F18:A-:B1 Plasmids Carrying blaCTX-M-55 Are Prevalent among Escherichia coli Isolated from Duck–Fish Polyculture Farms

We determined the prevalence and molecular characteristics of blaCTX-M-55-positive Escherichia coli (E. coli) isolated from duck–fish polyculture farms in Guangzhou, China. A total of 914 E. coli strains were isolated from 2008 duck and environmental samples (water, soil and plants) collected from four duck fish polyculture farms between 2017 and 2019. Among them, 196 strains were CTX-M-1G-positive strains by PCR, and 177 (90%) blaCTX-M-1G-producing strains were blaCTX-M-55-positive. MIC results showed that the 177 blaCTX-M-55-positive strains were highly resistant to ciprofloxacin, ceftiofur and florfenicol, with antibiotic resistance rates above 95%. Among the 177 strains, 37 strains carrying the F18:A-:B1 plasmid and 10 strains carrying the F33:A-:B- plasmid were selected for further study. Pulse field gel electrophoresis (PFGE) combined with S1-PFGE, Southern hybridization and whole-genome sequencing (WGS) analysis showed that both horizontal transfer and clonal spread contributed to dissemination of the blaCTX-M-55 gene among the E. coli. blaCTX-M-55 was located on different F18:A-:B1 plasmids with sizes between ~76 and ~173 kb. In addition, the presence of blaCTX-M-55 with other resistance genes (e.g., tetA, floR, fosA3, blaTEM, aadA5 CmlA and InuF) on the same F18:A-:B1 plasmid may result in co-selection of resistance determinants and accelerate the dissemination of blaCTX-M-55 in E. coli. In summary, the F18:A-:B1 plasmid may play an important role in the transmission of blaCTX-M-55 in E. coli, and the continuous monitoring of the prevalence and transmission mechanism of blaCTX-M-55 in duck–fish polyculture farms remains important.


Introduction
Antimicrobial resistance is a serious global public health problem associated with significant clinical, economic and social impacts. Escherichia coli exists as part of the commensal microbiota in the mammalian digestive tract, as a zoonotic pathogen responsible for intestinal and extraintestinal infections in both humans and animals [1,2]. Globally, the emergence of multidrug-resistant (MDR) E. coli producing extended-spectrum β-lactamase (ESBL) enzymes has led to empirical therapy failure, leading to high morbidity and mortality, which has raised great public concern [1,2].
Currently, CTX-M-bearing E. coli is the most common species related to ESBLs and more than 220 CTX-M family members have been identified. These variants were divided into five major groups (groups 1, 2, 8, 9 and 25) based on their amino acid homology. Among these groups, 1 and 9 were the most common globally [2][3][4]. Over the past decade, CTX-M-55, as a variant of CTX-M-15, from animal-origin E. coli, which was first discovered in Thailand in 2006, has spread rapidly in dozens of countries around the world, especially in China [4][5][6]. The dissemination of bla CTX-M-55 was mainly caused by plasmid-mediated gene horizontal transfer, and epidemic self-mobilizable F33:A-:B, IncI1, IncI2 and IncHI2 plasmids played an important role in the transmission of bla CTX-M-55 [6][7][8]. In addition to animals, bla CTX-M-55 is also distributed in food products and humans, and frequently co-localized with other resistance genes, such as fosA3, rmtB, mcr-1, bla TEM , tet(A) and floR [7][8][9][10]. The wide distribution of bla CTX-M-55 and the co-transfer of bla CTX-M-55 with different resistance genes worldwide represent a growing threat to public health.
As the largest producer and consumer of cultivated duck in the world, duck production plays a major role in the agricultural economy of China [11]. Duck farming in China is practiced on a large and diverse scale, and the integrated culture of fish-duck farming using untreated duck manure as fish feed, is the typical farming method throughout coastal areas of China, particularly in Guangdong Province. Previous studies have shown that fish-duck integrated farming systems have become a hotspot environment for the occurrence and proliferation of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB), which can facilitate the spread of resistance genes and have the potential to be the reservoir of novel ARGs [12][13][14]. Owing to the threat of bla CTX-M-55 to public health and the risks of ARG transmission in fish-duck farming, monitoring the prevalence of bla CTX-M-55 -positive E. coli isolates from duck-fish polyculture farms should receive more attention.
However, CTX-M-55 has been widely reported in E. coli isolated from food animals, pets and humans in China [6,8,15]. Current data on the prevalence, genetic information and the transmission mechanism of CTX-M-55-producing E. coli from duck-fish polyculture farms are still limited. In this study, we investigated the prevalence of bla CTX-M-55 and illustrated the characteristics of bla CTX-M-55 -bearing E. coil and plasmids recovered from ducks and the environment in duck-fish polyculture farms. Our findings emphasize the importance of the surveillance of ESBL-producing E. coli in duck-fish polyculture farms in China and provide knowledge for further One Health studies to control the spread of resistant bacteria from food animals to humans.
The antimicrobial susceptibility results showed that the 177 E. coli isolates were highly resistant to ceftiofur, ciprofloxacin and florfenicol, with resistance rates of 98.3%, 96.7% and 95.4%, respectively. The rates of resistance to colistin, ceftazidime and fosfomycin were 35.0%, 31.7% and 29.9%, respectively. However, these strains were less resistant to tigecycline, meropenem and amikacin, and the resistance rates were less than 10% (Table S2).
Based on the above results, we selected the 37 F18:A-:B1-plasmid-carrying strains and 10 F33:A-:B-plasmid-carrying strains for further study of the characteristics and transmission mechanism of plasmids carrying bla CTX-M-55 -positive E. coli. Among the 47 isolates, the number of strains from duck fecal samples was the largest, accounting for 81% (38/47), followed by the strains from water samples, accounting for 13% (6/47). The remaining 6.4% (3/47) of strains were isolated from soil samples ( Figure 1).

Figure 1.
Pulsed-field gel electrophoresis fingerprinting patterns of XbaI-digested total DNA preparations from 47 strains harboring bla CTX-M-55 . In yellow is indicated the PBRT classification, in red are indicated antimicrobial resistance genes.

Molecular Characterization of bla CTX-M-55 -Positive E. coli
PFGE was successfully performed for all 47 selected strains, and the PFGE results were divided into 15 different clusters according to similarity >85%, indicating the genetically different backgrounds of strains from different sources. In addition, two clusters of strains with the same PFGE spectrum were derived from the different types of samples from farms B, C and D (TC5, UD5, RD1, B1W1, UD9 and D7), suggesting that there is clonal transmission among different farms ( Figure 1).
The conjugation results indicated that 17 of the 47 strains could successfully transfer bla CTX-M-55 from the donor strain to the recipient strain E. coli C600, and the transfer frequency was between 2.6 × 10 −5 and 4.65 × 10 −1 (Table S3). There were four strains that could transfer bla CTX-M-55 from the donor strain to the recipient strain E. coli J53 with conjugative transfer frequencies between 3.89 × 10 −6 and 8.35 × 10 −5 . The remaining 26 strains showed unsuccessful transfer of bla CTX-M-55 from the donor strain to the recipient strain after multiple attempts (Table S3). To further determine the location of the bla CTX-M-55 gene in the non-conjugatively transferable strains and the genetic environment of the bla CTX-M-55 gene, whole-genome sequencing was performed on all 47 strains. Sequence analysis showed that among the 26 non-conjugable strains, the bla CTX-M-55 gene was located on the chromosome of 11 strains, while the bla CTX-M-55 gene of the remaining 15 strains was located on plasmids.

Genomic Analysis of bla CTX-M-55 -Positive E. coli
Whole-genome sequencing data were generated for the 47 bla CTX-M-55 -positive E. coli isolates. The results of WGS demonstrated that these isolates belonged to seventeen distinct strains (STs). Among them, the most dominant ST type was ST602, with a total of nine (9/47, 19%) strains belonging to this ST type; followed by ST155, ST410 and ST2179 (five, 10.6% each); ST48, ST162 and ST354 (three, 6.4% each); and ST165 and ST224 (two, 4.3% each). Of the remaining eight ST types, only one strain belonged to each ST type.
We identified 14 ARGs that mediated resistance to 9 types of antibiotics that coexisted with bla CTX-M-55 in these 47 E. coli isolates. These included genes that mediated resistance to fosfomycin, colistin, tetracycline, aminoglycosides, chloramphenicol, quinolones, macrolides, sulfonamides and lincomycin. Among these, 26% (12/47) and 13% (6/47) of the strains carried the quinolone resistance genes oqxAB and qnrS, respectively. In addition, 38% (18/47) of the strains carried the colistin resistance gene mcr-1, and 26% (12/47) and 21% (10/47) of the strains carried the fosfomycin resistance genes fosA3 and fosA7.5, respectively. Eleven percent (5/47) of the strains carried the chloramphenicol resistance gene floR, and thirteen percent (6/47) of the strains carried the lincomycin resistance gene lnuF. Interestingly, our results showed that all strains belonging to ST602 carried the fosfomycin resistance gene fosA7.5, and fosA7.5 was located on the chromosome. Additionally, all ST410 strains carried the lnuF gene, and the bla CTX-M-55 gene carried by the ST410 strains in the current study could not be transferred by conjugation ( Figure 1 and Table S3).
Genetic environment analysis showed that bla CTX-M-55 was present in four genomic contexts, including types I, II, III and IV. The structure of type I was ISEcp1-bla CTX-M-55 -orf477, which was the most prevalent, and was isolated in a total of 21 (45%) strains originating from feces, soil and water (Table S3 and Figure S1). The structure of type II was ∆IS26-∆ISEcp1-bla CTX-M-55 -orf477-∆Tn2, and there were 12 (26%) strains belonging to this genetic environment. There were 10 (21%) strains belonging to the type III genetic environment, and the structure of type III was ∆IS26-∆ISEcp1-bla CTX-M-55 -orf477-∆Tn2-∆IS26. The type IV genetic environment was more complex, and its structure was ∆Tn2-ISEcp1bla CTX-M-55 -orf477-∆Tn2-∆IS26. Only three isolates belonged to this genetic environment. Notably, among the strains belonging to the type I genetic structure, the bla CTX-M-55 gene of the ten strains was located on the plasmid, and the bla CTX-M-55 gene of the other eleven strains was located on the chromosome. The bla CTX-M-55 gene in the strains belonging to the type II and type III genetic structures were all located on the plasmid. Among the strains with type IV genetic structure, one strain had the bla CTX-M-55 gene located on the chromosome, and bla CTX-M-55 in the other two strains was located on plasmids (Table S3 and Supplementary Figure S1).

Complete Sequence Analysis of bla CTX-M-55 -Carrying F18:A-:B1 Plasmids
To better understand the characteristics of the bla CTX-M-55 -bearing F18:A-:B1 plasmid, S1-PFGE and Southern blotting were performed on the 12 conjugants successfully obtained from the F18:A-:B1 plasmid harboring bla CTX-M-55 -positive strains. The results showed that bla CTX-M-55 from 12 strains was located on F18:A-:B1 plasmids with sizes betweeñ 76 kb and~173 kb (Supplementary Figure S2). To further explore the sequence features of the F18:A-:B1 plasmid, four representative strains (PBS4, B1W1, B1S11 and KW21) were subjected to long-read sequencing to obtain the complete sequence of different F18:A-:B1 plasmids. The complete sequences of the F18:A-:B1 plasmids pPBS4, pB1W1, pB1S11 and pKW21 were obtained by long-read combined with short-read sequencing, and detailed information on the four plasmids is shown in Supplementary Table S4.
The MDR regions of pB1W1, pB1S11 and pPBS4 were~46.0 kb,~26.5 kb and~38.9 kb, respectively. Even though the resistance genes in the three plasmids were varied, they all contained floR, tet(A) and tet(R) and bla CTX-M-55 , except pB1S11. In addition to the mentioned genes, the pB1S1 MDR region also contained the quinolone resistance gene qnrS1, which was highly similar to pGSH8M-2-1 (GenBank accession no. AP019676.1) and shared 99.8% identify with 100% coverage. pGSH8M-2-1 was recovered from the effluent of a wastewater treatment plant in Tokyo Bay, and the only difference in the MDR region between pGSH8M-2-1 and pB1S11 was that an IS26 was inserted between two ∆Tn2 in pB1S11 (Figure 3b). Compared with pB1S11, pB1W1 and pPBS4 contained more resistance genes and carried multiple copies of IS26 and transposons. BLAST analysis did not detect plasmids with high homology to the MDR region of pPBS4 and pB1W1, which may suggest that multiple copies of IS26 and transposons formed the distinctive MDR regions of pPBS4 and pB1W1 through multiple complex recombination events. Notably, although pPBS4 and pB1W1 were derived from the same farm (Farm B), the genetic environments of bla CTX-M-55 in pPBS4 and pB1W1 were different, indicating that the bla CTX-M-55 in these two plasmids originated from different sources (Figure 3b). In addition, we also found a truncated class I integron in the plasmid pB1W1 (Figure 3b). Figure 2. Circular sequence alignments of the plasmids pKW21, pB1W1, pB1S11, pPBS4 and pTREC8. Genes depicted in the outer circle belong to plasmid pKW21, which was included as a reference, and the image was generated using BRIG.    Table S1 in the Supplemental Material). All samples were screened for cefotaxime-resistant E. coli by a selective isolation procedure. In brief, each sample was suspended in 10 mL of buffered peptone water (BPW; BD Difco, Sparks, MD, USA) and incubated at 37 • C for 24 h. Then, subsequent selective cultivation on MacConkey (MC; BD Difco) agar supplemented with 1 mg/L cefotaxime (CTX) was performed. For each sample, only one red colony was selected and identified as E. coli by MALDI-TOF MS AximaTM (Shimadzu-Biotech Corp., Kyoto, Japan) and 16S rRNA sequencing by Sanger sequencing. In all cefotaxime-resistant E.coli, bla CTX-M-1G was detected by PCR using previously reported primers [16] and sequencing (Supplementary Table S1).

Antimicrobial Susceptibility Testing
Antibiotic susceptibility testing was performed by the agar dilution method and interpreted according to the Clinical and Laboratory Standards Institute guidelines (CLSI M100-S29) for the following antimicrobials: amikacin, meropenem, cefotaxime, ceftazidime, ceftiofur, florfenicol, ciprofloxacin and fosfomycin [17]. Susceptibility to colistin and tigecycline was assessed by broth microdilution as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST Version 9.0) [18]. E. coli ATCC 25922 was used as the quality control strain.

Molecular Typing
The incompatibility (Inc) groups of all bla CTX-M-55 -producing E. coli were assigned by PCR-base replicon typing (PBRT) [19]. To better characterize IncFII plasmid, replicon sequencing typing (RST) was performed, according to protocols described previously [20]. Based on the results of PBRT analysis, a total of 47 strains (37 strains harboring the F18:A-:B1 plasmid and 10 strains harboring the F33:A-:B-plasmid) were selected for further study to explore the transmission characteristics and molecular mechanism of bla CTX-M-55 in these strains.
The genetic typing of the 47 selected bla CTX-M-55 -producing E. coli isolates was performed by digestion with restriction endonuclease XbalI and pulsed-field gel electrophoresis (PFGE) according to our previous study [21]. The band patterns were analyzed with BioNumerics software version 5.10 (Applied Maths, Austin, TX, USA).

Conjugation Assay and Southern Blotting
To investigate the transferability of the resistance genes, a conjugation assay was performed for all selected bla CTX-M-55 -positive E.coli isolates with streptomycin-resistant E. coli C600 or sodium-azide-resistant E. coli J53 as the recipient strain. For E. coli C600, donor strains and E. coli C600 were mixed and applied to a 0.22 µm filter in Luria-Bertani (LB) plates for 16~18 h. The mixed culture was then diluted and spread on selective MacConkey agar plates containing both 1 mg/L of cefotaxime and 2 g/L of streptomycin to recover transconjugants. For E. coli J53, donor strains and E. coli J53 were mixed and applied to a 0.22 µm filter in LB plates for 16-18 h. The mixed culture was then diluted and spread on selective MacConkey agar plates supplemented with 0.5 mg/L cefotaxime and 0.2 g/L sodium azide. Transconjugants were confirmed by PCR. S1-PFGE and Southern blotting were performed to determine plasmid size according to previous study, and the Salmonella enterica serotype, Braenderup H9812, was used as the standard size marker [22].

DNA Extraction and Whole-Genome Sequencing
Total DNA was extracted from 47 bla CTX-M-55 -producing E. coli isolates using a Genomic DNA Purification Kit (TIANGEN, Beijing, China) according to the manufacturer's instructions. WGS was performed with the Illumina HiSeq 2500 System (Novogene Guangzhou, China) using the paired-end 2 × 150-bp sequencing protocol. The draft genome was assembled using the tools available at EnteroBase (https://enterobase.warwick. ac.uk/species/ecoli, accessed on 28 November 2020) with default parameters. All genome assemblies of the 47 sequenced E. coli isolates were deposited in GenBank and are registered with BioProject number PRJNA934699. Then, the sequence types, replicon types and antibiotic resistance genes of all the sequenced isolates were identified by the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/, accessed on 20 March 2021). Four representative strains (PBS4, B1W1, B1S11 and KW21) were further selected for whole-genome sequencing on the PacBio RS II sequencing platform (Biochip Company, Tianjin, China) to obtain the complete sequence of the F18:A-:B1 plasmid. Sequences of those strains were assembled using HGAP version 4.0. to analyze the genetic features.

Nucleotide Sequence Accession Numbers
The complete sequences of the plasmids (pPBS4, pB1W1, pB1S11 and pKW21) have been deposited in GenBank under accession numbers CP117716, CP117722, CP117718 and CP117673.

Discussion
Duck-fish polyculture is a common circular farming model in the Pearl River Delta in southern China, specifically in Guangdong Province. In this model, duck manure is discharged directly without treatment, and a large number of ARGs or residual agents can directly contaminate fish ponds, promoting the transmission of ARGs between ducks and fish [14,23,24]. Previous studies have shown that fish-duck integrated farming systems have become a hotspot environment for the occurrence and proliferation of ARGs and ARB, and both ARGs and pathogen-related ARB have been detected in the water and sediment of this culture system [12][13][14]. Moreover, fish-duck integrated aquaculture farms have significantly higher levels of antibiotic resistance compared to monoculture fish farms, suggesting a higher risk of transmission of ARGs and mobile genetic elements (MGEs) to humans or the environment [13]. The Pearl River Delta water system is intricate, which provides a unique opportunity to develop freshwater aquaculture, but there is also the risk of contaminating river or sea areas on a large scale by ARG and ARB dissemination via aquatic water or sediment [24,25]. Based on the "one health" concept, considering the common duck-fish freshwater aquaculture system in the Pearl River Delta in southern China, greater attention should be given to the transfer risk of ARGs in integrated duck-fish farming to promote the healthy development of Chinese aquaculture and the environment.
In this study, the antibiotic susceptibility of 177 bla CTX-M-55 -positive strains was tested with 10 antibiotics commonly used in both veterinary and human medicine. The results showed that the bla CTX-M-55 -positive strains were highly resistant strains, and almost all strains were multidrug-resistant. In addition to resistance to cephalosporins, they were also highly resistant to ciprofloxacin and florfenicol (with resistance rates at 96.7 and 95.4%, respectively). These strains also had a high resistance rate to ceftazidime (31.7%), colistin (35.0%) and fosfomycin (29.9%), and the resistance rate to tigecycline, meropenem and amikacin was less than 10%. Two recent studies also showed that E. coli strains isolated from ducks and the environment were not only resistant to cephalosporins but also resistant to chloramphenicols, aminoglycosides, quinolones and tetracyclines. Our results were similar to those reported in their study. Notably, the rate of detection of strains carrying the bla CTX-M-55 gene was higher than that of strains without this gene for both the antibiotic resistance spectra and ARGs [10,26].
Previous studies have shown that bla CTX-M-55 is mainly transmitted by the horizontal transfer of epidemical plasmids, and IncI1 and F33:A-:B-plasmids were the most important types of plasmids that mediated the spread of bla CTX-M-55 in human and animal E. coli in China [6,15]. In the present study, we selected 37 bla CTX-M-55 -positive strains carrying the F18:A-:B1 plasmid and 10 bla CTX-M-55 -positive strains carrying the F33:A-:B-plasmid to explore the transmission mechanism and molecular characteristics of bla CTX-M-55 among these strains. The PFGE results showed that the 47 strains could be divided into 15 different clusters, and the genetic backgrounds were relatively different. However, strains from different farms also had the same PFGE profile. CTX-M-positive E. coli isolates from ducks in Korea also had significant differences in PFGE profiles, but the same PFGE profiles were found in different livestock farms and slaughterhouses, which was consistent with the results of our study [27].
Multilocus sequence typing results indicated that ST602, ST155, ST410 and ST2179 were the prevalent ST types in our study, and the internationally prevalent ST10 and ST131 clones were not detected. ST602 has not been widely reported in previous studies, but a recent epidemiological surveillance of ESBL E. coli from human and food-chain-derived samples from England, Wales and Scotland found that the CTX-M-1G-positive ST602 strain was widely present in chicken samples [28]. Notably, all ST602 strains carried the bla CTX-M-55 gene chromosomally, and co-carried the chromosomal fosfomycin resistance gene fosA7.5. fosA7.5 is a new member of the fosfomycin resistance gene fosA7 gene family recently reported in E. coli isolates in Canadian hospitals. Its distribution is limited to E. coli, and it can be located on both plasmids and chromosomes [29]. Because fosfomycin was effective as a first-line therapy for urinary tract infections, the emergence of fosA7.5 and bla CTX-M-55 co-carrying ST602 clone needs more attention.
ST410 E. coli is an emerging multidrug-resistant pathogen. Two major sublineages are currently circulating in Europe and North America, one is a fluoroquinolone-and extended-spectrum cephalosporin-resistant clade that emerged in the 1980s, and the other is a carbapenem-resistant clone that emerged in 2003. ST410 has been considered a "highrisk" clone similar to ST131 owing to its high transmissibility, its capacity to cause recurrent infections and its ability to persist in the gut [30,31]. This clone has also been found to harbor mcr-1 in isolates recovered from food and human samples worldwide, and tet(X)carrying ST410 E. coli in China and South Asia has been recently reported [10,[31][32][33]. Given the potential of ST 410 E. coli to acquire resistance to last-resort antimicrobials, this clone should arouse regional and global concern.
The genetic contexts of bla CTX-M-55 were divided into four types in the current study. In the genetic context of type III (∆IS26-∆ISEcp1-bla CTX-M-55 -orf477-∆Tn2-∆IS26) and type IV (∆Tn2-ISEcp1-bla CTX-M-55 -orf477-∆Tn2-∆IS26), ∆Tn2 and ∆IS26 were located both downstream and upstream of ∆ISEcp1/ISEcp1-bla CTX-M-55 -orf477's structure. This structure was found not only in E. coli but also in Klebsiella, Vibrio parahaemolyticus and Salmonella. This structure, possible formed by a copy of IS26 and an ISEcp1-mediated transposon carrying bla CTX-M-55 and orf477, was inserted into a tnpA gene; this finding stresses the need for further assessment of the mobility of IS26 or its variant [34].
Plasmids play a key role in the horizontal transfer of the ESBL gene among E. coli strains. Our results suggest that the F18:A-:B1 plasmid may play an important role in the transmission of the bla CTX-M-55 gene in E. coli isolated from duck farms and the environment. The F18:A-:B1 plasmid was first reported in avian pathogenic E. coli in 2010. It is characterized by the lack of an iron uptake gene (eitABCD), hemagglutinin and a survival gene. The bla CTX-M-55 -bearing F18:A-:B1 plasmid was first reported in E. coli isolated from patients with urinary tract infections in the United States in 2016, and carried the mcr gene [20,33]. Subsequently, the F18:A-:B1-plasmid-carrying bla CTX-M-55 was reported in E. coli isolated from human clinical samples in China [35]. Recently, a study in Southeast Asia reported that the highly pathogenic clone E. coli ST410 isolated from the environment and humans carried the bla CTX-M-55 -bearing F18:A-:B1 plasmid with a high prevalence [30]. In addition, studies from Tunisia and China also reported the high prevalence of the F18:A-:B1 subtype in animal-derived E. coli strains, which also contained fosA3, oqxAB, bla CTX-M-14 and other resistance genes [36][37][38]. These studies emphasize the possibility of horizontal transfer of the F18:A-:B1 plasmid between humans and animals.
However, to the best of our knowledge, only one recent study reported bla CTX-M-55 , floR and fosA3 carrying the F18:A-:B1 plasmid obtained from ducks [10]. Our study is the first to report the high prevalence of the bla CTX-M-55 -carrying F18:A-:B1 plasmid in E. coli isolated from duck-fish polyculture farms. Complete plasmid sequence analysis showed that bla CTX-M-55 was colocalized with tetA, floR, fosA3, bla TEM , aadA5, aph(3 )-Ib, aph(6 )-Id, CmlA, InuF and other ARGs on the F18:A-:B1 plasmid. These ARGs, linked by different kinds of insertion sequences and transposable sequences and multiple copies of IS26, constitute the complex multidrug resistance region of the F18:A-:B1 plasmid. IS26 is commonly associated with ARGs in multidrug-resistant Gram-negative and Gram-positive species, and is most widespread in Gram-negative bacteria. Clusters of ARGs can be generated by directly oriented IS26 interspersed in multiple resistant pathogens [34,39]. As the best-studied member of the IS26 family, IS26 is known to form cointegrates using conservative transposition, homologous recombination and replicative transposition [39,40]. Multiple copies of IS26 with different orientations located on the F18:A-:B1 plasmid are very likely to form cointegrates to promote the transmission of ARGs.

Conclusions
We reported the high prevalence of bla CTX-M-55 -carrying E. coli isolated from duckfish polyculture farms. Both horizontal transfer and clonal spread contributed to the dissemination of the bla CTX-M-55 gene among E. coli strains isolated from ducks and their environment, and the F18:A-:B1 plasmid may play an important role in the spread of bla CTX-M-55 . Coexistence of bla CTX-M-55 and other resistance genes (eg., tetA, floR, fosA3, bla TEM , aadA5 CmlA, InuF) on the same F18: A-: B1 plasmid may result in the co-selection of these resistance determinants and accelerate the dissemination of bla CTX-M-55 in E. coli. In addition, our study is the first to report the emergence of a fosA7.5 and bla CTX-M-55 co-carrying ST602 clone in E. coli isolated from ducks and soil. These findings emphasize the importance of the ongoing surveillance of bla CTX-M-55 -positive E. coli in duck-fish polyculture farms in China.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antibiotics12060961/s1, Table S1: Sample and isolate information from different sources; Table S2: The MIC results of 177 bla CTX-M-55 -positive E. coli strains; Table S3: Bacterial information and antimicrobial resistance profiles; Table S4: Characteristics of sequenced strains and obtained F18:A-:B1 plasmids and Figure S1: Comparison of four types of bla CTX-M-55 genomic contexts in 47 strains. Figure S2: Pulsed field gels of S1 digested genomic DNA and Southern