Escherichia coli is the most common conditional pathogen in the culture environment, which can cause varying degrees of infection in livestock and poultry. These Escherichia coli strains, which contain a large number of antibiotic resistant genes, accumulate in humans and farmed animals, and are released into the environment via feces. They continue to contaminate the human food environments (e.g., agricultural, aquatic, and livestock products) as well as livestock and poultry breeding environment (Sajjad et al., 2011; Chajȩcka-Wierzchowska et al., 2014; Yano et al., 2014; Blaak et al., 2015; Liao et al., 2015). Finally, multidrug-resistant E. coli accumulate in the human body through the food chain (Wellington et al., 2013).

Extended-spectrum β-lactamase (ESBLs) is currently considered one of the world’s major public health problems. In Enterobacteriaceae, E. coli causes the most infections and is the main species producing ESBL (Bradford, 2001). Many genes are able to produce ESBLs that they can hydrolyze cephalosporins, penicillin, and monocyclic β-lactam antibiotics aztreonam (Kanayama et al., 2010). β-lactamases have been divided into four major classes (A, B, C, and D) according to the amino acid similarity. β-lactamases of classes A, C, and D are serine β-lactamases, whereas those of class B are metallo-β-lactamases (Paterson and Bonomo, 2005). Initially, the most prevalent ESBLs were TEM or SHV variants possessing amino acid substitutions (Sivaraman et al., 2021). bla_CTX–M is one of the most important ESBL genes, which has been found in several conjugative plasmids of Enterobacteriaceae (Abera et al., 2016; Abraham et al., 2018). IncF plasmids are usually low copy number plasmids, > 100 kb in size, and typically carry fused replicons with multiple replication initiation genes (Laura et al., 2010). The multi-replicon status is described as a means by which a plasmid with a narrow host range can replicate in a wide host range. Integrons were initially discovered because they are often associated with drug-resistant genes (Stokes and Hall, 1989). Class 1 integrons are the most frequently identified integron classes within clinical environments. This class was the first integron found, which mainly distributed on plasmids of Gram-negative bacteria (Goldstein et al., 2001). bla_CTX–M gene was used as a query for BLAST search in the NCBI nucleotide sequence database, and it was found that it existed mainly in Enterobacterales. However, it is still unknown how frequently the gene occurs in the E. coli in Zhejiang Province, and the number of related IncFII plasmids containing bla_CTX–M is not very high in the NCBI database. Moreover, the genetic background of bla_CTX–M also needs to be further studied.

In this study, the relative abundance of the known ESBL genes in E. coli in livestock and poultry breeding environment was determined. This research provided beta-lactam resistance profile of bacteria isolated from livestock and poultry breeding environment in Zhejiang Province. Our study suggests that resistance to beta-lactam is too high in E. coli. Therefore, local farmers can possibly reduce the use of specific beta-lactam antibiotics, making a particular contribution to the reduction of local antibiotic resistance. Furthermore, two IncFII plasmids harboring bla_CTX–M along with other resistance genes were analyzed to clarify the basis for dissemination. This research enriched the study of plasmids containing bla_CTX–M resistance genes in the NCBI database.

In this study, E. coli strains isolated from the livestock and poultry breeding environment in Zhejiang Province showed high-level resistance (137/204) to extended-spectrum β-lactam antibiotics (Cefazolin, Ceftazidime, and Imipenem). These antibiotics are prevalently used for the treatment of infections caused by Enterobacteriaceae. Essentially, all ESBL producers demonstrated multi-drug resistance profiles and carried at least one ESBL resistance gene, especially bla_TEM (94.89%) and bla_CTX–M (92.7%). Among all the E. coli strains investigated in the current study, the prevalence of ESBL producers (67.2%) is quite close to that previously reported in Henan Province (60.8%) from 2007 to 2008 (Yuan et al., 2009). However, it is still lower than those reported in northeast China (Jilin, Liaoning, and Heilongjiang Provinces (100%) from 2011 to 2013 (Tong et al., 2015).

In this study, six extended-spectrum β-Lactamase resistance genes were detected in all antibiotic resistant strains, and bla_CTX–M was the second common resistance gene. However, Tong et al. (2015) found that CTX-M-type ESBL resistance genes were the most common genotype of E. coli isolated from chickens in China. These contradictories may be due to different regions where the strains were isolated and various poultry species. However, it might be related to mobile genetic elements such as plasmids and transposons as well. Previous reports showed that CTX-M-encoding genes in E. coli, isolated from food animals, have a high diversity in China, and CTX-M-14 was the most widespread CTX-M enzyme, followed by CTX-M-55 (Zheng et al., 2012). bla_CTX–M–55 has been widely reported in food-producing animals and pets in China (Rao et al., 2014). In this study, the strains carrying CTX-M-55-type ESBL showed different MDR profiles. In order to further study the mobile genetic elements related to CTX-M gene transfer, we selected two plasmids of two strains for sequencing and analysis.

We identified two conjunctive IncFII multi-resistance plasmids carrying bla_CTX–M–55 and analyzed the genetic context of bla_CTX–M–55 and MDR region. Most CTX-M genes were identified to be associated with IncFII or IncI1 plasmids (Carattoli, 2009; Cao et al., 2011). The most common IncFII type plasmid was F2:A-: B-, which had also been reported to be linked to bla_CTX–M genes in Enterobacteriaceae strains isolated from other regions (Laura et al., 2010; Kim et al., 2011). In the current study, the IncFII plasmid p11011-fosA contains another ESBL resistance genes bla_TEM–1B and fosfomycin resistance gene fosA3, while the other IncFII plasmid p417957-CTXM don’t have the two resistance genes. fosA3 is frequently co-transferred with bla_CTX–M genes, and the dissemination of fosA3 may be related to the co-selection of cephalosporins (Hou et al., 2012). In previous epidemic plasmids, it was observed that the fosA3 gene is frequently co-transferred with bla_CTX–M–14, bla_CTX–M–55, bla_CTX–M–65, floR, and rmtB genes. Thus, reducing the total use of antibiotics, especially cephalosporins, in livestock and poultry breeding environment contributes to controlling the dissemination of plasmid-mediated fosfomycin and β-Lactamase resistance genes (Yang et al., 2014).

In this study, we found that bla_CTX–M–55 was located on the ISEcp1-bla_CTX–M–55-orf477 transposition unit, which was similar to previous studies (Zheng et al., 2012). ISEcp1-bla_CTX–M–55-orf477 transposition unit is a significant element in the propagation of bla_CTX–M–55. ISEcp1 is bounded by 14-bp inverted repeats, with IR_L at the left end of the transcription direction of the transposase gene and IR_R at the right end. In addition, ISEcp1 uses IR_L in conjunction with alternative sequences like these IR to mobilize adjacent regions, forming a 5-bp direct repeat (DR) on transposition (Poirel et al., 2005; Wachino et al., 2006). The MDR region in pHN7A8 and p11011-fosA is mainly formed by the insertion of mobile elements. The insertion of Tn2-rmtB element into the insB of IS1R results in only 214 bp residue in insB. The insertion of the region of 9.4 kb [IS26, Δ Tn6377, the IS26-fosA3-IS26 unit (Ho et al., 2013) and IS1294] between tnpA and tnpR of Tn2 leads to the loss of res site of Tn2. Tn2-rmtB element (Doi et al., 2008) and the variant of this element have been widely found in Enterobacteriaceae. Tn6377 was originally identified on the plasmid pKP96 (Shen et al., 2008) in Klebsiella pneumoniae, and the transposon was formed by inserting Tn1722 (Zong et al., 2011) into the ISEcp1 – bla_CTX–M–65-IS 903D unit of the Tn3 family. Tn6377 in pHN7A8 is interrupted at both terminals by IS26.

The MGEs, such as transposons, play an essential role in the rapid spread of AMR by horizontal gene transfer due to the selective pressure imposed by antibiotics (Snesrud et al., 2018). Tn5403 found in MDR-2 region in plasmid p11011-fosA did not contain antibiotic resistance genes and was first identified in the plasmid of Klebsiella pneumoniae (Rinkel et al., 1994). Another transposon Tn1721 belongs to the transposon of the Tn21 family and has three 38 bp repeats and two transposase genes tnpA with the same sequence. There are tnpA, tnpR, res sites, and the gene mcp that encodes methyl-chemotactic proteins at one terminal, with 38 bp reverse repeats on both sides of this terminal. This terminal is named Tn1722 and can be moved independently. The rest of Tn1721 contains the tetracycline resistance gene tetA. Although Tn1721 in the MDR-2 region of p11011-fosA is interrupted by IS26 and Tn5403, the key tetracycline resistance gene in this transposon remains intact. IS26-mph (A)-mrx-mphR (A)-IS6100 unit is the carrier element for common macrolide resistance genes, which are usually located on resistant plasmids in E. coli (Partridge, 2011).

The presence of multiple mobile elements in a bla_CTX–M-carrying multi-resistance plasmid makes it flexible. These elements aid its persistence and dissemination among E. coli and potentially other Gram-negative bacteria. This study demonstrated the abundance of β-lactamase resistance genes in livestock and poultry breeding environment in Zhejiang Province. It also upgraded the research on IncFII plasmids containing bla_CTX–M antibiotic resistance genes in the NCBI database. This research also provided beta-lactam resistance profile of bacteria isolated from farms in Zhejiang Province, indicating that local farmers can possibly reduce the use of specific beta-lactam antibiotics to reduce the evolution of multi-resistant strains.

Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov/nuccore/NZ_MH454107.1 and https://www.ncbi.nlm.nih.gov/nuccore/NZ_MG764548. 1NCBI, GenBank accession numbers NZ_MH454107.1 and NZ_MG764548.1.

WZ: visualization, data curation, software, and writing—original draft preparation. EZ: conceptualization, methodology, formal analysis, and writing—original draft preparation. JZ: visualization and investigation. ZH: software and writing—review and Editing. YZ: software and validation. JH: formal analysis and resources. DQ: conceptualization, resources, supervision, and writing—review and Editing. All authors contributed to the article and approved the submitted version.

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.

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