Distribution and spread of tigecycline resistance gene tet(X4) in Escherichia coli from different sources

Tigecycline serves as a last-resort antimicrobial agent against severe infections caused by multidrug-resistant bacteria. Tet(X) and its numerous variants encoding flavin-dependent monooxygenase can confer resistance to tigecycline, with tet(X4) being the most prevalent variant. This study aims to investigate the prevalence and characterize tigecycline resistance gene tet(X) in E. coli isolates from various origins in Yangzhou, China, to provide insights into tet(X) dissemination in this region. In 2022, we tested the presence of tet(X) in 618 E. coli isolates collected from diverse sources, including patients, pig-related samples, chicken-related samples, and vegetables in Yangzhou, China. The antimicrobial susceptibility of tet(X)-positive E. coli isolates was conducted using the agar dilution method or the broth microdilution method. Whole genome sequencing was performed on tet(X)-positive strains using Illumina and Oxford Nanopore platforms. Four isolates from pig or pork samples carried tet(X4) and exhibited resistance to multiple antimicrobial agents, including tigecycline. They were classified as ST542, ST10, ST761, and ST48, respectively. The tet(X4) gene was located on IncFIA8-IncHI1/ST17 (n=2), IncFIA18-IncFIB(K)-IncX1 (n=1), and IncX1 (n=1) plasmids, respectively. These tet(X4)-carrying plasmids exhibited high similarity to other tet(X4)-bearing plasmids with the same incompatible types found in diverse sources in China. They shared related genetic environments of tet(X4) associated with ISCR2, as observed in the first identified tet(X4)-bearing plasmid p47EC. In conclusion, although a low prevalence (0.65%) of tet(X) in E. coli strains was observed in this study, the horizontal transfer of tet(X4) among E. coli isolates mediated by pandemic plasmids and the mobile element ISCR2 raises great concerns. Thus, heightened surveillance and immediate action are imperative to curb this clinically significant resistance gene and preserve the efficacy of tigecycline.


Introduction
Globally, the rapid increase of multidrug-resistant (MDR) bacteria poses a serious threat to clinical therapy and public health, a concern underscored by the World Health Organization (WHO, 2014).Tigecycline, a semisynthetic glycylcycline approved by the US Food and Drug Administration (FDA) in 2005, is considered as a last-resort antimicrobial agent for treating severe infections caused by MDR bacteria, particularly carbapenemresistant Enterobacteriaceae (Pournaras et al., 2016;Yaghoubi et al., 2022).It acts by binding reversibly to the 30S ribosomal subunit, impeding the entry of tRNA into the A site of the ribosome and thereby preventing the elongation of peptide chains (Pournaras et al., 2016;Yaghoubi et al., 2022;Stein and Babinchak, 2012).In contrast to first and second generation tetracyclines (e.g., tetracycline, doxycycline, and minocycline), tigecycline can evade classical resistance mechanisms of tetracyclines, such as tetracyclines efflux pump Tet(A) and the ribosomal protection protein Tet(M) (Pournaras et al., 2016;Yaghoubi et al., 2022).However, the clinical use of tigecycline has led to the gradual emergence of strains resistant to tigecycline (Deng et al., 2014;Pournaras et al., 2016;Yaghoubi et al., 2022).
Although tigecycline is not approved for use in animals, the emergence of tet(X) and tigecycline resistance in farmed animals may be linked to the long-term and extensive use of tetracycline drugs, such as oxytetracycline and doxycycline in veterinary medicine (He et al., 2019).The rise and prevalence of the horizontally transferable tigecycline resistance gene tet(X4) in E. coli derived from animals and food sources raises concerns, due to the limited therapeutic options available.Consequently, there is an urgent need to assess and monitor the prevalence of tet(X) in pathogens.This study aims to investigate the prevalence and characterize tet(X4) in E. coli isolates from various origins in Yangzhou, China, to provide insights into the dissemination of tet (X4) in this region.

Isolates and tet(X) detection
In 2022, a total of 618 E. coli isolates were collected from various sources, including patients (n=16), chicken meat (n=98), pork (n=108), chicken intestinal contents (n=86), pigs (n=241, feces and the pig farm environment), and vegetables (n=69) in Yangzhou, China (Supplementary Table S1).The samples were incubated in Luria-Bertani (LB) broth at 37°C for 18-24 h and then cultured on MacConkey agar at 37°C for 24 h.One pink colony per plate was further streaked on an eosin methylene blue (EMB) agar plate for 24 h at 37°C.One suspected E. coli isolate (metallic sheen color) was selected from each plate and subsequently confirmed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The presence of the tet(X) gene was detected through PCR and Sanger sequencing using the universal primers tet (X)-F (5′-CCGTTGGACTGACTATGGC-3′) and tet(X)-R (5′-TCAACTTGCGTGTCGGTAA-3′) (Wang et al., 2019), with anticipated product of 475 bp.The tet(X) gene was amplified by PCR as follows: 94°C for 5 min; 30 cycles of penetration at 94°C for 30 s, annealing at 46°C for 30 s, and extension at 72 °for 30 s; and a 5 min final incubation at 72°C.

Conjugation experiments
Conjugation experiments were conducted using tet(X)positive E. coli strains as donor strains and E. coli C600 (with high-level streptomycin resistance) or J53 (sodium azideresistant) as the recipient strain.In brief, donor and the recipient strains were inoculated in 2 mL of LB broth at 37°C, 180 r/min for 4 h, followed by mixing in a 1:4 ratio (v/v) and further incubation at 37°C for 24 h.Transconjugants were selected on MacConkey agar plates contained tigecycline (2 mg/mL) and streptomycin (3000 mg/mL) or sodium azide (100 mg/mL) and subsequently confirmed by detecting tet(X) through PCR as described above.Experiments were performed in triplicate.

Nucleotide sequence accession number
The whole-genome sequences of the four tet(X4)-positive isolates have been deposited in GenBank under accession numbers: PRJNA1039986.

Discussion
Since its discovery in E. coli in 2019, the plasmid-mediated tigecycline resistance gene tet(X4) has been identified in various bacterial species, such as E. coli, Proteus, Raoultella ornithinolytica, Acinetobacter baumannii, Aeromonas caviae, Citrobacter freundii, Klebsiella pneumoniae, and Shewanella xiamenensis, with E. coli being the primary host (He et al., 2019;Dong et al., 2022;Chen et al., 2019a;Dao et al., 2022;Zeng et al., 2021;Zhai et al., 2022a).In our study, we tested tet(X4) in E. coli from diverse sources, revealing the exclusive presence of tet(X4) in E. coli originating from pigs and pork.Conversely, tet(X4) was not detected in E. coli isolates derived from other sources, including chickens, chicken meat, vegetables, and clinical specimens.However, it is imperative to acknowledge that the number of isolates from these sources was relatively limited, posing a constraint on our study.Currently, tet(X4) is commonly detected in E. coli originating from food-producing animals and food products, particularly pigs and pork, but it is infrequently reported in E. coli from humans (He et al., 2019;Sun et al., 2019aSun et al., , 2020;;Dong et al., 2022;Li et al., 2021a;Sun et al., 2023).The detection rate of tet(X4) in this study was relatively low (0.65%), aligning closely with the findings of Sun et al. (0.22%) (Sun et al., 2019a), Yu et al. (0.17%) (Yu et al., 2022), and Zhang et al. (0.32%) (Zhang et al., 2020) in their research on E. coli collected from China.However, in contrast, significantly higher detection rates of tet(X4) were reported in retail pork samples from Shandong and Sichuan provinces (20.6%, 7/34) (Bai et al., 2019) and across ten regions in China (39.56%, 55/139) (Li et al., 2021a).
Currently, a variety of E. coli strains with different sequence types (STs) carrying tet(X4) have been identified in animals, food, the environment, and humans (Li et al., 2020b(Li et al., , 2023;;Sun et al., 2019bSun et al., , 2020;;Dong et al., 2022;Li et al., 2021a, b;Cui et al., 2022).Additionally, tet(X4)-positive plasmids with the same Inc type have been detected in E. coli sharing the same or different STs (Sun et al., 2019b(Sun et al., , 2020;;Li et al., 2021a;Cui et al., 2022).These observations suggest that plasmids are the primary means by which tet(X4) is disseminated among diverse E. coli strains.However, it is worth noting that the clonal spread of certain ST-type E. coli strains may also contribute to the dissemination of tet(X4).For instance, E. coli ST542, ST10, ST48, and ST761, which were identified in our study, represent common ST types known to carry tet(X4) in China and have been detected in animals, food products, the environment, and patients (Dong et al., 2022;Bai et al., 2019;Chen et al., 2019c;Zhang et al., 2020;Li et al., 2021a;Cui et al., 2022;Wang et al., 2022;Zhai et al., 2022b).Globally, ST10 and ST48 are the most prevalent clones in the tet(X)-positive E. coli isolates, detected in multiple countries and over 10 hosts (Li et al., 2023).Notably, certain tet(X)positive E. coli isolates (e.g., ST10) from different geographical locations or hosts shared high genetic similarity (<200 SNPs), indicating possible clonal transmission (Li et al., 2023).
Plasmids are crucial vectors for tet(X4) transmission.Presently, tet(X4) is associated with diverse plasmids, such as IncX1, IncFIA/ IncHI1, IncFIA18/IncFIB(K)/IncX1, IncFII, IncQ1, ColE2, IncA/C, and IncN plasmids (Fang et al., 2020;Li et al., 2020b;Li et al., 2021b;Cui et al., 2022;Wang et al., 2022).The tet(X4)-bearing plasmids identified in this study exhibit high similarity to those tet(X4)carrying plasmids found in Enterobacteriaceae, particularly E. coli, of various origins.This observation underscores the role of horizontal transmission of prevalent plasmids in the dissemination of tet(X4) within Enterobacteriaceae.Certain plasmids with a complete conjugal transfer region carrying tet (X4) possess conjugative capabilities, allowing them to be horizontally transferred between distinct bacterial strains, greatly facilitating the widespread distribution of tet(X4).Conversely, plasmids lacking a conjugal transfer region, such as the tet(X4)positive IncFIA18-IncFIB(K)-IncX1 plasmids in this study, are rendered non-conjugative.For instance, the IncX1 plasmid has been identified in E. coli from various sources and has demonstrated its capacity to transfer and persist within a range of Enterobacteriaceae (Li et al., 2020b;Cui et al., 2022;Chen et al., 2019c).However, our study found that the IncX1 plasmid pYUYZPE244-2 was incapable of conjugative transfer, possibly due to the absence of the conjugal transfer region compared to other tet(X4)-positive IncX1 plasmids.On the contrary, the presence of IncI2, IncX4, and IncFII conjugative plasmids carrying mcr-1 could serve as helper plasmids, enabling the conjugative transfer of non-conjugative tet(X4)-positive IncX1 or IncQ plasmids (Sun et al., 2019b;He et al., 2020).Furthermore, the non-conjugative tet(X4) plasmid can undergo homologous recombination with conjugative IncFII plasmid mediated by IS26, resulting in the formation of a novel fusion plasmid, thus, achieving the conjugative transfer of tet(X4) (Du et al., 2020).In our investigation, we also discovered that plasmids carrying tet(X4) often bear a variety of drug resistance genes, such as bla TEM-1b , qnrS1, and floR, which confer resistance to b-lactams, quinolones, and florfenicol.It suggests that these commonly used antibiotics in livestock may exert a co-selective pressure on tet(X4).
The horizontal transfer of tet(X4) between different plasmids, plasmids or chromosomes is associated with the insertion sequence ISCR2 (He et al., 2019;Chen et al., 2019b;Liu et al., 2022).ISCR2 is present upstream and/or downstream of tet(X4) and other tet(X) variants such as tet(X3), tet(X5), and tet(X6) (He et al., 2019;Wang et al., 2019;He et al., 2020;Liu et al., 2022).As revealed in prior research, ISCR2 carries adjacent regions including tet(X4) to form a 4,608 bp RC-TU, and it could also capture more resistance genes such as floR to form a larger RC-TU, although the ISCR2-tet(X4) element was frequently truncated by other insertion sequences such as IS26 (Liu et al., 2022).Additionally, the insertion sequence IS1 can also mediate the transfer of tet(X4) (Yu et al., 2022).

Conclusions
While the prevalence of tet(X4) within E. coli isolates in this study was relatively low, the horizontal transfer of this resistance gene within E. coli strains associated with pandemic plasmids (IncFIA8-IncHI1, IncFIA18-IncFIB(K)-IncX1, and IncX1) and mobile elements such as ISCR2 is a matter of great concern.Mobile elements and plasmids play a pivotal role in rapid dissemination of this clinically crucial resistance gene in E. coli originating from livestock and animal-derived food products.It has the potential for this gene to be transmitted to humans through the food chain.The detection of tet(X4) among E. coli isolates from animals and food products raises substantial public health concerns, necessitating enhanced surveillance and immediate action to control this medically significant resistance gene and uphold the efficacy of tigecycline.

FIGURE 2
FIGURE 2 Genetic organization of the tet(X4) gene in this study and comparison with p47EC (MK134376).The extents and direction of antibiotic resistance (red arrows) and other genes (black arrows) are indicated.ISs are shown as boxes labeled with their name.labeled vertical arrows with IS box indicate the insertion site of IS element.D indicates a truncated mobile element.Direct repeats are indicated by arrows and sequences.

TABLE 1
Characterization of tigecycline-resistant E. coli strains in this study.