic, the rate of multidrug resistance (MDR, resistance to three or more classes of antimicrobials) in *Enterobacteriaceae* such as ETEC, is increasing alarmingly ([@B22]). So far, due to this increase in MDR, the usage of successively stronger antimicrobial agents belonging to the tetracycline, sulfonamide, macrolide, and fluoroquinolone families to treat ETEC infections has increased ([@B10]; [@B31]). Fluoroquinolones are one of the most recently developed, powerful, and broad-spectrum antibiotics used for treating a wide variety of bacterial infections ([@B25]). Unfortunately, since their introduction in the late 1980s, bacterial resistance to fluoroquinolones has increased markedly ([@B1]). Colistin, which is a polymyxin antibiotic, is currently used as a last-resort antibiotic for treating infections caused by Gram-negative bacteria exhibiting MDR. However, a study conducted by [@B18] on *E. coli* strains from China, has described a new plasmid-mediated colistin-resistance mechanism conferred by the phosphoethanolamine transferase-encoding gene *mcr-1* ([@B18]). Since then, numerous studies on *mcr-1* have been undertaken worldwide to investigate the presence of this gene in *E. coli* strains isolated from various animals, environments, food samples, and even human subjects ([@B11]; [@B14]; [@B26]; [@B37]; [@B46]).

Since the discovery of *mcr-1*, several variants of the gene, such as *mcr-1.2, mcr-2, mcr-1.3, mcr-1.4, mcr-1.5, mcr-1.6, mcr-1.7*, and *mcr-1.8* have been discovered in Italy, Belgium, China, and Brunei ([@B9]; [@B43]; [@B20]; [@B38]; [@B44]; [@B47]). The variants, *mcr-2, mcr-1.3, mcr-1.4, mcr-1.5, mcr-1.7*, and *mcr-1.8* are known to have been identified from *E. coli* strains isolated from animal, sewage, or human urinary tract samples (the source for *mcr-1.8* has not been indicated) (**Table [1](#T1){ref-type="table"}**). In this study, we report a new variant of the *mcr-1* gene, which we term *mcr-1.9*, identified from an *E. coli* strain (an ETEC) isolated from a diarrheal fecal sample. Apart from analyzing the molecular and AMR characteristics of this ETEC strain, we have also identified the plasmid type in which *mcr-1.9* is located, and compared it with other similar *mcr-1*-harboring plasmids described elsewhere. We believe that this is the first report that identifies a new *mcr-1* variant from an ETEC strain isolated from a human fecal sample.

###### 

List of currently known *mcr-1* gene variants and *mcr-2*.

  Variants    Accession numbers   Nucleotide mutations   Amino acid variations   Host strain        Source              Country     Reference
  ----------- ------------------- ---------------------- ----------------------- ------------------ ------------------- ----------- ------------
  *mcr-1*     KP347127            --                     --                      *E. coli*          Pig                 China       [@B18]
  *mcr-2*     LT598652            --                     --                      *E. coli*          Calves              Belgium     [@B43]
  *mcr-1.2*   KX236309            A8T                    Q3L                     *K. pneumoniae*    Human rectal swab   Italy       [@B9]
  *mcr-1.3*   KU934208            A111G, A112G           I38V                    *E. coli*          Chicken             China       [@B44]
  *mcr-1.4*   KY041856            G1318T                 D439N                   *E. coli*          Sewage              China       [@B47]
  *mcr-1.5*   KY283125            C1354T                 H452Y                   *E. coli*          Human urine         Argentina   [@B38]
  *mcr-1.6*   KY352406            A1263G, A1607G         T215A, R536H            *S. Typhimurium*   Human rectal swab   China       [@B20]
  *mcr-1.7*   KY488488            G643A                  A215T                   *E. coli*          Sewage              China       [@B47]
  *mcr-1.8*   KY683842            A8G                    Q3R                     *E. coli*          --                  Brunei      --
  *mcr-1.9*   MG946761            G1036A                 D346N                   *E. coli*          Human fecal         China       This study

Materials and Methods {#s1}
=====================

Bacterial Isolation, Serotyping, and *mcr-1* Gene Screening
-----------------------------------------------------------

A fecal sample was obtained from a 15-month-old male child diagnosed with watery diarrhea and fever, cured with ceftriaxone in 2014 at the Shanghai Children's Hospital, China. The fecal sample was streaked onto MacConkey agar and incubated overnight at 37°C. Colonies having a pink coloration and a round, neat appearance were selected as suspected *E. coli* colonies. These were again streaked directly onto MacConkey agar, and incubated overnight at 37°C, following which, the colonies so obtained were further sub-cultured on Luria--Bertani (LB) agar plates at 37°C. Genomic DNA was prepared using the boiling method from the isolated bacteria, and a Diarrhoeagenic *E. coli* (DEC) detection PCR kit (SSI Diagnostica, Denmark) was used to identify the type of *E. coli* strain obtained from the fecal sample. The PCR reactions were performed in a 20-μl volume containing 10 μl of PCR ReadyMix, 6 μl of Primer mix, and 4 μl of DNA template. The cycling conditions were 95°C for 2 min, followed by 35 cycles of denaturation at 94°C for 50 s, annealing at 62°C for 40 s, extension at 72°C for 50 s, and a final extension at 72°C for 3 min in an Applied Biosystems PCR cycler. The PCR products are then loaded into 2% agarose gel and electrophoresis. The type of DEC was then identified by the comparison on the fragment size of PCR products and marker in the kit. We screened for the presence of the *mcr-1* gene in this isolate (termed SH66) without prior knowledge of its colistin-resistance. The screening was carried out with PCR and Sanger sequencing using previously described primers ([@B19]).

Informed and written consents were obtained from the patient's parents for sample collection and usage, as well as for the publication of obtained results in this study. All experiments were approved and authorized by the Ethics Committees of the Institute of Disease Prevention and Control, People's Liberation Army, China.

Antimicrobial Susceptibility Testing
------------------------------------

Minimum inhibitory concentrations (MICs) of colistin for all isolates obtained from the fecal sample were determined using the 96-well plate dilution method, with a maximum colistin concentration of 32 μg/mL, and a minimum concentration of 0.5 μg/mL; the results were interpreted according to the conventions set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)^[1](#fn01){ref-type="fn"}^. For the *mcr-1*-positive isolates, we also tested for antimicrobial susceptibility to other antimicrobials via the broth microdilution method using a 96-well microtiter plate (Sensititre, Trek Diagnostic Systems, Thermo Fisher Scientific Inc., West Sussex, United Kingdom). MICs were determined according to the clinical and laboratory standards institute ([@B5]) recommendations ([@B3]). The *E. coli* strain ATCC 25922 was used for quality control.

Plasmid Profiling and Southern Blotting
---------------------------------------

The DNA of ETEC strain for plasmid profiling was obtained and embedded into agarose according to the pulsed-field gel electrophoresis (PFGE) plug preparation method ([@B30]). Then the plug was cut into slices and treated with S1 endonuclease, following which, the DNA fragments were subjected to PFGE according to the PulseNet standard protocols ([@B30]). Following this, the membrane transfer, molecular hybridization, and probe detection steps were performed according to a previously reported method ([@B48]). The *mcr-1* probe sequences used in the southern blotting analysis were identical to the *mcr-1* primer sequences mentioned in Section "Bacterial Isolation, Serotyping, and *mcr-1* Gene Screening."

Conjugation Experiments
-----------------------

Conjugation experiments to test the transferability of the *mcr-1*-positive plasmid were carried out using the *E. coli* strain J53 (LacZ^-^, AzrR, RifR) as a recipient, and the *mcr-1*-positive *E. coli* strain (identified in the fecal sample and named SH66) as a donor. Overnight cultures of donor and recipient bacterial strains (3 mL of each) were mixed together, harvested, and resuspended in 80 μl of LB with a donor:recipient ratio of 1:3. The resultant mixture was spotted into 5 mL of liquid LB broth and incubated 37°C for 12 to 18 h for mating to occur. Following this, a small amount of the mating mixture was spotted on to a Muller-Hinton agar plate (BD Biosciences, San Jose, CA, United States) containing 100 mg/L sodium azide and 2 mg/L polymyxin B; this was used as a selective medium to identify *E*. *coli* J53 transconjugants. The identities of putative transconjugants were confirmed using antimicrobial susceptibility testing and PCR detection of the *mcr-1.9* gene as described before. The conjugation transfer rate was calculated through the formula "R = number of transconjugants/number of recipients."

Whole Genome Sequencing and Bioinformatic Analysis
--------------------------------------------------

The genomic DNA of the *E. coli* isolate SH66 was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany), and sequenced using the Illumina MiSeq platform (Illumina Inc., San Diego, CA, United States) according to the manufacturer's instructions. A DNA library was constructed with inserts of length 600 bp using the MiSeq Reagent Kit v2. The raw sequence data were assembled *de novo* via the CLC Genomics Workbench 9.0.1 (CLC Bio, Qiagen Bioinformatics, Germany). The *mcr-1* plasmid sequence was assembled through the above software and the gaps were filled through combinatorial PCR and Sanger sequencing. The point mutation in *mcr-1.9* was verified by the PCR amplification and Sanger sequencing with the primers MCR1-3-F (5′-GGCGTATTCTGTGCCGTGTA-3′) and MCR1-3-R (5′-CGTGATCGCGTCATGGGT-3′) (**Supplementary Data Sheet [S2](#SM5){ref-type="supplementary-material"}**). Resistance genes, virulence genes, and other genetic structures were identified through the center for genomic epidemiology (CGE) website^[2](#fn02){ref-type="fn"}^. Sequence alignments were performed using the software Mauve ([@B7]), and further rounds of PCR amplification and Sanger sequencing were used to fill in the gaps. Complete plasmid sequences were annotated using the online annotation server RAST ([@B2]). The plasmid replicons, IS sequences, and AMR genes were identified using the online software PlasmidFinder^[3](#fn03){ref-type="fn"}^, ISfinder^[4](#fn04){ref-type="fn"}^, and ResFinder^[5](#fn05){ref-type="fn"}^, respectively. Sequence comparisons and map generation were performed using BLAST^[6](#fn06){ref-type="fn"}^ and Easyfig ([@B36]), respectively.

Results
=======

*mcr-1* Gene Screening and Antimicrobial Susceptibility Testing
---------------------------------------------------------------

One *E. coli* strain (termed SH66) was isolated from the fecal sample used in this study, and identified as an ETEC using the DEC PCR detection kit. This strain was subsequently transferred to and preserved in our laboratory, where it was recently screened for the presence of the *mcr-1* gene with no prior knowledge of its colistin-resistance characteristic. The screening indicated that this strain harbored an *mcr-1* gene. According to medical records, the patient who harbored this strain was not treated with polymyxin antibiotics.

The results of the antimicrobial susceptibility testing indicate that the ETEC strain identified in this study is resistant to colistin (MIC, 4 μg/ml), ampicillin (\>32 μg/ml), chloramphenicol (\>32 μg/ml), trimethoprim/sulfamethoxazole (\>4 μg/ml), tetracycline (\>32 μg/ml), ciprofloxacin (\>4 μg/ml), sulfonazo isoxazole (\>256 μg/ml), nalidixic acid (\>32 μg/ml), streptomycin (\>64 μg/ml), and azithromycin (\>16 μg/ml), though it was found to be susceptible to amoxicillin/clavulanic acid (4 μg/ml), ceftriaxone (≤0.25 μg/ml), ceftiofur (0.25 μg/ml), cefoxitin (8 μg/ml), and gentamicin (1 μg/ml) (**Table [2](#T2){ref-type="table"}**). According to the classification standards of the WHO ([@B6]), SH66 is an MDR strain showing resistance to nine classes of antimicrobials, including aminoglycosides, amphenicols, folate pathway inhibitors, macrolides, penicillins, polymyxins, quinolones, sulfonamides, and tetracyclines.

###### 

Comparisons of antimicrobial resistance levels between the donor *E. coli* strain SH66, recipient strain J53 and the J53 transconjugants.

  Antimicrobial class         Antimicrobial         Donor strain SH66   Transconjugants J53   Recipient strain J53                
  --------------------------- --------------------- ------------------- --------------------- ---------------------- -------- --- --------
  Polymyxins                  PolymyxinB/Colistin   R                   4                     R                      4        S   0.125
  Aminoglycosides             Gentamicin            S                   1                     S                      ≤0.25    S   ≤0.25
                              Streptomycin          R                   \>64                  R                      \>64     S   ≤2
  Amphenicols                 Chloramphenicol       R                   \>32                  R                      \>32     S   ≤8
  Cephalosporins              Ceftriaxone           S                   ≤0.25                 S                      ≤0.25    S   ≤0.25
                              Ceftiofur             S                   0.25                  S                      ≤0.5     S   ≤0.5
  Cephamycins                 Cefoxitin             S                   8                     S                      ≤8       S   ≤8
  Folate pathway inhibitors   TRI/SUL               R                   \>4                   R                      \>4      S   ≤2
  Macrolides                  Azithromycin          R                   \>16                  R                      \>16     S   ≤4
  Penicillins                 AMO/CLA               S                   4                     S                      ≤4       S   ≤4
                              Ampicillin            R                   \>32                  R                      \>32     S   ≤8
  Quinolones                  Ciprofloxacin         R                   \>4                   S                      ≤0.015   S   ≤0.015
                              Nalidixic acid        R                   \>32                  S                      ≤4       S   ≤4
  Sulfonamides                Sulfisoxazole         R                   \>256                 R                      \>256    S   ≤16
  Tetracyclines               Tetracycline          R                   \>32                  R                      \>32     S   ≤4

The classifications of antimicrobial agents are based on the classification standards of WHO (World Health Organization) (

Collignon et al., 2009

). The unit of MICs isμg/ml. S, susceptible; R, resistant; TRI/SUL, trimethoprim/sulfamethoxazole; AMO/CLA, amoxicillin/clavulanate.

Conjugation Experiments and Southern Blotting
---------------------------------------------

In order to determine the transferability of the *mcr-1* gene carried by SH66, conjugation experiments were carried out using the *E. coli* strain J53 as a recipient, and SH66 as the donor strain. All transconjugants obtained in this experiment tested positive for the *mcr-1* gene via PCR amplification and sequencing analysis. The conjugation transfer rate is 1.6 × 10^-5^. Colistin susceptibility testing showed that the MIC values for the transconjugants (the J53 strains carrying the *mcr-1* gene) increased to 4 μg/ml (**Table [2](#T2){ref-type="table"}**). This is much higher than the colistin resistance levels of the original J53 strains (which have MIC values of 0.125 μg/ml), and indicates that the transconjugants acquire the same colistin resistance gene as the donor strain.

The S1 endonuclease-PFGE and southern blotting analysis revealed that the *mcr-1* gene in SH66 was located on a plasmid approximately 60 kb in length (**Supplementary Figure [S1](#SM1){ref-type="supplementary-material"}**).

Virulence and Resistance Factors Carried by the *mcr-1*-Harboring ETEC Strain SH66
----------------------------------------------------------------------------------

The DNA of the *E. coli* strain SH66 identified in this study were subjected to 600-bp paired-end whole genome sequencing with 73.4-fold coverage using the MiSeq sequencer. A total of 1,364,690 reads and 405,565,025 clean bases were generated, which were assembled into 107 contigs with an N50 length of 216,913 bp. Bioinformatics analyses showed that this strain carries the virulence genes *eltA, eltB, astA, eilA, lpfA, ltcA*, and *stb*. Additionally, SH66 also carries several resistance genes, namely, *bla*~TEM-1~, *mphA, ermB, aph(3')-Ia, aadA5, strA, strB, sul1, sul2, tetA, tetM, floR, catA1, dfrA17, oqxA*, and *oqxB*, along with the *mcr-1* gene, all of which could mediate resistance to β-lactams, macrolides, aminoglycosides, sulphonamides, tetracyclines, phenicols, trimethoprim, quinolones, and colistin (**Supplementary Table [S1](#SM3){ref-type="supplementary-material"}**). The presence of these genes also explains the increase in resistance to several antibiotics observed in the *E. coli* J53 transconjugants after the conjugation experiment (**Table [2](#T2){ref-type="table"}**). Analyses of the *gyrA* and *parC* point mutations carried by SH66 also showed that *gyrA* contained the Ser83→Leu and Asp87→Asn substitutions, along with the *parC* mutations Ser57→Thr and Ser80→Ile.

Identification and Analysis of the Plasmid Carrying the Novel Variant, *mcr-1.9*
--------------------------------------------------------------------------------

An *mcr-1*-carrying plasmid of length 64,005 bp, which we have named pEC26, was identified in SH66 through sequence analysis. Interestingly, the *mcr-1* gene identified on this plasmid was found to be a variant of the original *mcr-1* due to an SNP (**Supplementary Figure [S2](#SM2){ref-type="supplementary-material"}**). The SNP was located at position 1036 (where a G→A transition had occurred), which results in the replacement of an aspartic acid residue with an asparagine residue (Asp346→Asn). Since the position of this SNP is different from those of all previously discovered *mcr-1* variants, we named this variant *mcr-1.9*. The sequence analysis shows that there is no resistance gene in addition to *mcr-1.9* located in plasmid pEC26.

The plasmid pEC26 belongs to the IncI2 group, the same plasmid incompatibility group as pHNSHP45, which was the first plasmid reported to harbor *mcr-1* ([@B18]); pEC26 also harbors 86 predicted open reading frames (ORFs), and has a GC content of 42.55% with a plasmid backbone very similar to that of other IncI2-type plasmids, such as pmcr1-IncI2 (99% identity and 98% coverage, GenBank accession no. KU761326) ([@B15]) and pWF-5-19C (99% identity and 99% coverage, KX505142) ([@B17]). A comparison of these plasmids and pHNSHP45 (KP347127) ([@B18]) shows that pEC26, pmcr1-IncI2, and pWF-5-19C contain an IS200 element before the *mok* gene instead of the IS683 found in pHNSHP45. However, the IS200 element in these plasmids is inserted before a 363 bp fragment that precedes the IS683 element in pHNSHP45 (**Figures [1](#F1){ref-type="fig"}, [2A](#F2){ref-type="fig"}**). This indicates that although these two IS elements are inserted into the same plasmid backbone, their positions are not the same. Like all the other plasmids mentioned here, pEC26 also contains the 2,607 bp DNA segment that includes the *mcr* (*mcr-1* or *mcr-1.9*) and *pap2* genes, which are integrated downstream of the *nikB* gene. Besides this, the *mcr-1*-associated IS*Apl1* element was not found in the pEC26 plasmid (**Figures [1](#F1){ref-type="fig"}, [2B](#F2){ref-type="fig"}**). The sequence of pEC26 (**Supplementary Data Sheet [S1](#SM4){ref-type="supplementary-material"}**) has been deposited with GenBank under the accession number [MG946761](MG946761).

![Linear comparisons of complete sequences of the IncI2-type plasmids pHNSHP45 (accession no. KP347127), pmcr1_IncI2 (KU761326), pWF-5-19C (KX505142), and pEC26 (MG946761). Colored arrows represent open reading frames, with black, yellow, green, red, and orange arrows representing replication genes, mobile elements, plasmid transfer genes, the *mcr-1* gene, and plasmid backbone genes, respectively. Gray shading denotes regions of shared homology amongst the different plasmids.](fmicb-09-00815-g001){#F1}

![A comparison of the genetic environments of IS683 and IS200 **(A)**, along with *mcr-1* and *mcr-1.9* **(B)** in the plasmids pHYEC7-mcr1 ([@B16]), pHNSHP45, pWF-5-19C, pmcr1_Incl1 and pEC26. Gray shading indicates \>99% nucleotide identity.](fmicb-09-00815-g002){#F2}

Discussion
==========

This study reports the identification of a novel *mcr-1* gene variant, *mcr-1.9*, which was harbored in an ETEC strain isolated from the diarrheal fecal sample of a 15-month-old male child. We find that the variant *mcr-1.9*, differs from the *mcr-1* gene at position 1036 (where a G→A transition had occurred), which results in the replacement of an aspartic acid residue with an asparagine residue (Asp346→Asn). The MCR-1 protein consists of a transmembrane domain and a soluble catalytic domain. The transmembrane domain is located on the N-terminal, and is composed of residues 1--214, whereas the soluble catalytic domain is located on the C-terminal, and is composed of residues 215--541 ([@B35]). Therefore, the Asp346→Asn change detected in *mcr-1.9* is located in the catalytic domain. Since four of the known *mcr-1* gene variants, *mcr-1.4--1.7*, also contain point mutations in the catalytic domain (**Table [1](#T1){ref-type="table"}**), it appears that this domain has a higher mutation rate than the transmembrane domain. However, although the currently identified mutations in the *mcr-1* gene have so far not showed obvious increases in colistin resistance, the presence of two or more point mutations in AMR genes can occasionally lead to changes in the strength of resistance imparted by the gene, such as the resistance increase to fluoroquinolones by the additional mutations in the *gyrA* and/or *parC* genes ([@B39]; [@B32]).

Apart from mutations causing heightened resistance to known antimicrobials, another concern regarding resistance genes is their spread through bacterial populations via horizontal transfer. The occurrence of the *mcr-1* variant *mcr-1.9* in an ETEC strain isolated from a human fecal sample is a cause of much concern; as the human intestine contains a complex collection of microflora where the incidence of horizontal gene transfer can occur at a high rate, the spread of this gene can cause widespread resistance to colistin. Added to this, several studies have shown that the *mcr-1* gene can be ubiquitously disseminated in the *Enterobacteriaceae* ([@B12]; [@B23]; [@B45]). Furthermore, the *mcr-1* gene is usually associated with the highly active transposon element *ISApl1* that can occur either on one, or both sides of the gene ([@B16]; [@B33]) (**Figure [2B](#F2){ref-type="fig"}**). The presence of *ISApl1* could further enhance the probability of this gene being transferred horizontally; *in vitro* mobilization assays have already proved that the *mcr-1* gene can be mobilized by *ISApl1*-mediated transposition ([@B27]). Moreover, the transfer of *mcr-1* via *ISApl1*-mediated transposition has been shown to generate mutations, leading to the formation of new *mcr-1* gene variants ([@B27]). We therefore hypothesize that *mcr-1.9* and perhaps other variants of *mcr-1* may have been generated during horizontal transfer events involving transposable elements.

The first reported *mcr-1* gene was identified in an IncI2 plasmid ([@B18]). As of now, the *mcr-1* gene and its variants have been reported in various plasmid backbones, including the IncI2, IncHI2, IncF, IncP, IncFIP, and IncX4 ([@B18]; [@B11]; [@B13]; [@B24]), all of which are known to be vehicles disseminating antibiotic resistance genes among the *Enterobacteriaceae.* The *mcr-1.9* gene identified by us was found on an IncI2-type plasmid, which has a broad host range ([@B21]). Furthermore, three \[*mcr-1.3* (KU934208), *mcr-1.5* (KY471308), and *mcr-1.7* (KY829117)\] of the six *mcr-1* variants \[*mcr-1.8* (KY683842) has no description of plasmid type\] previously discovered are located on IncI2-type plasmids. Besides this, the plasmid most similar to pEC26 is pWF-5-19C (KX505142), which is found in *Cronobacter sakazakii* isolated from chicken ([@B17]). Based on this information, we speculate that pEC26 and plasmids similar to it have a strong ability to facilitate horizontal transfer of the *mcr-1* gene, and that this gene may have a higher mutation rate when carried by IncI2-type plasmids than when they are present in other plasmid types. However, since only a few such instances have been identified so far, this hypothesis needs much further study for verification and confirmation.

Besides harboring the novel *mcr-1* gene variant, *mcr-1.9*, the ETEC that we have isolated and described in this study was found to carry various virulence genes, namely, heat-stable enterotoxin (*estA*), heat-labile enterotoxin (*eltB*), EAST1 (enteroaggregative *E. coli* heat-stable toxin 1) gene (*astA*), adherence related gene (*eilA*), long polar fimbriae (*lpfA*), heat-labile enterotoxin A subunit (*ltcA*), and heat-stale enterotoxin II (*stb*). The strain was also found to carry several antimicrobial-resistance genes and shows MDR to at least nine classes of antibiotics (**Table [2](#T2){ref-type="table"}**). These results indicate that this strain, called SH66, probably has high AMR and pathogenicity. Since *mcr-1* variants have begun to appear in human intestinal bacteria, especially in diarrheal *E. coli* strains, it is very possible that the spread of this gene in human intestinal microflora may cause issues in the effective treatment of diarrheal conditions induced by bacterial infections.

Overall, our findings highlight the possibility of a widespread dissemination of *mcr-1* gene variants, along with various virulence factors and resistance genes in the bacterial flora of the human intestine. Therefore, we believe that it is of utmost importance to track and identify mutations in the *mcr-1* gene, and to control its transmission. We also believe that the judicious use of antibiotics by physicians is crucial to reduce the rate of emergence of MDR bacterial strains.

Author Contributions
====================

HS, YS, and CY designed the experiments. HL and BZ performed the experiments. SQ, YX, LJ, and BL analyzed the data. PL, YL, and LY prepared the tables and figures. CY, YX, JX, and LW prepared the manuscript. All authors read and approved the final manuscript.

Conflict of Interest Statement
==============================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Funding.** This work was supported by the National Key R&D Program of China (no. 2017YFC1600105), Mega-projects of Science and Technology Research, China (no. 2018ZX10714002), and the National Nature Science Foundation of China (nos. 81673237 and 81473023).

<http://www.eucast.org/clinicalbreakpoints/>

<http://www.genomicepidemiology.org/>

<https://cge.cbs.dtu.dk/services/PlasmidFinder/>

<https://www-is.biotoul.fr/search.php>

<https://cge.cbs.dtu.dk/services/ResFinder/>

<http://blast.ncbi.nlm.nih.gov>

Supplementary Material
======================

The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fmicb.2018.00815/full#supplementary-material>

###### 

Plasmid profiling and southern blotting of SH66. The red box marks the plasmid pEC26. The genome DNA of *Salmonella* ser. Braenderup H9812 were restriction digested with *Xba*I enzyme and the products were used as the markers. The sizes of products are showed on the left with the unit "kb."

###### 

Click here for additional data file.

###### 

Nucleotide and protein sequence alignments of the *mcr-1* gene with its different variants. Only the alignment blocks carrying mutations found in *mcr-1.9* are shown. The mutations in the base and amino acid sequences are denoted by the use of a different color. In *mcr-1.9*, G was mutated to A in nucleotide position 1036, leading to the replacement of an Aspartic acid residue with an asparagine residue at amino acid position 346.

###### 

Click here for additional data file.

###### 

List of resistance and virulence factors/genes found in the *mcr-1.9*-harboring ETEC identified in this study.

###### 

Click here for additional data file.

###### 

The complete sequence of *mcr-1.9* harboring plasmid pEC26 (GenBank accession number is [MG946761](MG946761)).

###### 

Click here for additional data file.

###### 

The verification result files of the point mutation in *mcr-1.9* by the PCR amplification and Sanger sequencing.

###### 

Click here for additional data file.

[^1]: Edited by: Dongsheng Zhou, Beijing Institute of Microbiology and Epidemiology, China

[^2]: Reviewed by: Xinglin Zhang, Zhejiang University, China; Meiying Yan, National Institute for Communicable Disease Control and Prevention, China; Séamus Fanning, University College Dublin, Ireland; Xianming Shi, Shanghai Jiao Tong University, China

[^3]: ^†^These authors have contributed equally to this work.

[^4]: This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology
