Genomic characterization revealing the high rate of tet(X4)-positive Escherichia coli in animals associated with successful genetic elements

Introduction The rapid spread of plasmid-mediated tet(X4) conferring high tigecycline resistance poses a significant threat to public health. Escherichia coli as the most common pathogen which carries tet(X4) has been widely disseminated in China. Thus, comprehensive investigations are required to understand the mechanism of transmission of tet(X4)-positive E. coli. Methods In this study, a total of 775 nonduplicate samples were collected in Guangdong, China from 2019 to 2020. We screened for tet(X4)-positive E. coli by PCR amplification and species identification. Furthermore, we analyzed the phylogenetics and genetic context of tet(X4)-positive E. coli through whole-genome sequencing and long-reads sequencing. Results Overall, 146 (18.84%) tet(X4)-positive E. coli were isolated, comprising 2 isolates from humans and 144 isolates from pigs. The majority of tet(X4)-positive E. coli exhibited resistance to multiple antibiotics but all of them were susceptible to amikacin and colistin. Phylogenetic analysis showed that ST877, ST871, and ST195 emerged as the predominant sequence types in tet(X4)-positive E. coli. Further analysis revealed various genetic environments associated with the horizontal transfer of tet(X4). Notably, a 100-kbp large fragment insertion was discovered downstream of tet(X4), containing a replicon and a 40-kbp gene cluster for the bacterial type IV secretion system. Discussion The high colonization rate of tet(X4)-positive E. coli in animals suggests that colonization as a key factor in its dissemination to humans. Diverse genetic context may contribute to the transfer of tet(X4). Our findings underline the urgent need for controlling the spread of plasmid-mediated tigecycline resistance.


Introduction:
The rapid spread of plasmid-mediated tet(X4) conferring high tigecycline resistance poses a significant threat to public health.Escherichia coli as the most common pathogen which carries tet(X4) has been widely disseminated in China.Thus, comprehensive investigations are required to understand the mechanism of transmission of tet(X4)-positive E. coli.

Introduction
With the overuse of antibiotics, the problem of bacterial resistance has become increasingly serious, followed by the emergence and prevalence of various superbugs, including carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter baumannii, which pose a huge threat to human health annually and places a huge burden on healthcare systems (Hsu et al., 2017;Tacconelli et al., 2018).Tigecycline, as third generation tetracycline drug, exerts antibacterial effects by binding to the 30S subunit of the ribosome and inhibiting peptide elongation (Olson et al., 2006).It is recognized as a crucial option against multi-drug resistant bacteria, often referred to as the "last resort." However, recently, a large number of tetracycline resistance genes have emerged and become popular (Fang et al., 2020).The plasmidmediated tet(X4) gene, which mediates the high level of tigecycline resistance, was discovered in 2019 (He et al., 2019).In addition, tet(X4) is prevalent worldwide, primarily in China (Zhang et al., 2022), and has been found in a variety of sample sources, including patients, farms, slaughterhouses, sewage, and migratory birds (Chen et al., 2019;Cui et al., 2022;Dong, 2022).Thus, the spread of the tet(X4) is a public health concern that cannot be ignored.
An important reason for the pandemic of resistance genes is that they tend to transfer in individual samples, which is associated with a variety of transfer elements (Partridge et al., 2018).Previous studies have shown that the transfer of tet(X4) is related to several transfer elements, including ISCR2, IS26, and IS1R (Liu et al., 2022).The transmission mode of tet(X4) requires further investigation.The type IV secretion system (T4SS), which mediates the horizontal transfer of antibiotic resistance and virulence genes (Christie, 2016), has been found in the tet(X4)-positive plasmid (Du et al., 2020).However, the insertion of large fragments containing T4SS associated genes into tet(X4) plasmids has rarely reported.
In this study, we described the genomic characteristics of tet(X4)positive E. coli from animals and humans in China and the potential element insertions related to T4SS which may associated with highfrequency transfer of tet(X4).Therefore, it emphasizes that the sustainability surveillance of tet(X4) is imperative.

Epidemiological study
We undertook a retrospective observational cross-sectional study to assess the prevalence of tet(X4)-positive E. coli in animals, farmers, inpatients and healthy volunteers.A total of 775 nonduplicate samples were collected from two farms and one hospital in Guangdong province, China from 2019 to 2020, including 475 nasal swab samples of pigs in two farms, with farm 1 contributing 419 samples and farm 2 contributing 56 samples, 67 fecal swab samples of pigs, 48 skin swab samples of pig farm workers in farm 1, 172 fecal swab samples of inpatients and 13 fecal swab samples of healthy volunteers.

Long-reads sequencing and plasmid analysis
To analyze the tet(X4)-carrying plasmids, long-reads sequencing was performed using the PacBio platform.Raw data were assembled using Unicycler v0.4.8.The method of gene annotation and identification was the same as that used for the whole genome analysis mentioned above.Insertion sequences were identified using Isfinder.4T4SS genes were identified using oriTfinder. 5The comparison of the genetic environments of tet(X4) were performed using Easyfig v2.1 and R package "gggenes."

Phylogenetic analysis of tet(X4)-positive Escherichia coli strains from various sources
Fifty-six tet(X4)-positive E. coli including 2 isolates from humans and 54 isolates randomly chosen from 144 tet(X4)-positive E. coli from pigs were further sequenced by whole genome sequencing.Among these, 54 strains were derived from two different farms (farm 1, n = 53 and farm 2, n = 1).To analyze the genomic differences between tet(X4)positive E. coli strains from different sources, 8 tet(X4) positive E. coli strains of different sources obtained from GenBank were included in the dataset (Supplementary Table S2A).As mentioned above, 64 tet(X4)-positive E. coli strains used for phylogenetic analysis were sourced from healthy individual's stool, inpatient fecal samples, pig swabs, chicken, pork, and wastewater (Figure 2A).The origins of these samples varied and clustered into 30 distinct STs (Figures 2B,C).In the strains collected in this study, the STs with the highest prevalence were ST877 (8/56, 14.29%), ST871 (6/56, 10.71%), ST195 (7/56, 12.50%), ST10 (4/56, 7.14%), and ST398 (4/56, 7.14%).The sample collected from inpatient belongs to ST9867.The ST871 strain S221-6 from inpatients in the public dataset showed close proximity in the phylogenetic tree to the corresponding ST871 strains collected in our study.Similarly, the ST10 strain YPE10 from pork in the public dataset exhibited a close relationship in the phylogenetic tree with the ST10 strains collected in our study.This finding indicated that tet(X4) had undergone extensive dissemination among multiple lineages of E. coli.S2A.(B) Minimum spanning tree based on MLST (Multilocus Sequence Typing).Each node in the tree represents a specific MLST type.The colors assigned to the nodes correspond to the strain's origins, following the same color scheme as presented in (A).(C) Phylogenetic tree and heatmap of resistance genes among the strains.The reference strains obtained from GenBank are highlighted in the tree.
BLAST analysis of p47P1FS3-2-1 against the NCBI database revealed that the plasmid p1919D3-1 (CP046004.1)had 98% coverage and 100% identity, whereas the plasmid pTKPN_3-186k-tet(X4) (MZ773211.1)had 100% coverage and identity.Their replicon types were all IncFIA(HI1)-IncHI1B(R27)-IncHI1A, suggesting that these strains shared a plasmid backbone with our strains.BLAST alignment results showed that these 4 plasmids were highly conserved at both ends, and had different fragment insertions downstream of tet(X4) mediated by variable insertion sequences, such as IS26, IS1R, ISCR2, and IS15DI (Figure 5A).This region was diverse, including a multidrug resistance region in p1919D3-1 and a region containing a plasmid replicon in pTECO1-1.Comparison of tet(X4)-positive plasmids showed a 100-kbp large fragment insertion at the back end of tet(X4), containing a replicon and a 40-kbp length of the T4SS transfer-related gene cluster (Supplementary Figure S1).Klebsiella quasipneumoniae pJZ2-tet(X4) (ON390804.1)was identified by performing BLAST of pTECO25-1 against the NCBI database, with 100% coverage, 99.8% identity (Figure 5B), indicating that crossspecies transmission of this plasmid has occurred.S2C.The IS1R' in this figure indicates that the IS1R gene fragment is incomplete, potentially due to truncation caused by certain insertion sequences.The representation method mentioned above is equally applicable to the incomplete genes present in this figure and Figure 5, including ISCR2', ISPst2', TnAs1', and TnAs3'.

Discussion
The environment serves as a vast reservoir for drug resistance genes and plays a crucial role in their transmission (Lin et al., 2021).Since its discovery, tet(X4) has been extensively detected in environment samples (Zhang et al., 2022).Regarding the transmission of tet(X4) from the environment to human, potential routes have been proposed in previous studies.These include: contamination of agricultural land or water sources such as reservoirs and wetlands through animal manure, subsequently leading to human exposure via crops or aquatic organisms, such as freshwater fish and shrimp (Li et al., 2019;Dao et al., 2022).Additionally, the presence of contaminated retail meat into the market had been identified as a potential transmission pathway (Sun et al., 2021).Direct contact or fecal-oral transmission among individuals involved in the food processing chain is also considered a possible route (Wang et al., 2020).Our study highlights the possibility of transmission from pork processing plants to workers, emphasizing the importance of implementing prevention and control measures among individuals working in relevant industries.The plasmids p1919D3-1 and pTKPN_3-186k-tet(X4) were derived from strains isolated from swab samples of pig feces from Henan province and pig nasal swabs from Guangdong province, China, respectively.The similarity of these plasmid backbones to the plasmid p47P1FS3-2-1 originating from the patient in our study suggests the potential transmission and evolution of tet(X4)-positive plasmids across different regions in China and between animals and humans.
tet(X4) is highly prevalent in China, with the detection of strains carrying tet(X4) including E. coli, Klebsiella pneumoniae, Citrobacter braakii, Enterobacter cloacae (Li et al., 2021;Wu et al., 2022).The prevalence of tet(X4)-positive E. coli in China exhibited significant diversity in STs with regional variations.In two provinces in northwestern China, Shanxi and Ningxia, the dominant STs of tet(X4)-positive E. coli were ST6704, ST2035, ST48, ST1602, and ST877 (Sun et al., 2020).In western China, ST10, ST34, ST48, and ST195 have been identified as predominant STs (Feng et al., 2022), while in the southern region, ST10, ST48, ST877, and ST2144 were relatively more prevalent (Cui et al., 2022).In our study, we isolated tet(X4)-positive E. coli strains from Guangdong Province in southern China, where we observed high proportions of ST877, ST871, ST195, and ST10.The diverse range of STs indicates that the horizontal transfer of plasmids carrying tet(X4) has occurred, contributing to the genetic variation and dissemination of this resistance gene.Moreover, the two tet(X4)-positive E. coli strains isolated from the patients in this study exhibited a unique ST (ST9867), which has not been previously reported, indicating the transfer of tet(X4) to a novel ST and its subsequent spread to human populations.
A transposable unit is formed when tet(X4) is transferred, and is generally composed of mobile elements, tet(X4) and partner gene catD, in which mobile elements are the key mediators of the transfer.Previous studies have demonstrated that ISCR2, through its upstream and downstream oriIS and terIS elements, plays a crucial role in mediating the transfer of tet(X4) by forming a transposon unit that facilitates rolling-circle transposition (Toleman et al., 2006;Sun et al., 2020).Additionally, under the influence of other IS elements and homologous recombination events, ISCR2 generates a highly diverse genomic environment.This includes the formation of tandem multicopies due to transposase misreading of terIS, capture of other resistance genes, and truncation by other IS elements (Liu et al., 2022;Zhai et al., 2022).In this study, all the isolated strains carrying a single copy of tet(X4).Using the long-reads sequencing, we identified three transposon units comprising the different mobile elements.
Combining our data with available online resources, we found that, in addition to ISCR2, IS1R and IS26 elements were also frequently involved in mediating the transfer of tet(X4).Furthermore, we discovered that the presence of the florfenicol resistance floR gene frequently accompanied tet(X4) downstream, indicating that the dissemination of tet(X4) plasmids may be influenced by the selective pressure exerted by florfenicol.
One way in which antibiotic resistance plasmids acquire additional resistance genes is through the frequent insertion of genetic fragments.During horizontal gene transfer, antibiotic resistance plasmids can acquire additional resistance genes by inserting new fragments, such as transposons or other mobile elements.In this study, four plasmids shared the same plasmid backbone, IncFIA(HI1)-IncHI1B(R27)-IncHI1A.However, compared to plasmid p47P1FS3-2-1, the other three plasmids underwent insertion of resistance gene fragments, including aadA2, qnrS1, and Inu(F).The insertion of diverse resistance genes provided the tet(X4)-positive plasmid with increased adaptability.In addition, the acquisition of an exogenous sequence containing a substantial number of T4SS-related genes by pTECO1-1 was a notable event.However, the significance of these findings requires further investigation.This study has several limitations.First, sample size was relatively small, as it included samples collected in Guangdong, China.Additionally, this study focused primarily on pigs, which lacked diversity in sample sources.This study also revealed the insertion of T4SS genes into a tet(X4)-positive plasmid, however, their functional significance requires further experimental verification.

Conclusion
In this study, we described the high colonization rate of tet(X4)positive E. coli in animals, along with its presence among the farm workers.Comparative analysis revealed that the successful genetic elements and the insertion of T4SS-associated genes in tet(X4)positive plasmids, which may contribute to the rapid dissemination of tet(X4).Overall, this study serves as a timely warning regarding the potential acceleration of tigecycline resistance spread and underscores the critical importance of comprehensive surveillance spanning from animals to humans for effective global control of antibiotic resistance.

FIGURE 2
FIGURE 2 Population structure and resistance gene characteristics of 65 tet(X4)-positive E. coli.(A) Pie chart illustrating the distribution of sample sources.GenBank data accession numbers mentioned in this figure are listed in Supplementary TableS2A.(B) Minimum spanning tree based on MLST (Multilocus Sequence Typing).Each node in the tree represents a specific MLST type.The colors assigned to the nodes correspond to the strain's origins, following the same color scheme as presented in (A).(C) Phylogenetic tree and heatmap of resistance genes among the strains.The reference strains obtained from GenBank are highlighted in the tree.

FIGURE 3
FIGURE 3Comparison of genetic contexts of tet(X4).The schematic diagram provides a comparative analysis of genetic contexts surrounding tet(X4) in three strains subjected to short-read sequencing (TECO25, TECO1, and 47P1FS3-2).The symbols flanking the ISCR2 represent the putative oriIS and terIS of the ISCR2, respectively.

FIGURE 4
FIGURE 4 Different types the genetic environments of tet(X4).The figure represents a comparison of the genetic environments of tet(X4) among 41 tet(X4)positive E. coli obtained from GenBank.GenBank data accession numbers mentioned in this figure are listed in Supplementary TableS2C.The IS1R' in this figure indicates that the IS1R gene fragment is incomplete, potentially due to truncation caused by certain insertion sequences.The representation method mentioned above is equally applicable to the incomplete genes present in this figure and Figure5, including ISCR2', ISPst2', TnAs1', and TnAs3'.