Multidrug-Resistant Escherichia coli Isolate of Chinese Bovine Origin Carrying the blaCTX-M-55 Gene Located in IS26-Mediated Composite Translocatable Units

Elevated detection rates of the blaCTX-M-55 gene in animals have been reported as a result of antibiotic misuse in clinics. To investigate the horizontal transfer mechanism of blaCTX-M-55 and its associated mobile genetic elements (MGEs), we isolated 318 nonrepetitive strains of Escherichia coli (E. coli) from bovine samples in Xinjiang and Gansu provinces, China. All E. coli strains were screened for the CTX-M-55 gene using PCR. The complete genomic data were sequenced using the PacBio triplet sequencing platform and corrected using the Illumina data platform. The genetic environment of the plasmids carrying the resistance blaCTX-M-55 gene was mapped using the software Easyfig2.2.3 for comparison. The results showed that all blaCTX-M-55-positive strains were resistant to multiple antibiotics. Five strains of Escherichia coli carry the blaCTX-M-55 gene, which is adjacent to other resistance genes and is located on the IncHI2-type plasmid. Four of the five blaCTX-M-55-harbor strains carried translocatable units (TUs). All the donor bacteria carrying the blaCTX-M-55 genes could transfer horizontally to the recipient (E. coli J53 Azr). This study demonstrates that the transmission of blaCTX-M-55 is localized on IS26-flanked composite transposons. The cotransmission and prevalence of blaCTX-M-55 with other MDR resistance genes on epidemic plasmids require enhanced monitoring and control.


Introduction
Antimicrobial resistance, particularly multidrug resistance, has become one of the greatest threats to global health today.E. coli is one of the most common nosocomial and community-acquired Gram-negative pathogens that is resistant to third-and fourthgeneration cephalosporins [1][2][3].
The extended-spectrum β-lactamase (ESBL) family in E. coli is widespread and prevails, in which CTX-M is by far the most prevalent drug resistance gene [4,5].Today, more than 230 bla CTX-M variants are documented in GenBank.In China, the main prevalent drug resistance genes are bla CTX-M-9 and bla CTX-M-1 [6].Of these, the CTX-M-55 ESBL-positive rate is already overgrown in the clinic [7].Increasing reports indicate the transmission of the bla CTX-M gene alone or its cotransmission with other ARGs in humans, animals, and waterfowl [8][9][10][11].Recently, CTX-M-55-positive E. coli strains isolated from cattle feces were widely reported in China, Canada, South Korea, and France [8,[12][13][14], suggesting that bla CTX-M is an important drug resistance gene in cattle from food animal sources.
Furthermore, the cotransmission of CTX-M-55 with different resistance genes has also been reported [9].Therefore, the genetic environment of bla CTX-M-55 needs to be explored to analyze the evolutionary and transmission mechanisms of drug resistance.
As in previous reports, bla CTX-M is seldom on chromosomes but mainly localized on plasmids, such as IncHI2, IncFIA, and IncFIB [15][16][17][18].Plasmid-encoded CTX-M enzymes have a robust horizontal transmission capability between humans and food animals, implying that they can be transmitted directly from animal products to humans, thereby ultimately causing a potential contamination risk of drug resistance [4].Additionally, mobile genetic elements (MGEs) located on plasmids can widely spread antibiotic resistance genes (ARGs) [19].The insertion sequence (IS), one of the MGEs, can significantly facilitate the transfer of ARGs between chromosomes and plasmids [20].It has been reported that IS (such as IS26, ISEcp1, and ISCR1) is commonly found to be adjacent to bla CTX-M , which plays a vital role in promoting the horizontal transmission of bla CTX-M genes [18,21,22].MGEs on plasmids have been a worrying feature, leading to their global spread and evolution [4,23].
Although the inappropriate use of antibiotics contributes to the persistence and spread of antibiotic resistance, there is limited information available on this topic from developing countries.The misuse and abuse of cephalosporins in clinics have resulted in the high rates of detection of the bla CTX-M-55 gene in food animals.As a result, there is an increased risk of transmission from food animals to humans caused by bla CTX-M-55 -bearing plasmids.To investigate if the occurrence of cephalosporin resistance has also increased with the high frequency of bla CTX−M and the horizontal transfer frequency of bla CTX-M in food animal isolates in recent years, E. coli isolates of bovine origin collected during 2018 for cephalosporin resistance and plasmid-mediated cephalosporin resistance genes were screened, and the genetic environment of the positive strain was analyzed to explore the contribution of certain MGEs to the horizontal transfer of the bla CTX-M-55 gene.

Strain Isolation and Identification
The study collected 232 nonrepetitive fecal samples from cattle at a farm (n = 73) in the provinces of Xinjiang Province, China, and two farms (n = 159) in the provinces of Gansu Province, China, between August and December 2018.All samples were collected in sterile containers, transported to the laboratory at 6 • C ± 2 • C, and processed immediately for further assays.
All 318 E. coli isolates were selected on MacConkey agar plates (Huankai, China), and the species were identified with MALDI-TOF-MS and 16S rRNA sequencing using universal primers (see Table S1).Positive strains were screened for the presence of CTX-M-55 using PCR [24].

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing of strains toward 15 different antimicrobials was determined with broth microdilution using breakpoints specified by Clinical & Laboratory Standards Institute (CLSI) M100 guidelines (http://clsi.org)(accessed on 10 October 2020).Briefly, bacterial colonies were selected and cultured overnight in 1 mL of MH broth.The following day, a 10 µL aliquot of the bacterial solution was absorbed and re-inoculated into 1 mL of fresh MH broth, followed by incubation at 37 • C, to obtain a bacterial concentration of approximately 1 × 10 8 CFU/mL.A 180 µL volume of MH broth was added into the first column of a 96-well plate and an additional 100 µL was also added into each subsequent well.After adding 20 µL of the tested antibiotic stock solution to the first column, serial dilutions were performed using the double-dilution methodology.Column eleven served as the negative control, while column twelve served as the positive control.Bacteria were diluted one hundred times with fresh MH broth to obtain a final concentration of approximately 1 × 10 6 CFU/mL before adding it (100 µL) into antibiotics (200 µL).Finally, the plate was incubated at medium culture temperature (37 • C).The following antibiotics were used for antimicrobial susceptibility testing: cefotaxime (CTX), ceftazidime (CAZ), cefalotin (KF), ceftriaxone (CRO), tetracycline (TE), ampicillin (AMP), amikacin (AK), ciprofloxacin (CIP), doxycycline (DO), fosfomycin (FOT), kanamycin (K), chloramphenicol (C), sulfamethoxazole-trimethoprim (SXT), gentamicin (GN), and aztreonam (ATM).E. coli ATCC25922 was used as the quality control strain.

Conjugation Assay and Determination of Conjugation Frequency
Conjugation was performed using E. coli J53 Az r as the recipient strains as previously described and conducted with solid mating on a filter [25] (Whatman, Maidstone, UK).The donor-recipient ratio was 1:1 using Mueller-Hinton medium (MHA, Huankai, Guangzhou, China) supplemented with cefotaxime (2 µg/mL) and sodium azide (200 µg/mL) as the selective medium.The transconjugants were analyzed via PCR (bla CTX-M-1 primers, see Table S1), Sanger sequencing, and antimicrobial susceptibility testing to compare their consistency with the donor strain.Transfer frequencies were calculated as the number of transconjugants per recipient.

Whole-Genome Sequencing and Bioinformatics Analysis of CTX-M E. coli Producer
Five isolates identified as positive for bla CTX−M−55 using PCR and Sanger sequencing were further characterized with whole-genome sequencing.The total genomic DNA of five strains was extracted with the SDS method [26].The harvested DNA was detected using agarose gel electrophoresis and quantified using a Qubit ® 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA).Libraries for single-molecule real-time (SMRT) sequencing were constructed with an insert size of 10 kb using the SMRT bell TM Template kit, version 1.0.Briefly, the process involved fragmenting and concentrating DNA, repairing DNA damage and ends, preparing blunt ligation reactions, purifying SMRTbell Templates with 0.45× AMPure PB Beads, size selection using the BluePippin System, and repairing DNA damage after size selection.Finally, the library quality was assessed on a Qubit ® 2.0 Fluorometer (Thermo Scientific), and the insert fragment size was detected using Agilent 2100 (Agilent Technologies, Santa Clara, CA, USA).The whole genomes of selected strains were sequenced using the PacBio Sequel I platform and Illumina NovaSeq6000 at Beijing Novogene Bioinformatics Technology Co., Ltd.(Beijing, China).

Plasmid Stability
Plasmid stability was determined according to Lv's [30].Briefly, without antibiotics, these strains were cultured continuously in daily refreshed LB broth with 1000-fold dilution for 15 days to detect their stability.Within each 5-day period, 20 colonies were randomly selected and confirmed using the PCR amplification of bla CTX-M-55 (bla CTX-M-1 primers, see Table S1).The plasmid retention rate was further calculated as mentioned above over a period of 15 days.

Statistical Analysis
Mean values and standard deviations were calculated using SPSS 17.0 version software.Student's t test was used to evaluate differences between means, with a significant probability at a p value of <0.05.

Characteristics of CTX-M-Positive E. coli
The presence of the bla CTX-M-55 gene was confirmed in five E. coli isolates (1.57%, 5/318), of which three (3/5) were from dairy cattle and two (2/5) were from beef cattle.These five isolates were recovered from beef and dairy cattle in three different farms located in two provinces (Gansu and Xinjiang).The MIC results showed that all five isolates were multidrug-resistant (MDR) and resistant to at least nine antibiotics.All five isolates showed resistance to KF, CTX, CAZ, CRO, AMP, C, GN, and ATM (Table 1).
To observe the similarity between the five plasmids harboring bla CTX-M-55 , the alignment of the complete nucleic acid sequence of these five plasmids was generated using BRIG.The backbone region of pXJ55-plas1 was almost identical to that of pZYB39-plas2, pZYB62-plas1, pXJ5.2-plas1, and pXJ6.1-plas1(Figure 3).Except for the backbone region, the major differences among the five plasmids were concentrated in the MRR and TraC (transfer protein) and ParB (N-terminal domain-containing protein) of the backbone.

Transconjugative Frequencies of Plasmids Carrying blaCTX-M-55
As a donor, the CTX-M-positive strain was transferred to recipient E. coli J53 according to the conjugation assay.Five plasmids carrying blaCTX−M−55 were all successfully transferred into the recipient strain.The conjugation frequencies of IncHI2 plasmids are listed in Table 3 as transconjugants per recipient.The conjugation frequencies of IncHI2 plasmids were 10 −5 ~10 −8 per recipient.

Transconjugative Frequencies of Plasmids Carrying bla CTX-M-55
As a donor, the CTX-M-positive strain was transferred to recipient E. coli J53 according to the conjugation assay.Five plasmids carrying bla CTX−M−55 were all successfully transferred into the recipient strain.The conjugation frequencies of IncHI2 plasmids are listed in Table 3 as transconjugants per recipient.The conjugation frequencies of IncHI2 plasmids were 10 −5 ~10 −8 per recipient.a The experiment was repeated three times.b Cefotaxime was used as the selection pressure.

Discussion
The detection of CTX-M-positive isolates from Western China has been conducted by our group, revealing that the most prevalent genotype of the CTX-M gene in Gansu beef cattle is CTX-M-55 (27.9%, 36/129) [32].However, the detection rate of CTX-M-55 in dairy cattle from Xinjiang Province was 11.1% (12/108).Extended-spectrum cephalosporin (ESC) resistance is a common prevalence in livestock [33], especially in E. coli strains producing CTX-M-type ESBLs, which has become a worrying issue.In the United States, bovine-derived ESC-resistant (ESC-R) E. coli resistance rates are as high as 95% [34], while in Asia, they range from 1% to 33% [35][36][37][38].All of these data suggest that E. coli serves as a reservoir for CTX resistance genes, and the spread of its resistance is a matter of serious concern.
In this study, five multidrug-resistant blaCTX-M-55-positive isolates were all resistant to cefotaxime.Furthermore, they displayed a wide spectrum of antibiotic resistance to additional common antibiotics in the clinical and breeding industry, such as β-lactams, aminoglycosides, and tetracycline.High-frequency therapeutic failures in the treatment of ESBL-producing microorganisms can be expected as these resistant strains spread.
The results of STs showed that the clones in this study did not involve high-risk clones (Table 1), such as ST131 or ST69 [39].However, ST58, which is a major extraintestinal pathogenic (causing urinary tract infection) E. coli lineage in humans, was found in clones carrying blaCTX-M-55 in our study and has also been reported in numerous countries [40][41][42].Furthermore, since XJ5.2 is serotype O45 with some pathogenicity and resistance, its transmission should be provided extra attention.
IS26, belonging to the IS6 family, has a simple organization and generates an 8 bp DR upon insertion [43].In this cointegration process, the second formed IS copy is directly

Discussion
The detection of CTX-M-positive isolates from Western China has been conducted by our group, revealing that the most prevalent genotype of the CTX-M gene in Gansu beef cattle is CTX-M-55 (27.9%, 36/129) [32].However, the detection rate of CTX-M-55 in dairy cattle from Xinjiang Province was 11.1% (12/108).Extended-spectrum cephalosporin (ESC) resistance is a common prevalence in livestock [33], especially in E. coli strains producing CTX-M-type ESBLs, which has become a worrying issue.In the United States, bovinederived ESC-resistant (ESC-R) E. coli resistance rates are as high as 95% [34], while in Asia, they range from 1% to 33% [35][36][37][38].All of these data suggest that E. coli serves as a reservoir for CTX resistance genes, and the spread of its resistance is a matter of serious concern.
In this study, five multidrug-resistant bla CTX-M-55 -positive isolates were all resistant to cefotaxime.Furthermore, they displayed a wide spectrum of antibiotic resistance to additional common antibiotics in the clinical and breeding industry, such as β-lactams, aminoglycosides, and tetracycline.High-frequency therapeutic failures in the treatment of ESBL-producing microorganisms can be expected as these resistant strains spread.
The results of STs showed that the clones in this study did not involve high-risk clones (Table 1), such as ST131 or ST69 [39].However, ST58, which is a major extraintestinal pathogenic (causing urinary tract infection) E. coli lineage in humans, was found in clones carrying bla CTX-M-55 in our study and has also been reported in numerous countries [40][41][42].Furthermore, since XJ5.2 is serotype O45 with some pathogenicity and resistance, its transmission should be provided extra attention.
IS26, belonging to the IS6 family, has a simple organization and generates an 8 bp DR upon insertion [43].In this cointegration process, the second formed IS copy is directly oriented as the original copy and flanked by DR sequences [44].In the process of study, we found that the bla CTX-M-55 genetic environment of pXJ55-plas1 and pZYB39-plas2 was flanked by directly oriented copies of IS26 with 8 bp DR sequences (TTTTGCTG).However, IS26 and potential IS26-mediated transposons do not necessarily generate the flanking DR, and intramolecular transposition will lead to the loss of the flanking repeat [43].Notably, pXJ6.1-plas1 is the plasmid containing the most TUs and the highest number of copies of IS26.This may be related to the increased activity and multiple copy number of IS26 in the plasmid [45].IS26 can enhance the expression of the bla CTX-M-55 gene, and its presence could explain the ease with which this gene is spreading among bacteria and different species.The TU contains a single IS26 copy and neighboring DNA, which plays a vital role in the diffusion of ARGs in Gram-negative bacteria to form complex resistance regions [46].sequences of strains were used in the study, and accession numbers and associated metadata can be found in the Nucleotide Sequence Accession Number Section as indicated.The whole-genome sequence was uploaded and registered in the NCBI database with accession numbers: CP074354 (Escherichia XJ5.2-chr1), CP074355 (Escherichia XJ5.2-plas1), CP074356 (Escherichia XJ6.1-chr1), CP074357 (Escherichia XJ6.1-plas1), CP074358 (Escherichia XJ6.1-plas2), CP098229 (Escherichia XJ55-chr1), CP098230 (Escherichia XJ55-plas1), CP098234 (Escherichia ZYB39-chr1), CP098235 (Escherichia ZYB39-plas1), CP098236 (Escherichia ZYB39-plas2), CP074366 (Escherichia ZYB62-chr1), CP074367 (Escherichia ZYB62-plas1), CP074368 (Escherichia ZYB62-plas2), and CP074369 (Escherichia ZYB62-plas3).

Table 1 .
Background information and characteristics of bla CTX-M-55 -carrying E. coli isolates.