Antimicrobial Resistance and Genomic Epidemiology of tet(X4)-Bearing Bacteria of Pork Origin in Jiangsu, China

The emergence of tigecycline-resistant bacteria in agri-food chains poses a public health concern. Recently, plasmid-mediated tet(X4) was found to be resistant to tigecycline. However, genome differences between tet(X4)-positive Escherichia coli of human and pork origins are still under-investigated. In this study, 53 pork samples were collected from markets in Jiangsu, China, and 23 tet(X4)-positive isolates were identified and shown to confer resistance to multiple antibiotics, including tigecycline. tet(X4)-positive isolates were mainly distributed in E. coli (n = 22), followed by Klebsiella pneumoniae (n = 1). More than half of the tet(X4) genes were able to be successfully transferred into E. coli C600. We downloaded all tet(X4)-positive E. coli isolates from humans and pork found in China from the NCBI database. A total of 42 known STs were identified, of which ST10 was the dominant ST. The number of ARGs and plasmid replicons carried by E. coli of human origin were not significantly different from those carried by E. coli of pork origin. However, the numbers of insertion sequences and virulence genes carried by E. coli of human origin were significantly higher than those carried by E. coli of pork origin. In addition to E. coli, we analyzed all 23 tet(X4)-positive K. pneumoniae strains currently reported. We found that these tet(X4)-positive K. pneumoniae were mainly distributed in China and had no dominant STs. This study systematically investigated the tet(X4)-positive isolates, emphasizing the importance of the continuous surveillance of tet(X4) in pork.


Introduction
In recent years, multidrug-resistant (MDR) Gram-negative bacteria have posed a serious threat to public health [1,2]. Because of its broad-spectrum antibacterial activity, tigecycline is considered the last resort in the clinical treatment of infection caused by MDR bacteria [3,4]. Tigecycline belongs to a class of drugs called glycylcyclines. Similar to tetracycline, it can reversibly bind to the 30 S subunit of the ribosome, interfering with amino acid translation and inhibiting bacterial growth [5,6]. However, He et al. discovered the plasmid-mediated mobile tigecycline resistance genes tet(X3) and tet(X4) in Enterobacteriaceae and Acinetobacter in 2019 [7]. The tet(X4) gene often possesses complex genetic environments and is distributed in plasmids of multiple plasmid replicon types [8]. Notably, previous studies have shown that pork is an important reservoir of tet(X4) [9,10]. However, studies on the genomic epidemiology of tet(X4) in pork are still lacking.
The tet(X4) gene has been identified in a variety of Enterobacteriaceae, such as E. coli, K. pneumoniae, Aeromonas caviae and Escherichia fergusonii [10,11]. However, the vast majority of reported tet(X4) are distributed in E. coli. Furthermore, the presence of tet(X4) usually does not result in a significant fitness cost to E. coli, which further exacerbates the spread of tet(X4) in E. coli [10]. In addition to E. coli, the tet(X4) gene was sporadically detected in K.
pneumoniae of different sources, including human sources and pork samples [10,12]. In this study, we analyzed the emerging tet(X4)-positive isolates isolated from pork samples in Yangzhou, China, in 2021. Meanwhile, we also compared the genomic differences of all reported tet(X4)-positive E. coli from human and pork sources in China using genomics methods, providing a genomic landscape of tet(X4)-positive isolates from various sources.

Bacterial Isolates
The 53 pork samples were randomly collected from markets in Yangzhou, China, in May 2021. Tigecycline-resistant isolates were selected on MacConkey agar plates with tigecycline (4 mg/L). 16S rRNA gene sequencing was used to perform bacterial species identifications of purified isolates. The tet(X4) gene was determined by PCR with reported primers [7].

Conjugation Experiments
The assessment of the transferability of the tet(X4) gene was conducted by conjugation experiments using tet(X4)-positive isolates as the donor strains and rifampicin-resistant E. coli C600 (RifR) as the recipient strain (1:1) at 37 • C [14]. The transconjugants were recovered on LB agar plates containing rifampicin (300 mg/L) and tigecycline (4 mg/L). PCR was used to further confirm the transconjugants. The plasmid replicon types carried in the original isolates and corresponding transconjugants were identified by PCR (Table S1).

Whole Genome Sequencing
According to the results of bacterial species identification and resistance phenotypes, six representative isolates were selected for WGS. The genomes of tigecycline-resistant strains were extracted with the FastPure bacteria DNA isolation Minikit (Vazyme, China) and quantified by a Qubit 4 Fluorometer. The genomic DNA samples were sequenced using the Illumina Hiseq 2500 platform with a 2 × 150 bp paired-end library. The paired-end reads were de novo assembled using SPAdes version 3.14.0 with the default parameters.

Data Availability
The sequences obtained in this paper have been deposited in the GenBank database under the BioProject number PRJNA900003.

Characterization of tet(X4)-Bearing Isolates among Pork
A total of 23 tigecycline-resistant isolates were collected from 53 pork samples. The 16S rRNA gene analysis showed that they were all E. coli (95.65%), except one that belonged to K. pneumoniae (4.35%). Antimicrobial susceptibility testing showed that these isolates all belonged to MDR isolates. Except for tigecycline (8-128 mg/L), these isolates were also resistant to other antibiotics such as florfenicol, chloramphenicol, streptomycin and tetracycline. However, all these isolates were susceptible to colistin and meropenem (Table S2).

Transferability of the tet(X4) Gene
To evaluate the transferability of tet(X4) in these isolates, conjugation assays were performed for these tet(X4)-positive isolates with E. coli C600 as the recipient. The tet(X4) gene in 14 isolates, including 13 E. coli isolates and 1 K. pneumoniae isolate, was successfully transferred to C600. The results of plasmid replicon typing showed that the tet(X4) gene was mainly located on IncX1-IncHI2A hybrid plasmids (35.71 %), followed by IncX1 plasmids (21.43 %) (Table S3).

Genome Sequence Features of tet(X)-Positive E. coli
In order to further elucidate the genomic characteristics of tet(X4)-positive E. coli isolated from pork and humans, we counted the ARGs, virulence genes, plasmid replicons and insertion sequences carried by these E. coli isolates. As shown in Figure 2, the number of ARGs carried by E. coli of human origin was close to that carried by E. coli of pork origin, with no significant difference (p > 0.5). Similar to the results of ARGs, there was also no significant difference in the number of plasmid replicons carried by E. coli from two different sources (p > 0.5). However, E. coli of a human source carries far more virulence genes (p < 0.5) and insertion sequences (p < 0.001) than E. coli of a pork source.

Genome Sequence Features of tet(X)-Positive E. coli
In order to further elucidate the genomic characteristics of tet(X4)-positive E. coli isolated from pork and humans, we counted the ARGs, virulence genes, plasmid replicons and insertion sequences carried by these E. coli isolates. As shown in Figure 2, the number of ARGs carried by E. coli of human origin was close to that carried by E. coli of pork origin, with no significant difference (p > 0.5). Similar to the results of ARGs, there was also no significant difference in the number of plasmid replicons carried by E. coli from two different sources (p > 0.5). However, E. coli of a human source carries far more virulence genes (p < 0.5) and insertion sequences (p < 0.001) than E. coli of a pork source.

Phylogenetic Analysis of tet(X)-Positive K. pneumoniae
In addition to E. coli, a tet(X4)-positive K. pneumoniae isolate X585-1 was isolated in this study. We downloaded all tet(X)-positive K. pneumoniae (n = 29) from the NCBI database and constructed a phylogenetic tree based on SNPs of the core genomes ( Figure 3, Table S5). We found that ST types and serotypes of the tet(X)-positive K. pneumoniae were diverse, and there were no dominant tet(X)-positive clones. These isolates were found in multiple countries but were mainly distributed in China (n = 18). Except for tet(X), these K. pneumoniae also carry multiple ARGs, including genes conferring resistance to β-lactams (bla TEM-1 , n = 14), sulfonamides (sul1, n = 18), aminoglycosides (aadA2, n = 14), tetracyclines (tetA, n = 25) and trimethoprims (drfA12, n = 10). The tet(X)-positive K. pneumoniae carried only a small number of the virulence genes compared to the ARGs.

Phylogenetic Analysis of tet(X)-Positive K. Pneumoniae
In addition to E. coli, a tet(X4)-positive K. pneumoniae isolate X585-1 was isolated in this study. We downloaded all tet(X)-positive K. pneumoniae (n = 29) from the NCBI database and constructed a phylogenetic tree based on SNPs of the core genomes ( Figure  3, Table S5). We found that ST types and serotypes of the tet(X)-positive K. pneumoniae were diverse, and there were no dominant tet(X)-positive clones. These isolates were found in multiple countries but were mainly distributed in China (n = 18). Except for tet(X), these K. pneumoniae also carry multiple ARGs, including genes conferring resistance to β-lactams (blaTEM-1, n = 14), sulfonamides (sul1, n = 18), aminoglycosides (aadA2, n = 14), tetracyclines (tetA, n = 25) and trimethoprims (drfA12, n = 10). The tet(X)-positive K. pneumoniae carried only a small number of the virulence genes compared to the ARGs.

The Genetic Context of tet(X4) Carried by K. Pneumoniae
The BLAST comparison results indicated that the sequence of K. pneumoniae X585-1 exhibited high similarity to the online IncFII (pCRY) plasmid pSDP9R-tetX4 (NZ_MW940621) found in K. pneumoniae (Figure 4). This result implies that the tet(X4) gene was also located on the pSDP9R-tetX4-like plasmid. In addition to tet(X4), the tet(X4)positive plasmid in X585-1 does not carry other ARGs. The core genetic environment of

The Genetic Context of tet(X4) Carried by K. pneumoniae
The BLAST comparison results indicated that the sequence of K. pneumoniae X585-1 exhibited high similarity to the online IncFII (pCRY) plasmid pSDP9R-tetX4 (NZ_MW940621) found in K. pneumoniae (Figure 4). This result implies that the tet(X4) gene was also located on the pSDP9R-tetX4-like plasmid. In addition to tet(X4), the tet(X4)-positive plasmid in X585-1 does not carry other ARGs. The core genetic environment of tet(X4) (ISCR2-abhtet(X4)-ISCR2) carried by plasmid pMX581-tetX was the same as the plasmid pSDP9R-tetX4.

Discussion
Our previous investigation suggests that pork is an important reservoir of the tet(X4) gene [10]. However, there is still a lack of research on whether the tet(X4) gene carried in pork can spread to humans and the genome differences between tet(X4)-positive E. coli of human and pork origins. In this study, we use genomics to answer the above questions and provide some theoretical basis for subsequent research. A total of 23 tet(X4)-positive isolates were isolated from 53 pork samples, mainly E. coli, demonstrating that E. coli is an important host of tet(X4) among pork samples, which is consistent with the previous study [9]. The tet(X4) gene is usually located on different plasmid Inc types and can spread to the same or different bacterial species [8]. The tet(X4) gene isolated from pork samples was mainly located on the IncX1-IncHI2 and IncX1 plasmids. In addition, the IncX1 plasmid carrying tet(X4) usually has no significant fitness cost to the host, suggesting that the IncX1 plasmid is an important vector of the tet(X4) gene [10]. More than half of these tet(X4) genes were able to be successfully transferred into C600, indicating that these tet(X4) genes are located on mobile elements, such as plasmids. Most of these transferable plasmids were IncX1-type plasmids, highlighting that this type of plasmid may be more easily transferable to other strains [21].
Although the tet(X4) gene is mainly present in animal-derived samples, it has also been detected in human clinics in recent years [19]. Comprehensive genomic analysis proved that there is a possibility of clonal transmission of tet(X4)-positive isolates between pork samples and clinical samples. This phenomenon will greatly limit the choice of clinical medication and pose great challenges to public health. We noticed that these tet(X4)-positive E. coli isolated from pork and clinical samples all belonged to MDR isolates and carried a variety of ARGs. However, there was no significant difference in the

Discussion
Our previous investigation suggests that pork is an important reservoir of the tet(X4) gene [10]. However, there is still a lack of research on whether the tet(X4) gene carried in pork can spread to humans and the genome differences between tet(X4)-positive E. coli of human and pork origins. In this study, we use genomics to answer the above questions and provide some theoretical basis for subsequent research. A total of 23 tet(X4)-positive isolates were isolated from 53 pork samples, mainly E. coli, demonstrating that E. coli is an important host of tet(X4) among pork samples, which is consistent with the previous study [9]. The tet(X4) gene is usually located on different plasmid Inc types and can spread to the same or different bacterial species [8]. The tet(X4) gene isolated from pork samples was mainly located on the IncX1-IncHI2 and IncX1 plasmids. In addition, the IncX1 plasmid carrying tet(X4) usually has no significant fitness cost to the host, suggesting that the IncX1 plasmid is an important vector of the tet(X4) gene [10]. More than half of these tet(X4) genes were able to be successfully transferred into C600, indicating that these tet(X4) genes are located on mobile elements, such as plasmids. Most of these transferable plasmids were IncX1-type plasmids, highlighting that this type of plasmid may be more easily transferable to other strains [21].
Although the tet(X4) gene is mainly present in animal-derived samples, it has also been detected in human clinics in recent years [19]. Comprehensive genomic analysis proved that there is a possibility of clonal transmission of tet(X4)-positive isolates between pork samples and clinical samples. This phenomenon will greatly limit the choice of clinical medication and pose great challenges to public health. We noticed that these tet(X4)-positive E. coli isolated from pork and clinical samples all belonged to MDR isolates and carried a variety of ARGs. However, there was no significant difference in the number of ARGs carried by these two different sources of E. coli. In addition, we found that clinical samples carried significantly more virulence genes than pork samples. E. coli isolated from clinical samples carry more mobile elements. Mobile elements such as ISCR2 and IS26 play an important role in the spread and transfer of tet(X4), further exacerbating the spread of tet(X4) between different pathogens [23,24].
At present, K. pneumoniae has become the most important pathogen of nosocomial infections in China [25]. Some K. pneumoniae-evolved carbapenem-resistant K. pneumoniae and carbapenem-resistant hypervirulent K. pneumoniae have emerged, and tigecycline is regarded as the last choice for clinical treatment [26]. Although only a small number of tet(X)-positive K. pneumoniae are currently detected [12], they are detected in animal, environmental, as well as human-derived samples and require global vigilance. In addition, we found that tet(X)-positive K. pneumoniae had no dominant clones, indicating that mobile elements such as plasmids as well as insertion sequences play a key role in the spread of tet(X) genes. In addition to the tet(X) gene, we found that these K. pneumoniae also carry multiple ARGs, which are at risk of co-transmission. This phenomenon suggests that we need to revisit the importance of mobile elements in mediating the spread of ARGs.

Conclusions
In conclusion, tet(X4)-positive E. coli and K. pneumoniae in pork samples were systematically analyzed in this study. tet(X4)-positive E. coli isolates in pork samples were all MDR isolates. There is a possibility of the clonal transmission of tet(X4)-positive isolates between pork samples, as well as between pork and clinical samples. Notably, mobile elements may play a key role in the spread of tet(X) genes, which suggests that we should pay more attention to the role of these mobile genetic elements in the spread of ARGs.