International Spread of Tet(X4)-Producing Escherichia coli Isolates

Tigecycline resistance in bacteria has become a significant threat to food safety and public health, where the development of which is attributed to plasmid-mediated tet(X4) genes. In this study, the genomes of 613 tet(X4)-producing Escherichia coli (E. coli) isolates, available from public databases, are evaluated to determine their international prevalence and molecular characterization. These E. coli isolates have been disseminated in 12 countries across Asia and Europe. It was found that pigs and their products (n = 162) were the most common vehicle, followed by humans (n = 122), chickens (n = 60), and the environment (n = 49). Carbapenems-resistant genes blaNDM-5 (1.3%) and blaNDM-1 (0.2%) were identified, as well as colistin-resistant genes mcr-1.1 (12.6%) and mcr-3.1 (0.5%). It was noted that the tigecycline-resistant gene cluster tmexC-tmexD-toprJ1 was identified in seven (1.1%) isolates. Phylogenomic results indicated that tet(X4)-producing E. coli isolates fell into seven lineages (lineages I, II, III, IV, V, VI, and VII), and international spread mainly occurred in Asian countries, especially China, Pakistan, Singapore, and Malaysia. Four forms of tet(X4) transposon units were found, including the I-type (IS26-tet(X4)-ISCR2), II-type (ΔIS1R-tet(X4)-ISCR2), III-type (ISCR2-tet(X4)-ISCR2), and IV-type (ISCR2-tet(X4)-ΔISCR2). These findings underline further challenges for the spread of E. coli bearing tet(X4) gene.


Introduction
Tigecycline is considered to be a drug of last resort for the treatment of multidrugresistant (MDR) and even extensively drug-resistant bacteria. Tigecycline resistance in bacteria primarily has resulted from chromosome-mediated overexpression of efflux pumps and ribosome protection [1,2]. Until 2019, novel plasmid-mediated high-level tigecycline resistance genes tet(X4)/tet(X3) were discovered in Enterobacteriaceae and Acinetobacter isolates from animals and humans in China [3]. It was of note that Tet(X4) could degrade all tetracyclines, including tigecycline and the USFDA newly approved eravacycline [4], which poses a new threat to public health.
Currently, tet(X4)-producing E. coli isolates have been disseminated in meat products such as pork and chicken, food animals, animal feces, and farm soil [3][4][5][6][7]. In a previous study, tet(X4)-producing E. coli isolates in pigs and chickens accounted for 1.3% and 0.8%, respectively, which was disseminated in five provinces in southern and eastern China including Guangdong, Fujian, Jiangsu, Jiangxi, and Guangxi [4]. However, tet(X4) had a relatively high prevalence (33.3-50.0%) in both pigs and chickens in some provinces of China such as Shaanxi and Ningxia [8]. These results suggested that food animals and their meat products were still an important reservoir of tet(X4). It was noted that tet(X4)producing E. coli isolates also emerged in humans in recent years, where the detection rate of tet(X4)-producing E. coli isolates from hospital patients was 4.5% [6]. The genetic link between meat/animal-borne tet(X4)-producing E. coli isolates and human-borne isolates requires urgent investigation. Furthermore, tet(X4)-positive plasmids are highly transferable in E. coli [4], which could facilitate their dissemination among Enterobacteriaceae bacteria. Furthermore, the tet(X4) gene has also been found in existence with the colistin-resistant gene mcr-1 and/or the carbapenem-resistant gene bla NDM-1 [3,4,9], which has resulted in concurrent resistance to tigecycline and colistin/carbapenem. Therefore, the emergence of the plasmid-mediated tet(X4) gene poses a significant threat to public health which requires urgent surveillance in terms of its prevalence.
Here, we characterize an international distribution of tet(X4)-producing E. coli isolates using a data set of 613 genomes from the public database. Antimicrobial resistance (AMR) genotypes, virulence genotypes, plasmid replicon types, and phylogenomic characteristics are further analyzed here, as well as the genetic environment of tet(X4).

E. coli Genomes Collected
We searched two important words, "tet(X4)" and "E. coli", in the public NCBI database on 2 April 2022. A total of 613 E. coli isolates were positive for the tet(X4) gene, and the detailed genome information including sample sources, countries, and years was downloaded. A total of 585 genomes were available for phylogenomic analysis because the rest had not been released.

Phylogenomic Analysis
A total of 585 tet(X4)-producing E. coli genomes were used for phylogenomic analysis. Single-nucleotide polymorphisms (SNPs) were extracted using Snippy (https://github. com/tseemann/snippy (accessed on 2 June 2022) to generate the core genomic alignment. Gubbins [13] was then used to identify and remove recombination regions using an algorithm that iteratively identifies loci containing elevated densities of base substitutions, then resulting in pairwise SNP differences that could be calculated. The core SNP alignment was used to generate a maximum-likelihood phylogeny using RAxML v8.1.23 [14] with the GTR nucleotide substitution model. The display, annotation, and management of phylogenetic trees were performed by the ITOL tool [15].

Local Database Construction and BLAST+ Comparison
In this study, 585 genome sequences were used as a local BLAST database to search the arrangement forms of tet(X4). First, BLAST+ was downloaded from NCBI (https: //ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ (accessed on 2 June 2022), and then a database folder (585 × 4 db) was created and added as a path for the environment variant. Custom reference genome files were created using makeblastdb. The gene sequence of tet(X4) was used as the query file, and then the instruction "blastn-query tet(X4).fasta-db585X4db-out tet(X4)out-outfmt6" was input to generate the alignment sequence (.txt).

Data Analysis
The bar charts of the AMR genotypes, virulence genotypes, and plasmid replicon types were generated with GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). The world map marked with the distribution of tet(X4)-producing E. coli was drawn by Inkscape 0.92 (Inkscape Software, Brooklyn, NY, USA).

Phylogenomic Analysis of tet(X4)-Producing E. coli
Phylogenomic analysis was performed for 585 tet(X4)-producing E. coli genomes from 10 countries to provide their evolutional features. A total of 31,260 core SNPs were identified to construct a maximum likelihood tree ( Figure 5). Phylogenomic results indicated that tet(X4)-producing E. coli isolates might have emerged early in China because they were the base of the phylogenetic tree, then evolving into lineages I, II, III, IV, V, VI, and VII ( Figure 5). All these seven lineages were mixed clusters, which were composed of isolates from different countries. Lineage I was composed of isolates from China, Pakistan, Singapore, and Malaysia. Lineage II was composed of isolates from seven countries including China, Pakistan, Thailand, Singapore, Malaysia, Vietnam, and Italy. Isolates from Turkey fell into lineage IV, together with those from China, Pakistan, and Vietnam. Isolates from China clustering with those from Pakistan could be found in lineages I, II, III, IV, V, and VII. Isolates from China clustering with those from Thailand could be found in lineages II, III, V, VI, and VII. Similar results were also found in isolates from China with those from Malaysia and Vietnam, Furthermore, some isolates from European countries were also found in these lineages, such as Italy (lineage II), Norway (lineage VI), and Switzerland (lineage V), suggesting a close genetic relationship with those from Asia. These results indicated that the international spread of tet(X4)-producing E. coli isolates has occurred in Asian countries, especially China, Pakistan, Singapore, and Malaysia. It was noted that the tet(X4)-producing E. coli might have been introduced into Europe from Asia. Clone spread of tet(X4)-producing E. coli isolates was found among different sourc For example, some isolates from stool, blood, pork, and unknown animal feces in linea IV shared a high similar genetic type ( Figure S1). Similar results were also found in som stool, blood, pig, pork, pig fecal swab, cow, and unknown animal feces isolates in linea VII ( Figure 5). It was noted that the genetic types of some chicken cloacal isolates could highly similar to those from pig fecal swabs in lineage III, which suggested that the spre of tet(X4)-producing E. coli isolates was likely to occur between chickens and pigs.

Genetic Environment of tet(X4) in E. coli
In this study, four forms (I, II, III, and IV) of tet(X4) transposon units were identifi (Figure 6). The I-type of tet(X4) was linked to the transposable elements IS26 upstrea and ISCR2 downstream. The I-type pattern of IS26-tet(X4)-ISCR2 was identified in 63 is lates, and its size was approximately 4.9 kb in length. The II-type of tet(X4) was linked the truncated ΔIS1R upstream and ISCR2 downstream. The II-type pattern of ΔIS1  Clone spread of tet(X4)-producing E. coli isolates was found among different sources. For example, some isolates from stool, blood, pork, and unknown animal feces in lineage IV shared a high similar genetic type ( Figure S1). Similar results were also found in some stool, blood, pig, pork, pig fecal swab, cow, and unknown animal feces isolates in lineage VII ( Figure 5). It was noted that the genetic types of some chicken cloacal isolates could be highly similar to those from pig fecal swabs in lineage III, which suggested that the spread of tet(X4)-producing E. coli isolates was likely to occur between chickens and pigs.

Genetic Environment of tet(X4) in E. coli
In this study, four forms (I, II, III, and IV) of tet(X4) transposon units were identified (Figure 6). The I-type of tet(X4) was linked to the transposable elements IS26 upstream and ISCR2 downstream. The I-type pattern of IS26-tet(X4)-ISCR2 was identified in 63 isolates, and its size was approximately 4.9 kb in length. The II-type of tet(X4) was linked to the truncated ∆IS1R upstream and ISCR2 downstream. The II-type pattern of ∆IS1R-tet(X4)-ISCR2 was identified in 46 isolates, and its size was approximately 4.8 kb in length. The III-type of tet(X4) was linked to the ISCR2 upstream and ISCR2 downstream. The III-type pattern of ISCR2-tet(X4)-ISCR2 was the most common arrangement profile (n = 78), and its size was approximately 6.1 kb in length. The IV-type of tet(X4) was linked to the ISCR2 upstream and truncated ∆ISCR2 downstream. The IV-type pattern of ISCR2-tet(X4)-∆ISCR2 was identified in 32 isolates, and its size was approximately 4.8 kb in length. These results indicated that ISCR2 was the most common transposable element associated with tet(X4).

Discussion
In this study, the prevalence of tet(X4)-producing E. coli was mainly observed in Asia, especially in China (n = 465, 75.9%). Furthermore, the international spread of tet(X4)-producing E. coli isolates also mainly occurred in Asian countries, especially in China, Pakistan, Singapore, and Malaysia. The tet(X4) gene was first reported in China in 2019 [3,4]. The tet(X4) gene was first reported in Pakistan in 2021, which was detected in E. coli isolates from poultry, chicken meat, wild bird, and slaughterhouse wastewater [9]. In a retrospective survey of animal-derived tigecycline-resistant E. coli across China in 2018, the tet(X4) gene was not present at a high prevalence across China but was highly endemic in northwestern China [8]. It was noted that tet(X4)-producing E. coli isolates had emerged in several European countries, such as Italy, Norway, and Switzerland, where the genetic relationships of which were close to those from Asia. It is urgent to prevent the international spread of tet(X4)-producing E. coli isolates through timely intervention strategies.
Pigs have been found to be important vehicles of tet(X4)-producing E. coli isolates, which is consistent with the results from previous studies [4,5,8,16]. Furthermore, the pig tet(X4)-producing E. coli isolates from Shaanxi differed by only 119 SNPs from the human

Discussion
In this study, the prevalence of tet(X4)-producing E. coli was mainly observed in Asia, especially in China (n = 465, 75.9%). Furthermore, the international spread of tet(X4)producing E. coli isolates also mainly occurred in Asian countries, especially in China, Pakistan, Singapore, and Malaysia. The tet(X4) gene was first reported in China in 2019 [3,4]. The tet(X4) gene was first reported in Pakistan in 2021, which was detected in E. coli isolates from poultry, chicken meat, wild bird, and slaughterhouse wastewater [9]. In a retrospective survey of animal-derived tigecycline-resistant E. coli across China in 2018, the tet(X4) gene was not present at a high prevalence across China but was highly endemic in northwestern China [8]. It was noted that tet(X4)-producing E. coli isolates had emerged in several European countries, such as Italy, Norway, and Switzerland, where the genetic relationships of which were close to those from Asia. It is urgent to prevent the international spread of tet(X4)-producing E. coli isolates through timely intervention strategies.
Pigs have been found to be important vehicles of tet(X4)-producing E. coli isolates, which is consistent with the results from previous studies [4,5,8,16]. Furthermore, the pig tet(X4)-producing E. coli isolates from Shaanxi differed by only 119 SNPs from the human isolates in a previous study from China [8]. It was found in this study that the high genetic similarity of tet(X4)-producing E. coli isolates from animals (pigs and chickens) and humans (stools and bloods) was also observed, which suggested the possibility of its spread from animals to humans.
Importantly, E. coli isolates co-harboring tet(X4) and mcr-1 have been reported [4,17,18]. In addition to mcr-1, carbapenems-resistant genes bla NDM-5 and bla NDM-1 were also identified in tet(X4)-producing E. coli isolates in this study. Furthermore, a gene cluster of tmexC-tmexD-toprJ1 was identified in 7 isolates, which was a newly plasmid-mediated RND-type tigecycline resistance mechanism widespread among Klebsiella Pneumoniae isolates from food animals [19]. This co-existence of tet(X4) and other antibiotic resistance genes such as mcr-1, bla NDM, and tmexC-tmexD-toprJ1 would increase greatly the challenge for the treatment of pathogen infections, and this co-existence phenomenon might result from the co-selected pressure of extensively used antibiotics in animals and humans.
In previous studies, the IncX1-type plasmid was a common vector for tet(X4), which has a strong transmission ability and a wide host range [6,8]. It was interesting that no IncX1 replicon gene was found in this study, but that the IncX4 (4.5%) and IncX3 (1.8%) plasmid replicon genes were identified. In addition, multiple plasmid replicon genes were common in tet(X4)-producing E. coli isolates in this study. Indeed, tet(X4) has been reported in multi-replicon plasmids, such as IncX1-IncFIA/B-IncY, IncX-IncFIA/B-IncHI1A/B, IncFIA/B-IncHI1A/B, IncX1-IncN, and IncX1-IncR [8]. The combination of multiple plasmid replicons might contribute to their adaptation to a broad range of hosts through preventing plasmid incompatibility.
This study features some limitations. First, the number of genomes (n = 585) used in this study was limited, although they represent the full genome containing tet(X4) dataset in NCBI. Second, numerous genomes were associated with the development of sequencing technology. Third, the majority of the tet(X4)-positive E. coli isolates originated from China, and limited data were gathered from other countries. Therefore, the above limitations would bias the results found here.

Conclusions
In conclusion, tet(X4)-producing E. coli isolates have emerged in Asia and Europe. The international spread of these isolates can be primarily attributed to Asian countries, especially China, Pakistan, Singapore, and Malaysia. Pigs and their products are the most common sample vehicle of tet(X4)-producing E. coli. The mobile genetic element ISCR2 might contribute to the spread of tet(X4). The spread of tet(X4) is a great concern for food safety and public health, and it is urgent to enhance surveillance and control its spread among Enterobacteriaceae.