Characterization of Tn7-like transposons and its related antibiotic resistance among Enterobacteriaceae from livestock and poultry in China

Background: This study was aimed to investigate the prevalence and structure of Tn7-like in Enterobacteriaceae from livestock and poultry as well as their possible role as reservoir of antibiotic resistance genes (ARGs). Methods: Polymerase chain reaction (PCR) and DNA sequencing analyses were used for the characterization of Tn7-like, associated integrons and ARGs. The antimicrobial resistance prole of the isolates was examined by using disc diffusion test. Results: Three hundred and seventy-eight Tn7-like-positive strains of Enterobacteriaceae were isolated, and included E. coli (128), Proteus(150), K. pneumonia(17), Salmonella(13), M. morganii (21) and A. baumannii(1), wherein high resistance was observed for Trimethoprim/Sulfamethoxazole and Streptomycin, and fty percent of the strains were multidrug-resistant. Integrons class 2 were detected in all of the isolates and there are high frequency mutation sites especially in 535, a stop mutation. Variable region of class 2 integrons carried same gene cassettes, namely aadA1-sat2-dfrA1. From the 378 isolated strains, we found a new type of Tn7-like on a plasmid, named Tn6765. Conclusions: These ndings proved that the Tn7-like can contribute to the horizontal transmission of antibiotic resistant genes in livestock and poultry. As potential vessels for antibiotic resistance genes (ARGs), Tn7-like could not be ignored due to their ecient transfer ability in environments.


Background
Enterobacteriaceae include a large group of gram-negative bacillus with similar biological characteristics, They are widely distributed in the natural environment and the intestinal tract of humans and animals. As an opportunistic pathogen, Enterobacteriaceae is associated with urinary tract infection (1), nosocomial infection (2), and food poisoning (3). The widespread and irrational use of antibiotics in humans and animals has given rise to numerous multidrug-resistant Enterobacteriaceae (MRE) via the dissemination of antimicrobial resistance genes (AMRg) (4). This multiresistance may be mediated by chromosomally located resistance determinants or mutations in a resident gene. however, it may also develop through the acquisition of resistance genes or an array of resistance genes by horizontal transfer. This latter phenomenon is currently thought to play an increasing role in the development of multidrug resistance in Enterobacteriaceae (5). These resistance determinants of Enterobacteriaceae are generally disseminated via mobile elements (integrons, transposons and plasmids) in bacterial populations from animals and humans (4). One signi cant nding over the past 10 years is the appreciation of the dissemination of Tn7, and related elements called Tn7-like elements that contain homologs of the Tn7 transposition proteins, in highly diverged bacteria adapted to a remarkable number of different environments (6-8).
Tn7 is typically large, often 14 kb in size (9). However, comparative analysis of some derivatives of the Tn7, Tn7-like, shows that these elements are very diverse in terms of genetic structure and have accumulated several accessory resistance regions (8, 10). Tn7-like carry the determinants for resistance not only to heavy metals such as mercuric ions, copper, silver ions, tellurite, arsenate and arsenite, but also to antibiotics such as β-lactams (including carbapenems), quinolones, aminoglycosides, tetracyclines, amphenicols, trimethoprim, sulfamethoxazole, streptomycin and fosfomycin (11). The Tn7-like, as an important mobile genetic element of transposons, transfers various resistance genes between bacteria through its transposase, promotes the horizontal spread of drug resistance in bacteria.
The sequences of the two ends of the Tn7 transposon are highly conserved, which are respectively transposition module and class 2 integron system. Transposition module encodes ve proteins required for two transposition path-ways, tnsA, tnsB, tnsC, tnsD, and tnsE (tnsABCDE) (12). The basic transposition machinery of Tn7 and how it directs transposition into the attTn7 site is well understood and has been the subject of multiple reviews (13,14). tnsABC are required for both Tn7 transposition pathways, but only allow transposition when they function with one of two target site selecting proteins, tnsD or tnsE (15,16). Transposition with tnsABC + tnsD directs transposition into the single attTn7 site located downstream of glmS (15). Transposition with tnsABC + tnsE occurs preferentially into mobile plasmids through the ability of the tnsE protein to recognize features found enriched during DNA replication on the lagging-strand template (17,18).
The class 2 integron of Tn7 has an organization similar to that of the class I integron and carries three resistance gene cassettes-aadA1, sat, and dfrA1 (19)(20)(21)-close to an open reading frame, IntI2*. IntI2* has premature translation termination due to mutation of base 535 encoding integrase from C to T, and has no function. Although these gene cassettes are xed in Tn7 due to mutations in the homologous recombinase, they can be rearranged in hosts expressing the relevant recombinase, resulting in other combinations of antibiotic resistance genes (22).
However, there is a paucity of data on the prevalence and role of Tn7-like transposons in antibiotic resistant Enterobacteriaceae in many countries such as china especially from livestock and poultry. The aim of this study was to determine the frequency of Tn7like transposons and its associated integrons in Enterobacteriaceae which from swine and chicken, and to investigate the association between the presence of Tn7-like and antibiotic resistance patterns, so as to better understand the characteristics of Tn7-like and resistant Enterobacteriaceae in farming animals. Additionally, we plan to analyze the skeletal structure of our Tn7-like and compared it with other Tn7-like to obtain insight into the horizontal transfer of resistance genes and the diversity and evolution of Tn7-like.

Bacterial isolates
A total of 1482 consecutive and unduplicated clinical isolates of Enterobacteriaceae were collected from livestock and poultry in China from 2018 to January 2020. The isolates were from the intestines of dead animals, feces, cloacal swab and drinking water (Table 1). All the isolates were presumptively identi ed through phenotypic methods, including colony morphology on MacConkey Agar or Eosin-Methylene Blue Agar. And the identi cation of these isolates was later con rmed using 16S rRNA gene sequencing.
All Enterobacteriaceae strains were stored at − 20 °C in 50% glycerol. The standard strain of Escherichia coli (ATCC25922) was used as a susceptibility control. Rifampin-resistant strain E. coli600 was used as the recipient strain in the conjugation experiments.

Characterization Of The Integrons And Associated ARGs
The isolated Tn7-like Enterobacteriaceae strains were checked for the presence of intl2 integrase genes by PCR, using primers (Table S1) and methodology as previously described by (24,25), and these mutation site sequence of intI2 were visualized with Weblogo (http://weblogo.berkeley.edu/logo.cgi). Subsequently, strains harboring intl2 genes were elaborated for the ampli cation of its three resistance gene cassettes (aadA1, sat2 and dfrA1). For this purpose, primer pairs which are listed in Table  S1, were used to amplify the gene cassette by targeting aadA1, sat2 and dfrA1, respectively.

WGS And Analysis
We also analysed genetic environment of Tn7-like transposons among strains exhibiting unique resistance phenotypes. The whole genome of the Tn7-like-positive strain was sequenced using Illumina MiSeq with a 200-fold sequencing depth and the nanopore platform with a 100-fold sequencing depth. Genome assembly was carried out by de novo assembly in SMRT portal version 3.2.0, and the sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline v4.2. Mobile elements, resistance genes, and other features were annotated by INTEGRALL (26), IS nder (27), ResFinder (28), PlasmidFinder (29), and the Tn Number Registry (30) online databases, and the analysis was conducted using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Horizontal Transfer Of Tn7-like Transposon
Conjugation was performed using rifampin-resistant E. coli EC600 as the recipient and the P. mirabilis SCBX1.1 isolate as the donor with selection on Salmonella Shigella agar plates containing 20 mg/liter rifampin and 8 mg/liter orfenicol. Successful horizontal transfer of plasmid p1.1.2 containing Tn7-like transposon was con rmed using antibiotic sensitivity test and PCR as mentioned above, then the conjugation frequency was calculated as transconjugants divided by number of donors (31). The stability of p1.1.2 in the positive transconjugants (designated EC-600-Pp1.1.2) was determined by passage in Brain Heart Infusion(BHI) broth lacking antibiotics as described previously (32).

Statistical analysis
Variables are expressed as percentages (%). Comparison of incidence of Tn7-like in Enterobacteriaceae isolates versus Tn7-like lacking isolates was assessed using the Chi square test. Statistical analysis was conducted with the GraphPad Prism 8 software.
P value of < 0.05 was considered as signi cant.

Genbank Accession Number
The complete sequences of p1.  (Fig. 1). The positive rate of Tn7-like transposon in proteus was signi cantly higher than that of other strains (p < 0.0001).

Antimicrobial Resistance Phenotypes
A total of 378 Tn7-like positive isolates were examined for their resistance against 10 antibiotics (Table 2). Two hundred and fourteen (56.61%) isolates showed resistance to three or more antibiotic classes and were considered as multidrug-resistance (MDR). Except for Acinetobacter baumannii, Proteus and Salmonella have the highest multidrug resistance rates of 65.69% and 63.90%, respectively. Notably, high resistance rates were observed for Streptomycin (88.7%) and Trimethoprim/Sulfamethoxazole (74.6%), followed by Ampicillin (63.23%). And low resistance rates to Ceftazidime (5.82%), Cefoxitin (5.82%) and Aztreonam (4.23%) were detected. Most of the Enterobacteriaceae isolates were highly susceptible to Imipenem. Prevalence And Cassette Gene Of Tn7-like Integrons The positive rate of class 2 integrons in 378 Enterobacteriaceae strains was 100%. Through sequencing and sequence comparison, the results showed that intI2 usually had mutation sites, and most of them were at 349,372,379,535,617,767 and 774 (Fig. 2). On ampli cation, 347 isolates were found to carry the resistance gene cassette (aadA1, sat2 and dfrA1) related to intI2, whereas 31 strains had different aadA1, sat2 and dfrA1 gene deletions, respectively. 24 isolates were missing one gene cassette, and 7 isolates were missing two gene cassette. A single S.enterica only harbored dfrA1 (Table 3). Structural Analysis Of Tn7-like Among the Tn7-like-carrying strains, three multi-drug resistant bacteria were randomly selected for whole-genome sequencing, and yielded three complete Tn7-like structures. According to the transposon nomenclature (http://transposon.lstmed.ac.uk/), we designated them Tn6763, Tn6764, and Tn6765, respectively. Tn6763 (GenBank accession number MN641830) and Tn6764 (GenBank accession number MN628641) were located on the chromosomes of Proteus and E. coli, respectively. They are typical Tn7 transposons comprising the transposition module (tnsA, tnsB, tnsC, tnsD and tnsE), three gene cassettes (aadA1, sat2 and dfrA1), and an inactive class 2 integrase gene. Besides, both of them inserted an insB-1 next to the aadA1 gene (Fig. 3).
Tn6765 was highly homologous with Tn6450 located on the chromosome detected by Chen (33). The similarity of nucleic acid sequence was more than 99%, and the coverage was 96%. The differences between the two are mainly in the following four aspects: (1) Tn6765 has a hypotheical protein gene before blaDHA-1; (2) The ampR gene of Tn6450 was replaced by lysR and HypA in Tn6765; (3) An incomplete aac(3)-IV was inserted between ISEc59 and aph(4)-Ia; (4) Tn6450 is inserted downstream of the glmS gene, encoding glucosamine 6-phosphate synthase and surrounded by 5-bp direct repeats (CCAAT), Whereas Tn6765 is located on the plasmid, there is no glms gene and repeated sequences at both ends (Figs. 3 and 4). The plasmid carrying Tn6765 was 138818 bp in size and had a GC content of 44.35%, which was named as p1.1.2. Blastn results showed that, except for the Tn6765 Tn7-like MDR region, other regions of plasmid p1.1.2. showed 99.98% similarity and 91% coverage with nucleic acid sequence of P.mirabilis plasmid pPm60 (GenBank accession number MG516911) that also carried the Tn7-like transposon published in NCBI database (Fig. 4).

Horizontal Transfer Of Tn7-like
Double-antibiotics Salmonella Shigella agar plates (300ug / ml rifampicin + 10.24ug / ml ufenicol) were used to screen the transconjugant. The results of drug sensitivity, electrophoresis, and sequencing showed that the plasmid p1.1.2 could be successfully transferred to E. coli EC600 (Table 4). A novel Tn7-like (Tn6765) transconjugant was obtained through the conjugative transfer test, and at a frequency of 1.5 × 10 − 1 transconjugants per donor (average of three independent determinations). The stability of Tn6765 was determined by continuous passage propagation lasting 10 days (20 passages) in the absence of antimicrobial pressure. One hundred clones in the 21st passage were picked to detect the presence of Tn6765. The results of all PCRs were positive. Showed that Tn6765 can be stably inherited in the recipient bacteria.

Discussion
Laboratory experiments have shown that Tn7 and its derivatives are capable of transposition in many different hosts, but few naturally occurring examples have been isolated (7) In this study, we examined the occurrence and prevalence of Tn7-like in Enterobacteriaceae isolates obtained from several farms of chicken and swine raised for meat purpose. Of 7 Enterobacteriaceae species in the collection, the most prevalent species included Proteus (150 isolates, 39.7%) and Escherichia coli (128 isolates, 33.9%). One hundred isolates (26.5%) were assigned to other Enterobacteriaceae species. Statistical analysis showed that the presence of Tn7-like was different among different bacteria genera (P < 0.0001) (Fig. 1). The result indicated Proteus malleable genomes contribute to facilitate high-frequency insertion of transposons like Tn7-like, which was similar to the ndings of other studies (31,34,35). Also, Escherichia coli is the representative strain of the genus Escherichia, which is the abundant type of bacteria in the intestine of humans and animals (36), so its proportion in Tn7-like positive Enterobacteriaceae is also large. Our results provide evidence of the pigs and chickens as a possible reservoir of Tn7-like carrying strains with the antibiotic resistance gene. Enterobacteriaceae, as a widespread intestinal bacterium, its carriage Tn7-like are likely to have a serious impact on human life through animals such as pigs and chickens, and is a risk that deserves our attention.
In multidrug-resistant Enterobacteriaceae bacteria, Tn7-like transposons play a very vital role due to their high capability for transferring antimicrobial resistance genes (37). Through drug susceptibility tests, we found that the multi-drug resistance of Enterobacteriaceae carrying Tn7-like is widespread in various strains. Except for M.morganii and K.pneumoniae, the multi-drug resistance rates of other strains are above 50%. Among them, Proteus (65.69%) and Salmonella (63.90%) are more prominent (Table 2). Proteus is an important drug-resistant storage bacteria because of its highly e cient genetic structure. It is usually considered commensal in the gut, but it can also cause food poisoning and is most commonly recognized clinically as a cause of urinary tract infections (38, 39). Salmonella, one of the most common and widely distributed foodborne pathogenic microorganisms, is a major cause of food-borne salmonellosis (40,41). And the food-borne salmonellosis is typically acquired through consumption of contaminated poultry meat and eggs (42). Obviously, for both Proteus and Salmonella, carriage of Tn7like, each containing a set of resistance genes, may increases the chances of horizontal transfer of multiple resistance determinants to susceptible strains, and may in turn confer unique advantages to the host and enable them survive a strong antimicrobial selection pressure especially in poultry and livestock farm settings.
Tn7-like isolates are particularly resistant to Trimethoprim/Sulfamethoxazole and Streptomycin because of the intI2-associated resistance gene cassette (aadA1,sat2 and dfrA1) carried by Tn7-like (43,44). The gene cassettes of intI2 contained the aminoglycoside adenyltransferase (aadA1 and aadA2), dihydrofolate reductase (dfrA1), and streptothricin acetyltransferase (sat2) encoding genes, which are responsible for streptomycin-spectinomycin, trimethoprim, and streptothricin resistance, respectively area (45). In this study, we used the PCR method to determined the occurrence of class 2 integrons and associated gene cassettes (aadA1, sat2 and dfrA1) in Tn7-like-positive isolates. Similar gene cassettes (aadA1, sat2, and dfrA1)have been obtained in the most of animals-derived Enterobacteriaceae isolates (46, 47).The presence of the gene cassette and its corresponding phenotypic resistance was matched (Tables 2 and 3). This explains why Tn7-like bacteria are highly resistant to Trimethoprim/Sulfamethoxazole and Streptomycin, and may pose the risk of widespread dissemination of large, single assemblage repositories of resistance genes and threatening the emergence of a post-antibiotic era (48). The high prevalence of the Tn7-like containing antibiotic resistance genes detected in the current study could be attributed to extensive use of antibiotics for disease prevention, treatment and growth enhancement in farm animals in the studied. Speci cally, spectinomycin is widely used against the gonorrhea and respiratory/enteric infections in humans and animals (25). Tn7-like have been so successful at spreading into diverse relevant taxa that they could be used as a proxy for anthropogenic pollution (49,50).
The intI2 downstream of Tn7-like transposons mediate the deletion of gene cassettes (aadA1,sat2, and dfrA1).Through sequencing, we found that there were multiple mutated sites in intI2, among which 535 base mutations (C mutated to T) were terminating mutations (Fig. 2), which led to premature termination of integrase gene translation, making its gene cassette sequence show a high degree of stability, usually aadA1, sat2 and dfrA1 (51). Karin Hansson et al. changed the stop codon 179 of the intI2 gene into the codon encoding glutamic acid (E), and found that intI2*179E promoted the speci c excision of each gene cassettes in Tn7 at different frequencies (22). Some of the gene cassettes deletions investigated in this study are precisely because the 535 base mutation produces a functional integrase gene intI2 ( Fig. 2 and Table 3). We show here experimentally that the second class of integrase and associated gene cassettes in Tn7 can promote recombination between resistant gene present in the host, but only after changing the termination codon to a sense codon (22). This makes Tn7-like a better reservoir of antibiotic resistance.
The structure of the Tn7-like transposon is changeable and interrelated. We have obtained a new transposon Tn6765. By comparison analysis, we know that Tn6765 and Tn6450 have a very high similarity. Both are Tn7-like transposons, which are generated by inserting sequences and transposons carrying anking drug resistance genes integrated into the Tn7 transposon variable region. Their transposable module was interrupted by the transposon Tn1721 at the transposase gene tnsD, and Tn1721 was interrupted by the downstream IS26 (33). The structures of Tn6765 and Tn6450 are complex and highly homologous, suggesting that they may have undergone similar evolutionary processes. Tn6765 carries ve IS26, two ISCR1, one IS1353, one IS1006, one ISCR2, one ISAba1, one ISEC59, and 2 defective transposons Tn1721 and Tn21, indicating that this transposon is generated by multiple mobile genetic elements carrying lateral resistance genes integrated into the variable region. There are a few genetic differences between Tn6450 and Tn6765 (Fig. 3). Noteworthily, Tn6450 is located on the chromosome of Proteus mirabilis but Tn6765 is located on the plasmid (Fig. 4), which con rmed that Tn7-like can be transmitted alternately on the chromosome and plasmid by cutting and inserting. Through the conjugative transfer test, Tn6765 was successfully transferred to EC600 with the plasmid. 41Ominously, like its distant relative Tn7, Tn6765 may exhibit the potential to jump bacterial species readily. To make matters worse, Tn6450 comes from a chicken source (33) and Tn6765 from a pig source, suggesting that Tn7-like can be transmitted from one animal to another with bacterial hosts.
Although the cfr section of Tn6765 and Tn6451 is slightly different, this also suggests an evolutionary direction of the Tn6765 carrying strain. By comparing the genetic structure of different Tn7-like transposons, we can speculate that the multidrug-resistant Tn7-like transposons have a certain evolutionary relationship, which con rmed that Tn7-like plays a vital role in the eld of storing resistance genes.
Unlike the DNA blueprints of higher organisms, Enterobacteriaceae bacterial genomes exhibit an extraordinary degree of plasticity and intraspecies diversity (53). A key feature of Enterobacteriaceae is the constant mobility of various segments of the genome both with in a single cell and between cells. This mobility plays a signi cant role in the evolution of bacteria through tuning of genetic architecture and the lateral acquisition of new genes that may provide a survival advantage, thus further adapting bacteria to new environments (54). Transposons as the major contributors to the acquisition and dissemination of antibiotic resistance genes (ARGs), and Tn7 are their central agents (55). One Tn7 relative may operate as a "founder element" that can locate and safely transpose into the bacterial chromosome, bringing with it other mobile elements and possibly attachment sites for bacteriophages, integrons, or other transposons. After multiple Tn7-like transposition events accumulate in the attachment site, recombination between these elements may enhance evolution by reassortment (7).

Conclusions
To the best of our knowledge, this study is the rst report showing the prevalence and baseline characteristics of Tn7-like in Enterobacteriaceae isolates from livestock and poultry in China. Our results showed that Tn7-like transposon has a great effect on bacterial resistance, and they can continuously accumulate ARGs in the evolutionary process to consolidate the bacteria's defense ability, which makes bacterial treatment more di cult. Animals are possible reservoirs and a source for the dissemination of Tn7like transposon associated ARGs in the environment. Multiple antibiotic resistance gene determinants in Tn7-like, especially when found in producing chickens and swine is highly worrisome and may become a serious threat by spreading associated resistance genes in other nearby animals and the humans connected therewith. Therefore, strict preventive measures should be taken to stop the spread and emergence of mobile genetic resistance determinants in food producing poultry and livestock in China and the world, and our study provides an important reference for this. The separation rate of Tn7-like in Enterobacteriaceae of different genera(n= 1482). Tn7-like positive; % of Enterobacteriaceae isolates, which are positive for tnsA gene via PCR, Tn7-like negative; % of isolates negative for tnsA genes via PCR. Comparison of the incidence of Tn7-like transposons in Enterobacteriaceae isolates versus Tn7-like lacking isolates was assessed using the Chis square test. The results are signi cant. p<0.0001<0.05.

Figure 3
Genetic structure of Tn6763, Tn6764 and Tn6765. The physical maps were generated using Easy g 2.