Identification of Novel tet(X3) Variants Resistant To Tigecycline in Acinetobacter Species

Abstract

The emergence of the tet(X) gene is a severe challenge to global public health security, as clinical tigecycline resistance shows a rapidly rising trend. In this research, we identified two tigecycline-resistant Acinetobacter sp. strains containing seven novel tet(X3) variants recovered from fecal samples from Chinese farms. The seven Tet(X3) variants showed 15.4% to 99.7% amino acid identity with Tet(X3). By expressing tet(X3.7) and tet(X3.9), the tigecycline MIC values for Escherichia coli JM109 increased 64-fold (from 0.13 to 8 mg/L). However, the other tet(X3) variants did not have a significant change in the MIC of tigecycline. We found that the 26th amino acid site of Tet(X3.7) changed from proline to serine, and the 25th amino acid site of Tet(X3.9) changed from glycine to alanine, which reduced the MIC of tigecycline by 2-fold [the MIC of tet(X3) to tigecycline was 16 mg/L] but did not affect its expression to tigecycline. The tet(X3) variants surrounded by mobile genetic elements appeared in the structure of gene clusters with tandem repeat sequences and were adjacent to the site-specific recombinase-encoding gene xerD. Therefore, there is a risk of horizontal transfer of resistant genes. Our study reports seven novel tet(X3) variants; the continuing emergence of tigecycline variants makes continuous monitoring of resistance to tigecycline even more critical. IMPORTANCE Although it is illegal to use tigecycline and carbapenems to treat bacterial infections in animals, we can still isolate bacteria containing both mobile resistance genes from animals, and tet(X) is currently an essential factor in degrading tigecycline. Here, we characterized two multidrug-resistant Acinetobacter sp. strains that contained vital resistance genes, such as sul2, a bla_OXA-164-like gene, floR, tetM, and multiple novel tet(X3) variants with different tandem structures. It is of paramount significance that their mechanism may transfer to other Gram-negative pathogens, even if their tandem structures have no cumulative effect on tigecycline resistance.

Keywords: Acinetobacter schindleri; Acinetobacter variabilis; tet(X); tet(X3); tigecycline resistance.

FIG 1

Genomic island structure of the BDT2044 chromosome and minicircle plasmid of JAT2044. Positions and transcriptional directions of the predicted open reading frames (ORFs) are denoted with arrows. Genes associated with antimicrobial resistance, mobile elements, resolvase, recombinase, and the transposon are highlighted in red, green, yellow, black, and blue, respectively. The repeat sequence was the ISVsa3-ΔISCR2-res-tet(X3)-xerD gene cassette.

FIG 2

Genetic structure in this study and comparison with similar regions in sequences deposited at GenBank. (a) Genetic structure of tet(X3) in this research compared with similar regions in sequences deposited under GenBank accession numbers CP060813, CP044446, CP084302, and MK134375. (b) Genetic structure of plasmid-borne tandem repeats in pBDT2091-4 was compared with similar regions in CP046044, CP059348, and CP063483. Regions of homology up to 100% are indicated by gray shading. Positions and transcriptional directions of the predicted ORFs are denoted by arrows. Genes associated with antimicrobial resistance, mobile elements, resolvase, recombinase, and transposon are highlighted in red, green, yellow, black, and blue, respectively.

FIG 3

Structure of plasmid pBDT2091-4. (Inner to outer circles) GC skew and GC content are indicated. Positions and transcriptional directions of the predicted ORFs are denoted with arrows. Genes associated with antimicrobial resistance, mobile elements, resolvase, recombinase, and transposons are highlighted in red, green, yellow, black, and blue, respectively. Four repeated sequences are labeled Tandem 1 to 4. The repeat sequence is the structure ISVsa3-ΔISCR2-res-tet(X3)-xerD-IS26-aph(3′)-Ia-IS26-aac(3)-IV-aph(4)-Ia-ISEc59-Tn5393-ISVsa3-floR.

FIG 4

BLAST alignments of amino acid sequences of eight Tet(X3) variants, constructed using ClustalW version 2.1 and ESPript version 3.0 (https://www.genome.jp/tools-bin/clustalw and http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The percentages represent the amino acid identity of the novel Tet(X3) variants to the previously reported Tet(X3). The structure found under PDB accession number 4A6N served as the reference for secondary structure depiction.

FIG 5

Phylogenetic analysis of amino acid sequences of different Tet(X) variants. Phylogenetic analysis of the amino acid sequences of all Tet(X) variants was conducted using the neighbor-joining method, using MEGA version 11.0.11 with default parameters and 500 bootstraps. The GenBank accession numbers of the Tet(X) variants are listed [those of the seven novel Tet(X3) variants in this study are shown in red].

FIG 6

Genetic environment of tet(X3) in plasmid pJAT2091 and comparison with the tet(X3)-carrying regions in pBDT2091-4. Regions of homology from 80% to 100% are marked by gray shading. The positions and transcriptional directions of the predicted ORFs are denoted with arrows. Genes associated with antimicrobial resistance, mobile elements, resolvase, recombinase, and other hypothetical proteins are highlighted in red, green, yellow, black, and azure, respectively.