Objectives To report a novel tigecycline resistance gene, tet(X6), and its variants in four bacterial species isolated from chickens and pigs in China. Methods WGS was conducted to identify the suspected resistance genes in the tigecycline-resistant Myroides phaeus 18QD1AZ29W. Functional cloning, homology modelling and molecular docking were performed to compare the function with other Tet(X) variants. Retrospective screening for tet(X6) was conducted for 80 isolates in our WGS data collection, and all genomic environments of tet(X6)-positive isolates were analysed. Results The tigecycline-resistant M. phaeus 18QD1AZ29W isolated from a pig farm in Shandong in 2018 was positive for tet(X2) and a novel tet(X) gene, designated tet(X6). Tet(X6) could increase the MICs of all tested tetracyclines/glycylcyclines for Escherichia coli only 2- to 4-fold, which was possibly due to a lower tetracycline binding capacity of Tet(X6) compared with that of other Tet(X) variants. Retrospective screening showed that seven other isolates (7/80, 8.8%), comprising four Proteus spp. and three Acinetobacter spp. from chickens and pigs in Shandong and Guangdong, were positive for three different variants of tet(X6). The analysis of the genomic environment revealed that two tet(X6)-positive isolates from M. phaeus and Proteus cibarius, respectively, contained ISCR2, which may play a role in tet(X6) transmission. Conclusions This study identified a novel type of tigecycline resistance gene, tet(X6), in Myroides, Acinetobacter and Proteus from chickens and swine. Tet(X6) conferred lower tetracycline/glycylcycline MICs than other Tet(X) variants, and ISCR2 may play a role in the transmission of tet(X6).

The wide dissemination of New Delhi metallo-β-lactamase genes (blaNDM) has resulted in the treatment failure of most available β-lactam antibiotics, with IncX3-type blaNDM-5-carrying plasmids recognised as having spread worldwide. In China, bacteria carrying these plasmids are increasingly being detected from diverse samples, including hospitals, communities, livestock and poultry, and the environment, suggesting that IncX3 plasmids are becoming a vital vehicle for blaNDM dissemination. To elucidate the fitness cost of these plasmids on the bacterial host, we collected blaNDM-negative strains from different sources and tested their ability to acquire the blaNDM-5-harboring p3R-IncX3 plasmid. We then measured changes in antimicrobial susceptibility, growth kinetics, and biofilm formation following plasmid acquisition. Overall, 70.7% (29/41) of our Enterobacteriaceae recipients successfully acquired the blaNDM-5-harboring p3R-IncX3 plasmid. Contrary to previous plasmid burden theory, 75.9% (22/29) of the transconjugates showed little fitness cost as a result of plasmid acquisition, with 6.9% (2/29) of strains exhibiting enhanced growth compared with their respective wild-type strains. Following plasmid acquisition, all transconjugates demonstrated resistance to most β-lactams, while several strains showed enhanced biofilm formation, further complicating treatment and prevention measures. Moreover, the highly virulent Escherichia coli sequence type 131 strain that already harbored mcr-1 also demonstrated the ability to acquire the blaNDM-5-carrying p3R-IncX3 plasmid, resulting in further limited therapeutic options. This low fitness cost may partly explain the rapid global dissemination of blaNDM-5-harboring IncX3 plasmids. Our study highlights the growing threat of IncX3 plasmids in spreading blaNDM-5.

T he rapid rise and dissemination of multidrug-resistant (MDR) bacteria are a major threat to public health worldwide and have narrowed the treatment options for infections caused by these bacteria (1). Colistin is one of the last-resort drugs for the treatment of infections caused by MDR Gram-negative bacteria. However, within the past 2 years, shortly after the report of the first plasmid-mediated colistin resistance gene, mcr-1, in 2015, four other mobile colistin resistance genes (mcr-2, mcr-3, mcr-4, and mcr-5) and multiple variants have been reported (2-6). Compared to mcr-2, mcr-4, and mcr-5, which have been detected solely in Europe so far, both mcr-1 and mcr-3 have been identified globally. Here, we report a novel mcr-3 variant in Aeromonas caviae, Proteus mirabilis, and Escherichia coli from a single domestic duck. A total of 15 cloacal samples were obtained from free-range domestic ducks near a river in a suburban area of Qingdao City, Shandong Province, China, in March 2017. Direct sample testing was then performed on all 15 samples to detect the mcr-3 gene, and only one sample was identified as mcr-3 positive. The sample positive for mcr-3 was further isolated on CHROMagar orientation agar plates (bioMérieux, Lyon, France) containing 2 mg/liter colistin. Three mcr-3-positive strains, A. caviae 17AC, P. mirabilis 17PM, and E. coli 17EC, were obtained, and their sequences were confirmed by using 16S rRNA gene sequencing and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis. A. caviae 17AC, P. mirabilis 17PM, and E. coli 17EC were then subjected to 150-bp paired-end whole-genome sequencing (WGS) using the Illumina HiSeq 2500 platform (Annoroad, Beijing, China). The draft genomes were assembled using CLC Genomics Workbench 9.0 (CLC Bio, Aarhus, Denmark), and all contigs were searched for mcr-3 by standalone BLAST analysis. WGS analysis identified mcr-3-carrying fragments in each genome, including a 26.2-kb contig from 17AC, a 17.8-kb contig from 17PM, and a 13.0-kb contig from 17EC. An mcr-3 variant gene in three contigs showed 98.83% nucleotide sequence identity to mcr-3 from porcine E. coli. The deduced protein sequence differed from MCR-3.1 by seven amino acid substitutions, one of which (V122G) was located in a putative transmembrane region, while the remaining six (R297L, I313V, E337K, H341Y, D358E, and Q468K) were in the catalytic domain. This novel mcr-3 variant was designated mcr-3.10. A. caviae 17AC and E. coli 17EC belong to novel sequence types (STs), ST513 and ST457, respectively. E. coli of ST457 carrying mcr-1 has been reported from humans in the United States and Vietnam (7, 8). Plasmid replicon typing revealed that the mcr-3.10-carrying plasmid in both 17EC and its transconjugant T-17EC belongs to the IncI2 replicon.

Tigecycline, the first member of glycylcycline class antibiotic, is often considered one of the effective antibiotics against multidrug-resistant (MDR) infections. However, the emergence and wide distribution of two novel plasmid-mediated tigecycline resistance genes, tet (X3) and tet (X4), pose a great threat to the clinical use of tigecycline.

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