Characterization of an Escherichia coli Isolate Coharboring the Virulence Gene astA and Tigecycline Resistance Gene tet(X4) from a Dead Piglet

tet(X4) is the critical resistance gene for tigecycline degradation that has been continually reported in recent years. In particular, pathogenic bacteria carrying tet(X4) are a severe threat to human health. However, information describing Escherichia coli coharboring tet(X4) with virulence genes is limited. Here, we isolated an E. coli strain coharboring tet(X4) and the heat-stable toxin gene astA from a dead piglet. The strain named 812A1-131 belongs to ST10. The genome was sequenced using the Nanopore and Illumina platforms. The virulence genes astA and tet(X4) are located on the chromosome and in the IncHI1-type plasmid p812A1-tetX4-193K, respectively. The plasmid could be conjugatively transferred to recipient E. coli J53 with high frequency. In vivo experiments showed that strain 812A1-131 is pathogenic to Galleria mellonella and could colonize the intestines of mice. In summary, pathogenic E. coli could receive a plasmid harboring the tet(X4) gene, which can increase the difficulty of treatment. The prevalence and transmission mechanisms of pathogenic bacteria coharboring the tet(X4) gene need more attention.


Introduction
Antimicrobial resistance (AMR) has become a global public health problem. The emergence of multidrug-resistant (MDR) bacteria is threatening human health. Tigecycline is a third-generation tetracycline known as the last resort to treat MDR bacterial infections in hospitals [1]. Unfortunately, the tigecycline resistance gene tet(X) has emerged and spread widely in animal-derived bacteria, which may be accelerated by the overuse and misuse of tetracycline antibiotics in livestock and poultry [2]. Tigecycline-resistant bacteria harboring the tet(X4) gene have also been discovered in patients, limiting the antibiotic treatment and threatening human health [3].
Multilocus sequence typing (MLST) is an important method for analyzing specific bacterial typing based on different housekeeping genes. MLST analysis could be helpful in understanding the processes of genomic evolution in diverse species and the characteristics of a specific clone [22]. E. coli ST types are extraordinarily diverse, including ST10, ST48, ST95, and so on [23]. E. coli ST10, as a non-host-restricted pathogenic bacterium, can spread in humans and animals [24]. ST10 strains are common among MDR E. coli and have always carried various virulence genes, including gad (glutamate decarboxylase), iss (increased serum survival), terC (tellurium ion resistance protein gene), and sitA (iron transport protein) [25,26]. It should be noted that mcr-1, tet(X4), and bla NDM-5 have been detected in ST10, which showed that ST10 strains are important carriers for ARGs [27]. Moreover, some ST10 strains belonging to the DEC have been discovered in diarrheal pigs and patients [4,28]. DEC ST10 carrying ARGs in sick animals and patients would increase the risk of antibiotic treatment failure.
In a routine antimicrobial resistance surveillance, we isolated an E. coli strain cocarrying the resistance gene tet(X4) and the virulence gene astA from a piglet of unknown cause of death. We tested the AMR of the strain and analyzed the genome characterization after whole-genome sequencing to elucidate the transmission mechanism of the tet(X4) gene and the virulence. The virulence of strain 812A1-131 was detected by in vivo experiments in Galleria mellonella and mice. This strain may pose a threat to other animals and humans.

Pathogen Detection
An anal swab of a dead pig (of unknown cause of death) at 18 days of age was sent from a farm. The RNA in the feces was extracted according to the instructions for the Column Stool RNAOUT kit (Yaji biological Co., Ltd., Shanghai, China). The RNA was immediately subjected to reverse-transcription PCR (Vazyme Biotech Co., Ltd., Nanjing, China) using the primer Oligo dT. Then, the RT-PCR amplified procedure and the detection primers for porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus (RV) were used as described in a previous study [29].

Bacterial Isolation and Identification
The swab was put into a sterile tube containing 5 mL BPW (Hangzhou Microbial Reagent Co., Ltd., Hangzhou, China) to enrich the bacteria and incubated at 37 • C at 200 rpm for 12-18 h. The bacterial solution was scribed onto MacConkey agar with a disposable inoculating loop and statically incubated at 37 • C for 12-18 h. A single colony on the McConkey agar plate was inoculated on eosin methylene blue (EMB) agar and Luria-Bertani (LB) medium and incubated overnight at 37 • C. The culture mediums were purchased from Beijing Land Bridge Technology Co. Ltd., Beijing, CHN. The strain was Pathogens 2023, 12, 903 3 of 13 identified by PCR using the primers described in a previous study [30]. The stain was named 812A1-131 and preserved in a cryovial containing 1 mL of 25% glycerol at −80 • C.
In short, the concentrated antibiotic solutions were diluted, and 100 µL volumes were added to 96-well plates by double dilution according to the CLSI instructions. Then, 2-5 single colonies of E. coli were scraped from a fresh LB agar plate and adjusted to 0.5 McFarland standard inoculum (1.5 × 10 8 CFU/mL) using saline solution. The suspension was added to MH nutrient broth at a ratio of 1:200, and then 100 µL of diluent was added to each well. The 96-well plate containing the antibiotics and bacterial suspension was placed in an incubator at a constant temperature of 37 • C for 16-18 h.
The MIC values of tigecycline were verified by the agar dilution method and E-test assay (Tang et al., 2022). E. coli ATCC 25922 was used as a quality control. Briefly, serial dilutions of bacterial suspensions (10 −1~1 0 −6 ) were inoculated onto LB agar plates with different concentrations of tigecycline and subsequently incubated at 37 • C for 12 h [32].

Conjugative Transfer
The sodium-azide-resistant E. coli strain J53 was used as the recipient, and the tet(X4)positive strain 812A1-131 was used as the donor. E. coli J53 is sensitive to tigecycline, as demonstrated in previous studies [5]. Strain 812A1-131 was sensitive to sodium azide and could not grow in LB agar plates containing sodium azide (100 mg/L). The bacteria were cocultured at two temperatures, 37 • C or 28 • C, for 16 h. The mixed culture was diluted to 10 −1~1 0 −6 using PBS, and 10 µL of the above diluted solution was inoculated on LB agar plates containing tigecycline and sodium azide. The LB agar plates were incubated at 37 • C for 12~16 h [31]. Single colonies of transconjugants were randomly selected, and the tet(X4) gene was verified by PCR [5].

S1-PFGE and Southern Blotting
The tet(X4) strain was embedded and subsequently lysed using SeaKem Gold Agarose (Lonza Rockland, Inc., Maryland, MD, USA), with Salmonella H9812 as the marker, as described previously [32]. The treated DNA fragments were then separated at 14 • C for 18 h in 0.5× Tris-borate EDTA buffer. A pulse-field electrophoresis system (CHEF Mapper, Bio-Rad Laboratories, California, CA, USA) was used with a voltage of 6 V, an electric field angle of 120 • , and a pulse time of 2.2 to 63.8 s. After the gel was removed at the end of the procedure, the gel blocks were stained and observed with a gel imager. The tet(X4)-specific probe was labeled according to the instructions of the DIG High Prime DNA marker and assay starter kit (Roche Diagnostics GmbH, Mannheim, Germany).

The Galleria mellonella Model and the Mouse Infection Model
To evaluate the virulence of 812A1-131, the Galleria mellonella infection model was used to conduct in vivo experiments. E. coli 649A1 containing the stx2 gene was used as the virulent control and stored in the laboratory. Sterilized PBS served as a blank control. The inactivated 812A1-131 strain (incubated at 65 • C for 30 min) and the avirulent strain DH5α were used as the negative controls to assess the virulence of the live strain 812A1-131 [43]. Larvae with symmetrical physiques were evenly divided into the PBS control group, the experimental group, the highly virulent control group, and the avirulent control group. Ten larvae were set in each group, and the larvae were injected with 10 µL PBS or 10 6 CFU/mL bacterial inoculum, as previously described [44]. The death of larvae in each group was recorded every 6 h and observed for 48 h. GraphPad software was used for the plotting and statistical analysis of the data.
The mouse infection model test was performed as described in a previous report [45]. Ten 5-week-old SPF ICR male mice (Hangzhou Qizhen Laboratory Animal Technology Co., Ltd., Hangzhou, China) were weighed (about 25 g per mouse) and randomly divided into two groups, with five animals in each group. The bacterial solution (10 9 CFU in 100 µL) or PBS containing 20% sucrose were injected into the stomachs of the mice. To obtain the highest number of E. coli, the mice were weighed and euthanized by cervical dislocation after 6 h, as previously described [46]. The ileum, cecum, and colon parts were collected. Moreover, the tissue was vigorously cleaned and ground with PBS. The homogenate was diluted 1000 times using PBS, and 100 µL was coated on a MacConkey plate (containing 2 µg/mL tigecycline) at 37 • C for 16 h. Then, colony counting was performed.

Pathogen Detection and Bacterial Isolation
PEDV, TGEV, and RV were not detected in the dead pig. The cause of the pig's death could not be determined. An E. coli strain was successfully isolated and identified by PCR.

Antimicrobial Susceptibility
Antimicrobial susceptibility testing showed that strain 812A1-131 was only sensitive to meropenem and trimethoprim/sulfamethoxazole and showed an intermediate reaction to colistin (Table 1). It was resistant to tigecycline, with an MIC of 16 µg/mL. The agar dilution method and E-test were used to further verify its resistance to tigecycline (Figure 1). Strain 812A1-131 is a multidrug-resistant (MDR) strain that is resistant to 10 different antibiotics from nine classes (AMP-A/C-CTX-AMK-GEM-CEF-CIP-TET-TIG-FFC).

Whole-Genome Sequencing and Sequence Analysis
The strain 812A1-131 has a genome consisting of one chromosome and three plasmids. The chromosome size was 4,665,801 bp, and the GC content was 50.9%. The MLST analysis showed that the strains belonged to E. coli ST10. There are three plasmids located in 812A1-131 that were named p812A1-tetX4-193K, p812A1-69K, and p812A1-65K. The plasmid p812A1-tetX4-193K has a size of 193,145 bp and a GC content of 46.21%, and it contains two types of replicons (IncHI1A and IncHI1B(R27)). p812A1-69K has no known replicon and is 69,262 bp with a 51.89% GC content. The size of p812A1-65K is 65,072 bp, and it contains the IncFIA(HI1)-and IncY-type replicons. In addition, the size of plasmid p812A1-tetX4-193K was verified using S1-PFGE, which was consistent with the whole-genome sequencing analysis (Figure 2). and it contains the IncFIA(HI1)-and IncY-type replicons. In addition, the size of plasmid p812A1-tetX4-193K was verified using S1-PFGE, which was consistent with the wholegenome sequencing analysis (Figure 2).  (Table 1). A total of 16 virulence genes were identified, including astA, hha, hlyE, traT, yehA, and traJ. These virulence genes are related to heat-stable toxins, hemolysin, outer membrane protein virulence factor, and invasion of the blood-brain barrier, respectively. Strains with close homology (<1000 SNP) from different hosts of ST10 types were screened from the NCBI database. These strains were identified in humans, cows, pigs, chickens, and the environment. The phylogenetic tree showed that strain 812A1-131 is relatively independent compared to the others (Figure 3). Some strains carried multiple ARGs, but the tet(X4) gene was rare. Except in strain 812A1-131, the tet(X4) gene was also found in strain ECSW_09, which was identified from pigs ( Figure 3). Moreover, these strains contained several virulence genes, alsA, csg, hlyE, nlpl, and traC ( Figure  4). Sixteen antimicrobial resistance genes (ARGs) were identified in 812A1-131, of which six were located on p812A1-tetX4-193K: tet(X4), bla TEM-1B , lnu(G), aadA22, qnrB, qnrS1, and floR. The plasmid p812A1-65K contains seven ARGs: fosA3, aph(4)-Ia, aac(3)-IV, sul2, tet(A), bla CTX-M-14 , and floR. In addition, rmtB, bla TEM-141 , bla CTX-M-55 , and bla TEM-1B were located on the plasmid p812A1-69K (Table 1). A total of 16 virulence genes were identified, including astA, hha, hlyE, traT, yehA, and traJ. These virulence genes are related to heatstable toxins, hemolysin, outer membrane protein virulence factor, and invasion of the blood-brain barrier, respectively. Strains with close homology (<1000 SNP) from different hosts of ST10 types were screened from the NCBI database. These strains were identified in humans, cows, pigs, chickens, and the environment. The phylogenetic tree showed that strain 812A1-131 is relatively independent compared to the others (Figure 3). Some strains carried multiple ARGs, but the tet(X4) gene was rare. Except in strain 812A1-131, the tet(X4) gene was also found in strain ECSW_09, which was identified from pigs ( Figure 3). Moreover, these strains contained several virulence genes, alsA, csg, hlyE, nlpl, and traC ( Figure 4).

Conjugative Transfer of 812A1-131
The recipient strain E. coli J53 was able to grow on an LB agar plate containing sodium azide and tigecycline after coculture with strain 812A1-131, confirming conjugative transfer. The tet(X4) gene was located in the transconjugants, which was verified by PCR. This result indicated that, by conjugation, strain 812A1-131 could transfer the plasmid harboring the tet(X4) gene to recipient bacteria. The conjugative transfer frequencies were different between different temperatures, being lower at 37 • C (1.87 ± 0.89 × 10 −5 ) than at 28 • C (4.71 ± 5.64 × 10 −3 ).

Conjugative Transfer of 812A1-131
The recipient strain E. coli J53 was able to grow on an LB agar plate containing sodium azide and tigecycline after coculture with strain 812A1-131, confirming conjugative transfer. The tet(X4) gene was located in the transconjugants, which was verified by PCR. This result indicated that, by conjugation, strain 812A1-131 could transfer the plasmid harbor-

Conjugative Transfer of 812A1-131
The recipient strain E. coli J53 was able to grow on an LB agar plate containing sodium azide and tigecycline after coculture with strain 812A1-131, confirming conjugative transfer. The tet(X4) gene was located in the transconjugants, which was verified by PCR. This result indicated that, by conjugation, strain 812A1-131 could transfer the plasmid harbor-

Pathogenic Testing of Strain 812A1-131 in Galleria mellonella and Mouse
Strain 812A1-131 and the highly virulent E. coli strain 629A1 killed 80% and 100% of Galleria mellonella at 20 h, respectively. All Galleria mellonella died by 24 h after infection with strain 812A1-131. However, only 10% of Galleria mellonella died in the PBS or avirulent groups by 20 h. The fatality rate of strain 812A1-131 was significantly higher than that of the PBS and avirulent strains ( Figure 6). The mice were subjected to gavage to observe the bacterial colonization ability. The change in weight was not significantly different between the different groups. E. coli 812A1-131 was isolated from the ileum, cecum and colon. The numbers of colonies were 2.58 × 10 3 , 2.79 × 10 8 , and 7.11 × 10 6 CFUs/g, respectively. This strain preferred to colonize the cecum and colon.

Pathogenic Testing of Strain 812A1-131 in Galleria mellonella and Mouse
Strain 812A1-131 and the highly virulent E. coli strain 629A1 killed 80% and 100% of Galleria mellonella at 20 h, respectively. All Galleria mellonella died by 24 h after infection with strain 812A1-131. However, only 10% of Galleria mellonella died in the PBS or avirulent groups by 20 h. The fatality rate of strain 812A1-131 was significantly higher than that of the PBS and avirulent strains ( Figure 6). The mice were subjected to gavage to observe the bacterial colonization ability. The change in weight was not significantly different between the different groups. E. coli 812A1-131 was isolated from the ileum, cecum and colon. The numbers of colonies were 2.58 × 10 3 , 2.79 × 10 8 , and 7.11 × 10 6 CFUs/g, respectively. This strain preferred to colonize the cecum and colon.

Discussion
Although tigecycline has not been used in breeding, reports of tigecycline-resistant bacteria carrying tet(X4) genes in animals have increased. This may be due to the widespread use of tetracycline in food animals. E. coli is an essential host of tet(X4) resistance genes. Notice that the host range of tet(X4) genes is increasingly extensive, and tet(X4) has even been found in pathogenic bacteria [47,48]. The risk of tigecycline therapy failure is increased when an MDR pathogen harboring tet(X4) infects humans. In this study, one E. coli strain belonging to ST10 carrying the tet(X4) resistance gene and the astA virulence gene was isolated from a dead piglet.
ST10 is a common E. coli sequence type that has been widely isolated from the environment, animals, and even humans [49]. E. coli ST10 is a potential food-borne pathogen that threatens human health and has been identified in the food chain [50,51]. There has been no direct evidence of the direct transmission of E. coli ST10 to humans through contact or the food chain [52]. However, previous studies have shown that the ST10 E. coli strain from the environment has a genome closely related to the strain from humans. The same ST10 was observed in human patients, pig feces, and pork samples [27,53]. This result suggested that ST10 has a broad host range and is a zoonotic pathogen. Moreover, ST10 strains are usually multidrug-resistant (MDR) bacteria [54]. The ST10 strain of E. coli has resistance to seven types of antibiotics, including colistin, and was isolated from Polish poultry [55]. MDR ST10 E. coli resistance to imipenem and colistin was identified in young clinical patients [56].
ARGs can be located on chromosomes or mobile genetic elements (MGEs) of bacteria. MGEs can help to rapidly spread ARGs that include integrons, transposons, insertion sequences, and plasmids [57]. Plasmids were the most common MGEs for transferring ARGs within the same family of bacteria. The tet(X4) gene was identified in an IncHI1 plasmid in this study. The IncHI1 plasmid is usually larger than 180 kb, and it always carries various ARGs and heavy-metal resistance genes [58]. It has a wide host range and has been widely isolated from various Enterobacteriaceae bacteria, especially Salmonella [59]. This plasmid plays an important role in tet(X4) transmission and has become the second mostprevalent plasmid for transferring the tet(X4) gene. The p812A1-tetX4-193K plasmids from the dead piglet were similar to those from the nonpathogen in different regions. This result indicated that the tet(X4)-positive IncHI1 plasmid is widespread and could transfer horizontally between bacteria in humans, animals, food, and the environment [60]. Furthermore, IncHI is a common plasmid type carrying the colistin resistance gene mcr [61].

Discussion
Although tigecycline has not been used in breeding, reports of tigecycline-resistant bacteria carrying tet(X4) genes in animals have increased. This may be due to the widespread use of tetracycline in food animals. E. coli is an essential host of tet(X4) resistance genes. Notice that the host range of tet(X4) genes is increasingly extensive, and tet(X4) has even been found in pathogenic bacteria [47,48]. The risk of tigecycline therapy failure is increased when an MDR pathogen harboring tet(X4) infects humans. In this study, one E. coli strain belonging to ST10 carrying the tet(X4) resistance gene and the astA virulence gene was isolated from a dead piglet.
ST10 is a common E. coli sequence type that has been widely isolated from the environment, animals, and even humans [49]. E. coli ST10 is a potential food-borne pathogen that threatens human health and has been identified in the food chain [50,51]. There has been no direct evidence of the direct transmission of E. coli ST10 to humans through contact or the food chain [52]. However, previous studies have shown that the ST10 E. coli strain from the environment has a genome closely related to the strain from humans. The same ST10 was observed in human patients, pig feces, and pork samples [27,53]. This result suggested that ST10 has a broad host range and is a zoonotic pathogen. Moreover, ST10 strains are usually multidrug-resistant (MDR) bacteria [54]. The ST10 strain of E. coli has resistance to seven types of antibiotics, including colistin, and was isolated from Polish poultry [55]. MDR ST10 E. coli resistance to imipenem and colistin was identified in young clinical patients [56].
ARGs can be located on chromosomes or mobile genetic elements (MGEs) of bacteria. MGEs can help to rapidly spread ARGs that include integrons, transposons, insertion sequences, and plasmids [57]. Plasmids were the most common MGEs for transferring ARGs within the same family of bacteria. The tet(X4) gene was identified in an IncHI1 plasmid in this study. The IncHI1 plasmid is usually larger than 180 kb, and it always carries various ARGs and heavy-metal resistance genes [58]. It has a wide host range and has been widely isolated from various Enterobacteriaceae bacteria, especially Salmonella [59]. This plasmid plays an important role in tet(X4) transmission and has become the second most-prevalent plasmid for transferring the tet(X4) gene. The p812A1-tetX4-193K plasmids from the dead piglet were similar to those from the nonpathogen in different regions. This result indicated that the tet(X4)-positive IncHI1 plasmid is widespread and could transfer horizontally between bacteria in humans, animals, food, and the environment [60].
Furthermore, IncHI is a common plasmid type carrying the colistin resistance gene mcr [61]. It carries the risk of coharboring the tet(X) and mcr genes, which would be a serious threat to human health.
The emergence of pathogens with important AMR genes and virulence factors seriously threatens public health. Pathogenic K. pneumoniae carrying virulence genes hvKp and tet(X4) were isolated from pork samples and caused 100% death in mice 12 h after inoculation [62]. Foodborne pathogens can infect humans, threatening human health [63]. The coexistence of the virulence factor tdh and the AMR gene mcr-1 was also discovered in Vibrio parahemolyticus [64]. Pathogenic E.coli (UPEC) containing the bla NDM gene has caused multiple typical urinary tract infections in patients [65]. This result indicated that bacteria coharboring resistance genes and virulence factors are widespread, limiting antibiotic treatment. The astA gene has been identified in various DEC strains with outbreaks of diarrhea [66][67][68][69]. This study indicated that the astA gene might increase the strain's virulence. The difficulty of treatment is increased when a pathogen acquires the tet(X4) gene. Therefore, monitoring the coexistence of resistance genes with virulence factors in bacteria is very important. Furthermore, limiting the use of tetracycline antibiotics in livestock and poultry may help reduce the spread of resistant bacteria. People should avoid contact with live animals and choose processed meats instead.
In conclusion, an E. coli strain coharboring tet(X4) and astA was isolated from a dead piglet in routine antimicrobial resistance surveillance. The 812A1-131 strain infection could lead to the death of Galleria mellonella, which indicates that astA might have virulence potential. The IncHI1 plasmid transferred tet(X4), which is increasingly common. The acquisition of tet(X4) by pathogenic bacteria would increase treatment difficulty in humans and animals, and the bacteria coharboring virulence and AMR genes need attention.

Accession Numbers
The whole-genome sequence of E. coli 812A1-131 has been submitted to GenBank with the accession numbers CP116046-CP116049.

Institutional Review Board Statement:
All animal experiments were carried out according to animal welfare standards and approved by the Laboratory Animal Welfare Ethics Committee of Zhejiang Academy of Agricultural Sciences (approval number 2023ZAASLA01).

Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets generated during and/or analyzed during the current study can be find in the main text.

Conflicts of Interest:
The authors declare no conflict of interest.