# Mechanisms of Transmission and Adaptation of tet(X4)-Positive IncHI1 Plasmids in XDR Escherichia coli from Pet Dogs: The Role of trhC, rsp, and the Tra1 Region

## TL;DR

This study explores how certain plasmids help spread antibiotic resistance in bacteria from pet dogs, focusing on mechanisms that enhance their adaptability and transmission.

## Contribution

The study identifies novel IncFII plasmids as conjugative helper plasmids that eliminate temperature sensitivity in tet(X4)-positive IncHI1 plasmids.

## Key findings

- IS26 from IncF18:A-:B- plasmids modifies IncHI1 plasmids by attacking trhC and rsp genes.
- Homologous recombination via ISCro1 and ISCR2 deletes the Tra1 region in IncHI1 plasmids.
- Fusion plasmids lose temperature sensitivity, increasing conjugation efficiency at both 28°C and 37°C.

## Abstract

Tigecycline is one of the last-resort drugs to treat serious infections caused by MDR Gram-negative bacteria. However, the emergence of the plasmid-mediated high-level tigecycline resistance gene tet(X4) is bound to create difficulties for clinical treatment. The IncHI1 plasmid serves as a crucial carrier for the transfer of the tet(X4) gene, playing a crucial role in the spread of tigecycline resistance. Nevertheless, the conjugative transfer of IncHI1 plasmids exhibits temperature sensitivity. In our study, we discovered that two novel types of IncFII plasmids can act as conjugative helper plasmids to fuse with IncHI1 plasmids through different mechanisms, eliminating temperature sensitivity and promoting their dissemination. Our findings provide new insights into the evolution and transmission of tet(X4)-positive IncHI1 plasmids, which are of significant importance for controlling the spread of multidrug-resistant plasmids among organisms.

tet(X4)-positive IncHI1 plasmids are widely prevalent in various bacteria. To understand their transmission characteristics, we analyzed two extensively drug-resistant (XDR) Escherichia coli strains isolated from pet dog feces in Henan Province, China. Strain T28R harbored tet(X4)-positive IncHI1, IncF18:A-:B-, and mcr-1-positive IncI2 plasmids, while T16R carried tet(X4)-positive IncHI1, F16:A-:B-, and mcr-1-positive IncX4 plasmids. Four representative fusion plasmids, pT28R-F1, pT28R-F2, pT28R-F3, and pT16R-F1, in transconjugants were analyzed using WGS and PCR mapping. The results showed that IS26 from the IncF18:A-:B--plasmid attacked the conjugative transfer-associated genes trhc and rsp on the IncHI1 plasmid, generating pT28R-F1 and pT28R-F2. pT28R-F3 was generated through ISCro1- and ISCR2-mediated homologous recombination, deleting the Tra1 region of the IncHI1 plasmid. T16R-F1 emerged from ISCR2- and IS1B-mediated homologous recombination, losing transfer regions of parental plasmids. Notably, fusion plasmids lost the temperature sensitivity of the IncHI1 plasmid, with conjugation frequencies between 1.57 × 10−4 and 3.84 × 10−5 at 28 °C and 37 °C. The findings suggest that tet(X4)-positive IncHI1 plasmids could be mobilized with the assistance of conjugative helper plasmids and that fusion events enhance the adaptability of these plasmids, thus facilitating the spread of antibiotic resistance, posing a growing public health threat.