Identification and Genomic Analyses of a Multidrug Resistant Avian Pathogenic Escherichia coli Coharboring mcr-1, blaTEM-176 and blaCTX-M-14 Genes

Wang, Zhiyang; Wang, Xinyu; Guo, Weiqi; Wang, Di; Hu, Jiangang; Zhang, Beibei; Qi, Jingjing; Tian, Mingxing; Bao, Yanqing; Li, Haihua; Wang, Shaohui

doi:https://doi.org/10.1155/2024/9332418

Transboundary and Emerging Diseases

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Abstract Introduction Materials and Methods Results Discussion Conclusion Data Availability Ethical Approval Disclosure Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

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Research Progress on Antibiotic Resistance or Drug-Resistant Pathogens

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Volume 2024 | Article ID 9332418 | https://doi.org/10.1155/2024/9332418

Identification and Genomic Analyses of a Multidrug Resistant Avian Pathogenic Escherichia coli Coharboring mcr-1, bla_TEM-176 and bla_CTX-M-14 Genes

Zhiyang Wang,^1,2Xinyu Wang,¹Weiqi Guo,¹Di Wang,¹Jiangang Hu,¹Beibei Zhang,¹Jingjing Qi,¹Mingxing Tian,¹Yanqing Bao,¹Haihua Li,²and Shaohui Wang^1,2

Academic Editor: Abdelfattah Selim

Received23 Dec 2023

Revised23 Jan 2024

Accepted03 Feb 2024

Published16 Feb 2024

Abstract

The emergence and transmission of the colistin-resistance gene mcr and extended-spectrum β-lactamase (ESBL) encoding genes pose a significant threat to global public health. In recent years, it has been reported that mcr-1 and ESBL genes can coexist in single bacteria strain. The objective of this study was to characterize a multidrug-resistant (MDR) avian pathogenic Escherichia coli (APEC) isolate carrying mcr and ESBL encoding genes in China. A total of 200 APEC isolates were collected for antimicrobial susceptibility testing by Kirby–Bauer (K–B) disk method. The MDR strain EC012 were then further analyzed for minimum inhibitory concentrations, antimicrobials resistance genes (ARGs) detection, conjugation, and whole-genome sequencing (WGS). Among all APEC isolates determined by K–B disk method, strain EC012 was resistant to almost all the antimicrobials, including polymyxin B, cefotaxime, and ceftazidime. Moreover, EC012 harbored ARGs mcr-1, bla_TEM-176, and bla_CTX-M-14. WGS analysis revealed that EC012 belonged to epidemic APEC serotype O1:H16 and multilocus sequence type ST295. EC012 consisted of one chromosome and six plasmids, encoding a broad ARGs. The bla_CTX-M-14, mcr-1 or bla_TEM-176 genes were located on conjugative plasmids pEC012-1 or pEC012-5, respectively. These plasmids were successfully transferred to transconjugants and resulted in the resistance to polymyxin B, cefotaxime, and ceftazidime. This study indicated that APEC was a potential reservoir of colistin-resistance gene mcr-1 and ESBL encoding genes, and highlighted the necessity for enhanced monitoring of ARGs dissemination among bacteria from different origins.

1. Introduction

Avian pathogenic Escherichia coli (APEC) is a kind of extraintestinal pathogenic E. coli (ExPEC), which can cause avian colibacillosis and lead to worldwide economic losses in the poultry industry [1]. The antimicrobials were used to treat APEC infection, however, the inappropriate use of antibiotics accelerated to the emergence of multidrug-resistant (MDR) bacteria. Previous studies revealed that APEC strains showed MDR and to be important reservoirs for antimicrobials resistance genes (ARGs), suggesting its potential risk for humans [2].

Colistin is a cationic amphiphilic lipopeptide antibacterial agent. As a last resort antimicrobial, colistin is an important antimicrobial for human treatment of Carbapenem-resistant Enterobacteriaceae (CRE) infections [3]. However, it has been widely used in livestock production, especially in pigs, for a long time [4]. With the global spread of colistin resistance gene mcr, a transferable plasmid-mediated colistin resistance gene encoding for phosphoethanolamine transferase, the clinical application of colistin is under serious threat [5].

Currently, multidrug resistance in E. coli is a major concern, especially resistance exhibited by extended-spectrum β-lactamase (ESBL)-producing strains to third- or fourth-generation cephalosporins, fluoroquinolones, aminoglycosides, tetracyclines, and trimethoprim–sulfamethoxazole [6, 7]. Liu et al. [8] indicated a plasmid-mediated colistin-resistance gene mcr-1 in E. coli, isolated from humans and livestock in China, which could be transferred among different bacteria. The rapid spread and evolution of the mobile colistin-resistance gene mcr has become a global concern. To date, mcr-1–mcr-10 have been widely identified around the world [9], and new genotypes are still being discovered. Since 2016, WHO has listed colistin as one of the crucially important antimicrobials [10]. Many countries have approved the withdrawal of colistin as a feed additive in animals [4, 11].

The gene mcr-1 is commonly carried by plasmids of various replicon types, among which IncHI2 is relatively common [12]. Similarly, many conjugative plasmids, such as IncF, IncI, IncK, and IncHI2, play important roles in the global dissemination of ESBL genes [12]. The most common ESBL-encoding genes are bla_CTX-M, bla_TEM, bla_SHV, and bla_OXA [13]. Insertion sequences (IS) were also involved in the mobilization of bla_CTX-M-type and bla_TEM genes via transposition or homologous recombination [14]. It was reported the horizontal transfer of conjugative plasmid is a crucial contributor to ARGs spread [15].

Recently, the resistance genes bla_CTX-M-type, bla_TEM, and mcr-1 were detected simultaneously in Enterobacteriaceae isolated from humans, food products, and animals [16]. A high prevalence of bla_CTX-M-type, bla_TEM, and mcr-1 in E. coli was found in both human and animal samples [4, 17]. It implies that a single pathogenic bacterial strain that carries a large number of ARGs could be a serious public health threat. In this study, the genomic features of a MDR APEC strain cocarrying the mcr-1, bla_CTX-M-14, and bla_TEM-176 genes in China was characterized.

2. Materials and Methods

2.1. Bacteria Collection

A total of 200 APEC isolates were used for the MDR bacteria screening. These APEC strains were isolated from diseased ducks or chicken with avian colibacillosis in Jiangsu, Anhui, Fujian, and Shandong provinces of China by our previous studies [4, 18,].

2.2. Antimicrobial Susceptibility Testing

The antimicrobial susceptibility of APEC strains was determined using Kirby–Bauer (K–B) disk method according to the protocol of Clinical and Laboratory Standards Institute (CLSI) [19, 20]. A total of eight different antimicrobials classes were tested, including β-lactams (ampicillin, amoxicillin, ceftriaxone, cephalothin, cefotaxime, and cefuroxime), aminoglycosides (streptomycin and kanamycin), quinolones (ciprofloxacin and enrofloxacin), sulfonamides (trimethoprim/sulfamethoxazole), phenicol (chloramphenicol and florfenicol), tetracycline (tetracycline and tigecycline), colistin (polymyxin B), and carbapenems (imipenem, meropenem, and ertapenem). APEC strain EC012 displayed resistance to almost all of the tested antimicrobials. Thus, the minimum inhibitory concentrations (MICs) of polymyxin B, cephalosporins, carbapenems, rifampicin, tigecycline, and tetracycline of strain EC012 were determined using a broth microdilution method recommended by the joint European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI guidelines [20]. The antimicrobial agents tested in this study were provided by Wenzhou Cont Biotechnology Co., Ltd., China. E. coli ATCC 25922 was used as a quality control strain.

2.3. Detection of Antimicrobials Resistance Genes

The APEC strains were detected for the presence of ARGs (including mcr-1, bla_CTX-M-9G, and bla_TEM) by PCR, using published primer sequences [8, 21]. Positive controls and negative controls were performed during the PCR amplification. The PCR amplicons were purified and sequenced to confirm the ARGs.

2.4. Whole Genome Sequencing and Analysis

Total DNA was extracted with the QIAmp DNA Mini Kit (Qiagen GmbH, Qiagen Strasse 1, Hilden, Germany) according to the manufacturer’s instructions. The genomic DNA was subjected to whole genome sequencing (WGS) using Illumina NovaSeq with paired-end reads of 150 bp length and the Oxford Nanopore platforms. Sequencing reads including short-read and long-read data were assembled with CANU v1.7.1 and HGAP v4 with the hybrid strategy [22]. The plasmid sequences were initially annotated using the RAST server (http://rast.nmpdr.org) and corrected manually.

Multilocus sequence type (MLST) was determined by analyzing the sequences of seven house-keeping genes using mlst.2 [23]. A maximum-likelihood tree based on single nucleotide polymorphism (SNP) differences of the 103 E. coli genomes (all belongs to ST295) was generated using EnteroBase [24]. Serotyping was performed using the SerotypeFinder 1.2 web tool [25]. The plasmid replicon genotypes were identified using PlasmidFinder 2.1 (https://cge.food.dtu.dk/services/PlasmidFinder/). OriTfinder was used to find the origin of transfer (oriT), relaxase, T4SS gene clusters, and the type IV coupling protein (T4CP; oriTfinder (https://tool-mml.sjtu.edu.cn/oriTfinder/oriTfinder.html)). IS elements were identified using ISfinder (https://isfinder.biotoul.fr/). Besides, the CARD-RGI online tool (https://card.mcmaster.ca/analyze/rgi) was used to search for ARGs and determine point mutations. The comparative analysis and plasmid maps were generated using Easyfig 2.2.5 and BLAST Ring Image Generator (BRIG) -0.95-dist.

2.5. Conjugation Assay

The transferability of ARGs and plasmid(s) from the donor strains APEC into recipient strain E. coli 600 was tested by a filter-mating conjugation experiment. Transconjugants were select on LB agar with rifampicin (500 μg/mL), cefotaxime (4 μg/mL), and polymyxin B (2 μg/mL) or rifampicin (500 μg/mL), cefotaxime (4 μg/mL), and ciprofloxacin (2 μg/mL). Colonies that grew on these selective plates were further confirmed by antimicrobial susceptibility tests. To demonstrate transferability of mcr-1, bla_CTX-M-14, and bla_TEM-176 genes along with the conjugative plasmid, transconjugants were PCR amplified.

2.6. Nucleotide Sequence Accession Numbers

The complete nucleotide sequences of six plasmids and chromosome in EC012 isolate were submitted to GenBank with the following accession numbers: chr-EC012 (CP119577.1), pEC012-1 (CP119578.1), pEC012-2 (CP119579.2), pEC012-3 (CP119580.2), pEC012-4 (CP119581.2), pEC012-5 (CP119582.2), and pEC012-6 (CP119583.2).

3. Results

3.1. Antimicrobial Susceptibility of APEC Isolates

These APEC isolates showed different antimicrobial resistance profiles, and all the isolates were resistant to at least three different classes of antimicrobials, especially showed highly resistant to quinolones, tetracycline, and sulfonamides. APEC isolates showed moderate resistance to phenicol and aminoglycosides. Among them, APEC EC012 was resistant to most of the tested antimicrobials, including aminoglycosides, quinolones, sulfonamides, chloramphenicol, and tetracyclines. Moreover, EC012 were coresistant to β-lactams and polymyxin B, including cefotaxime (>64 mg/L), cefuroxime (>32 mg/L), ceftriaxone (>64 mg/L), cefazolin (>64 mg/L), and polymyxin B (4 mg/L). However, this isolate was susceptible to carbapenem (meropenem and imipenem) and tigecycline (Table 1). PCR detection showed that EC012 carried ARGs mcr-1, bla_CTX-M-9G, and bla_TEM.

3.2. Whole Genome Analysis

The genome size of EC012 was 4,771,666 bp with GC content of 50.76%. Based on in silico EcOH typing and MLST, EC012 strain belonged to serotype O1:H16 and sequence type ST295, one of the epidemic serotypes and STs in APEC. The SNP analysis was performed on the 103 ST295 E. coli genomes, and the phylogenetic tree revealed that EC012 clustered with E. coli isolates from poultry and human (Figure 1). According to the Plasmidfinder, there were at least six replication subtypes in EC012, including IncHI2/HI2A/N (pEC012-1), IncN/FIA (pEC012-2), IncFIB (pEC012-3), IncFII (pEC012-4), IncX1_1 (pEC012-5), and ColRNAI (pEC012-6) (Figure 2).

Whole genome sequencing indicated that the quinolone resistance genes gyrA mutant (D87N and S83L) and parC mutant (S80I), as well as the fosfomycin resistance gene glpT mutant (E448K) were located in the chromosome. Whereas, genes mcr-1, qnrS, aadA2, cmlA1, sul3, and bla_CTX-M-14 were located in plasmid pEC012-1, bla_TEM-176, qnrS1, floR, and aph(3′)-Ia were located in plasmid pEC012-5. However, no ARG was found in other four plasmids.

3.3. Characterization of a Novel Hybrid mcr-1-Bearing Plasmid pEC012-1

The bla_CTX-M-14 and mcr-1 cobearing plasmid pEC012-1 was 257,014 bp in size and had an average guanine and cytosine (GC) content of 46.46%. The results of annotation indicated that pEC012-1 harbored 294 open reading frames (ORFs) and was a hybrid plasmid containing multiple replicons including IncHI2, IncHI2A, and IncN. CARD analysis identified a number of ARGs in pEC012-1, including aminoglycosides resistance genes aac(3)-IVa, aph(3′)-Ia, aph(4)-Ia, aadA1, and aadA2; macrolides resistance gene mph(A); β-lactamases resistance gene bla_CTX-M-14; phenicols resistance genes cmlA1 and floR; quinolones resistance gene qacH; tetracyclines resistance gene tet(M),; fosfomycin resistance gene fosA3,; and sulfonamides resistance genes sul2 and sul3 in addition to colistin resistance gene mcr-1 (Table 2).

Interestingly, these resistance genes were distributed in three distinct regions of pEC012-1, a 3,539 bp mcr-1-containing cassette ISApl1-mcr-1-pap2, a 12,032 bp tet(M)-harboring segment organized as IS1-IS6-tet(M)-tcpC-Tn3, partial-MFS transporter-mph(A)-IS26 were located in the backbone, and a 14,864 bp bla_CTX-M-14-haboring segment including the remaining resistance genes which were dispersed between multiple ISs. The comparative analysis showed that pEC012-1 aligned well with MG656414.1 and MW815279.1, but MG656414.1 and MW815279.1 lacked the 12,032 bp tet(M)-harboring segment. Additionally, genes responsible for plasmid maintenance including parB and higB (toxin–antitoxin system), conjugation, and transfer (tra-relative genes) were also observed in the backbone of pEC012-1 (Figure 3).

(a)

(b)

(c)

(d)

Figure 3

Comparative sequence analysis of bla_CTX-M-14 and mcr-1 carrying plasmids from E. coli isolates and genetic features of pEC012-1. (a) Comparison of mcr-1-harbouring IncHI2/HI2A/N plasmid pEC012-1 with MG656414.1 and MW815279.1. (b) Comparison of the mcr-1-haboring homologous fragment structure. (c) Comparison of the mph(A)-harboring homologous fragment structure. (d) Comparison of the bla_CTX-M-14-haboring homologous fragment structure. Green color indicates insertion sequences and transposase, blue color indicates antimicrobials resistance genes, red color indicates backbone of plasmid, and purple color indicates specific protein.

3.4. Characterization of a bla_TEM-176-Bearing IncX1 Plasmid pEC012-5

The bla_TEM-176-bearing plasmid pEC012-5 (42,886 bp) was found to contain 51 ORFs with a GC content of 45.73% (Figure 4). The plasmid pEC012-5 belonged to the IncX1_1 incompatibility group. Intriguingly, the comparative analysis showed that the MDR region was similar to pFT130-1 (Genbank accession no: CP040091.1), p1079-IncFIB-N (Genbank accession no: MG825383.1), and pPK8277-49kb (Genbank accession no: CP080137.1) from E. coli. Besides of lacking aphA1 or cmlA1, above bla_TEM-176 was a fragment of IS6 instead of Tn2 (Figure 4).

Sequence analysis by oriTfinder identified the type IV coupling protein (T4CP) gene, relaxase, and the T4SS gene cluster on plasmid pEC012-5, but oriT was absent (Figure 4). Given that the four components are necessary for conjugation in self-transmissible plasmids [26], indicating there were possible mutations of the oriT gene.

3.5. Transferability of Plasmids

In conjugation experiments, two plasmids (pEC012-1 and pEC012-5) could transfer from APEC EC012 into the recipient E. coli 600. The two transconjugants had similar antimicrobials profiles as parental clinical APEC isolate. In transconjugant E. coli 600 with pEC012-1, the mcr-1 and bla_CTX-M-14 genes were successfully transferred into the recipient E. coli 600 strain along the conjugative plasmid via transfer frequencies ranged from 10⁻³ to 10⁻⁴. The MIC values for polymyxin B in the transconjugant was 2 μg/mL, which was higher than the original MIC of the E. coli 600 strain (Table 1). The MIC values for cefotaxime, cefuroxime, and ceftriaxone in the transconjugants were much higher than the original MIC of the E. coli 600 strain (Table 1). In transconjugant E. coli 600 with pEC012-5, the bla_TEM-176 was successfully transferred to the recipient E. coli 600 strain as well. The MIC values for ceftriaxone is over eightfold than E. coli 600 and twofold than E. coli 600 with pEC012-1, as well as the MIC value of ciprofloxacin was thirty-two-fold higher than E. coli 600.

4. Discussion

As the last line defense against MDR E. coli infections, colistin has been widely used to treat infections caused by CRE [3]. Additionally, as the poultry and swine industries accounted for 96% of total colistin sulfate livestock use [4], the presence of mcr in them provides evidence that colistin treatment has promoted the transmission of mcr, with livestock as the primary reservoir. Colistin-resistant E. coli can then spread by contaminating animal-derived food or contaminating crops by excrement to threaten public safety [5].

The WGS analysis indicated that mcr-1 was located on IncHI2/HI2A/N hybrid plasmid. IncHI2 plasmids were first reported in Serratia marcescens in the United States in 1969 and were recovered from environmental and human Salmonella enterica serovar Panama in Chile in the 1980s [27]. A lot of evidence suggests that mcr-1 was located on a wide range of conjugative plasmids, IncI2, IncHI2, IncX4, IncF, and IncP with the potential to mediate the dissemination of mcr-1 genes into other gram-negative bacteria [28, 29]. In this study, mcr-1 was associated with only one copy of ISApl1 in pEC012-1 while ISApl1 is most likely an important factor responsible for the insertion and fixation of the mcr-1 gene into various classes of self-transmissible plasmids and host chromosomes [30]. There is increasing evidence that the mcr-1 gene is mobilized primarily as a composite transposon, which is made up of two copies of ISApl1 that bracket cassettes [31]. Snesrud et al. [30] analysis of the mcr-1 sequence environment showed that Tn6330 has a strong tendency to decay through deletion, removing parts of, or both copies of ISApl1, thus transfixing mcr-1 into a vector plasmid. The loss of ISApl1 elements results in the loss of transposability, stabilizing the mcr-1 cassette in plasmids, which facilitates the widespread dissemination of the colistin resistance gene in self-transmissible plasmids. Snesrud et al. also suggested that the transposase encoded by the upstream ISApl1 can recognize the downstream inverted repeat right and thereafter still be able to mobilize the mcr-1-pap2 region without a complete composite transposon [30].

The gene bla_CTX-M-14 belongs to bla_{CTX-M-9-group}, possessing superior hydrolytic activity against ceftriaxone and cefotaxime rather than ceftazidime. The coexistence of mcr-1 and ESBL genes in MDR E. coli isolates was first reported in China in 2016 [32]. Other reports have demonstrated that ESBL-producing E. coli were more likely to carry mcr-1 than non-ESBL-producing E. coli [5, 33].

Importantly, mcr-1 coexisted with other 14 ARGs in IncHI2 plasmid. IncHI2 plasmids play an important role in the dissemination of ARGs conferring resistance to β-lactams, cephalosporins, aminoglycosides, sulfonamides, quinolones, macrolides, fosfomycin, chloramphenicol, and tetracycline among Gram-negative bacteria as our research shows [34]. These ARGs can further exacerbate the spread of colistin-resistant isolates among animals, humans, and environments [35]. In this study, segment organized as IS1-IS6-tet(M)-tcpC-Tn3 and partial-MFS transporter-mph(A)-IS26 was found in the backbone of IncHI2-type conjugative plasmid different from MG656414.1 and MW815279.1. IS26 was indicated to be a reason for formation of mcr-1-bearing IncHI2/HI2A/N/FII/FIA hybrid plasmid [3]. The tetracycline resistance gene tet(M) encodes a ribosomal protection protein that confers tetracycline resistance to a variety of bacterial species. Although tet(M)-like genes are most commonly found in bacterial chromosome [36], they also exist in conjugative plasmids from bacteria of varies species. Tet(M) with multiple mutations have been reported to confer resistance to tigecycline in Streptococcus suis [37]. The IncHI2-type conjugative plasmids carrying mcr-1 and other 14 ARGs found in this study may spread between and across genera in the future, threatening antimicrobials treatment in clinical practice.

In the conjugation experiment, the plasmid pEC012-5 can be transferred from EC012 isolate to the E. coli 600. The MIC results indicated that the bla_TEM-176 can mediate high levels of resistance to third-generation cephalosporins. The β-lactamase gene bla_TEM-176 which was first reported in an E. coli strain D7111 isolated from the feces of a child in Peru (GenBank accession no: GU550123). TEM-176 differs by only one amino acid from TEM-1 (A222V). Spontaneous mutations occur in the bla_TEM-1 gene can lead to changes in enzyme activity causing resistance to third- or fourth-generation cephalosporins [38]. Nevertheless, since its first report in GenBank in 2010, bla_TEM-176 has been reported in microorganisms isolated from samples of different origins (e.g. human, companion animals or wild animals), and from several geographic locations, such as Austria, Singapore, or Japan, thus showing worldwide dissemination [39–43]. Although few data are available on its genomic location, previous studies have shown its presence in IncX-group plasmids [44], the plasmid pEC012-5 also confirms this fact.

In this study, WGS analysis revealed that EC012 strain belonged to the ST295 lineages, respectively. In China, E. coli ST295 lineage has been detected only once from a diarrhea human sample in 2018. This study reports for the first time the E. coli ST295 lineage from poultry in China. The EC012 isolate was nearly to an isolate from human in the United Kingdom (Genbank accession no: SRR12541334), it indicated ST295 may be emerging as a pathogen in humans. Plasmids carried by E. coli ST295 play an important role in the carriage and dissemination of resistance to clinically important antimicrobials, particularly ESBL genes such as bla_CTX-M, and possibly mcr genes. Besides, plasmid acquisition has played an important role in the evolution of E. coli ST295 [45].

Foodborne transmission is a possible route of transmission of mcr-positive E. coli from animals exposed to colistin [46]. A study found a positive correlation between mcr-1 producing E. coli carriage in the human normal flora and the consumption of colistin-exposed farm animals [8]. This data highlights the presence of mcr-positive E. coli in food-producing animals and the transmission route of mcr-positive E. coli from colistin-exposed animals to humans, which impacts public health care.

5. Conclusion

In summary, this study revealed the genomic features of a MDR APEC strain EC012 belonging to epidemic serotype O1:H16 and ST295. The EC012 carried multiple ARGs, including mcr-1, bla_CTX-M-14, and bla_TEM-176, which existed in transferable plasmids and mediated the resistance to colistin and third-generation cephalosporins. The results indicated that APEC was a potential reservoir of colistin-resistance gene mcr-1 and ESBL encoding genes. However, the detailed mechanisms and genetic diversity of these ARGs in different bacteria remain unclear. Thus, it is necessary to strengthen the surveillance of ARGs dissemination among bacteria from different origins.

Data Availability

Datasets used and/or analyzed during this study can be obtained from the corresponding author on reasonable request. All sequencing data are available at NCBI: chr-EC012 (https://www.ncbi.nlm.nih.gov/nuccore/CP119577.1), pEC012-1 (https://www.ncbi.nlm.nih.gov/nuccore/CP119578.1), pEC012-2 (https://www.ncbi.nlm.nih.gov/nuccore/CP119579.2), pEC012-3 (https://www.ncbi.nlm.nih.gov/nuccore/CP119580.2), pEC012-4 (https://www.ncbi.nlm.nih.gov/nuccore/CP119581.2), pEC012-5 (https://www.ncbi.nlm.nih.gov/nuccore/CP119582.2), and pEC012-6 (https://www.ncbi.nlm.nih.gov/nuccore/CP119583.2).

Ethical Approval

All clinical tissue samples used in this study were animal diagnostic samples, and therefore no animal handling activities related to ethical issues were involved in this study.

Disclosure

The funders played no roles in study design, data collection and interpretation, or submission for publication.

Conflicts of Interest

All of the authors declare that they have no conflicts of interest.

Authors’ Contributions

Shaohui Wang designed the study. Zhiyang Wang performed the experiments. Xinyu Wang, Weiqi Guo, Di Wang, Jiangang Hu, Beibei Zhang, Jingjing Qi, Mingxing Tian, and Yanqing Bao were involved in data analyses, interpreted, and reviewing. Shaohui Wang and Haihua Li directed the experiments. Zhiyang Wang wrote the original manuscript draft and Shaohui Wang revised the manuscript. All authors read and approved the final manuscript.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2021YFD1800402), the Guangxi Key Research and Development Program (AB23075145), National Natural Science Foundation of China (32172856, 31972654, and 32302881), the Natural Science Foundation of Shanghai (22ZR1476100 and 23ZR1476600), the Science and Technology Research Project of Henan Province (232102110102), and Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2021-SHVRI).

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Copyright © 2024 Zhiyang Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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