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 findings 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 efficient 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 Tn7-like, 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. Specifically, 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 specific 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 flanking 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 five 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 confirmed 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.
Proteus SCBX1.1 carrying Tn6765 also contains another cfr plasmid P1.1.1 (Fig. 6). The cfr-containing segment (corresponding to bases 8925 to 12726 to 4624 in GenBank accession number CP047113) harbours a genetic structure (Fig. 5), showing homology to the cfr segment characterized in P. cibarius G11(Genebank accession number CP047287), which is partially differ from the Chen et al. reported the existence of IS26-cfr-DTn554 tnpB-DTn3 family tnpA-IS26 section in another Tn7-like Tn6451 (52) (Fig. 5). 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 confirmed that Tn7-like plays a vital role in the field 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 significant 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).