Characteristics of the multiple replicon plasmid IncX1-X1 in multidrug-resistant Escherichia coli from Malayan pangolin (Manis javanica).

Potential zoonotic pathogens may be transmitted from wildlife to humans through the illegal wild meat trade, which has become a pressing issue. However, research on the antimicrobial resistance genes (ARGs) of Malayan pangolin (Manis javanica) intestinal bacteria is limited. Here, multidrug-resistant Escherichia coli M172-1 (ST354) isolated from Malayan pangolin feces in 2019 was found to be resistant to 13 antibiotics. BGWAS analysis revealed 4 plasmids, namely, pM172-1.1, pM172-1.2, pM172-1.3, and pM172-1.4, in the isolate. The pM172-1.2, pM172-1.3, and pM172-1.4 plasmids carried ARGs, namely, IncHI2-HI2A, IncX1-X1, and IncX1, respectively. pM172-1.3 and pM172-1.4 contained intact IntI1 integrons (Is26/IntI1/arr2/cmlA5/blaOXA-10 /ant(3″)-IIA/dfrA14/Is26). Notably, pM172-1.3 resulted from the fusion of 2 pM172-1.4 copies and carried many more ARGs. In addition to pM172-1.3 from the same host, other drug-resistant bacteria (E. coli M159-1 (ST48), E. coli S171-1 (ST206), and Klebsiella pneumoniae S174-1 (ST2354)) in the same Malayan pangolin fecal samples also carried 3 plasmids with 100% gene coverage of pM172-1.4 and 99.98% identity. Therefore, ARGs in IncX1 might spread in the intestinal flora of Malayan pangolin and between species via the illegal food chain, posing a potential threat to public health and safety.

Multiple-Replicon Resistance Plasmids of Klebsiella Mediate Extensive Dissemination of Antimicrobial Genes.

Multiple-replicon resistance plasmids have become important carriers of resistance genes in Gram-negative bacteria, and the evolution of multiple-replicon plasmids is still not clear. Here, 56 isolates of Klebsiella isolated from different wild animals and environments between 2018 and 2020 were identified by phenotyping via the micro-broth dilution method and were sequenced and analyzed for bacterial genome-wide association study. Our results revealed that the isolates from non-human sources showed more extensive drug resistance and especially strong resistance to ampicillin (up to 80.36%). The isolates from Malayan pangolin were particularly highly resistant to cephalosporins, chloramphenicol, levofloxacin, and sulfamethoxazole. Genomic analysis showed that the resistance plasmids in these isolates carried many antibiotic resistance genes. Further analysis of 69 plasmids demonstrated that 28 plasmids were multiple-replicon plasmids, mainly carrying beta-lactamase genes such as bla CTX-M- 15, bla CTX-M- 14, bla CTX-M- 55, bla OXA- 1, and bla TEM- 1. The analysis of plasmids carried by different isolates showed that Klebsiella pneumoniae might be an important multiple-replicon plasmid host. Plasmid skeleton and structure analyses showed that a multiple-replicon plasmid was formed by the fusion of two or more single plasmids, conferring strong adaptability to the antibiotic environment and continuously increasing the ability of drug-resistant isolates to spread around the world. In conclusion, multiple-replicon plasmids are better able to carry resistance genes than non-multiple-replicon plasmids, which may be an important mechanism underlying bacterial responses to environments with high-antibiotic pressure. This phenomenon will be highly significant for exploring bacterial resistance gene transmission and diffusion mechanisms in the future.