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. 2025 Jul 2:15:1594982.
doi: 10.3389/fcimb.2025.1594982. eCollection 2025.

A strain defined as a novel species in the Acinetobacter genus co-harboring chromosomal associated tet(X3) and plasmid associated bla NDM-1 from a beef cattle farm in Hebei, China

Qing Wang  1   2   3   4 , Yanming Wei  2 , Muhammad Shoaib  1   3   4 , Yanhua Qiu  1   3   4 , Chao Zhang  1   2   3   4 , Guonian Dai  1   2   3   4 , Honglin Lin  1   2   3   4 , Weiwei Wang  1   3   4 , Jiyu Zhang  1   2   3   4
Affiliations

A strain defined as a novel species in the Acinetobacter genus co-harboring chromosomal associated tet(X3) and plasmid associated bla NDM-1 from a beef cattle farm in Hebei, China

Qing Wang et al. Front Cell Infect Microbiol. .

Abstract

Introduction: The co-existence phenomenon of antibiotic resistance genes (ARGs), particularly of last-resort antibiotics in multi-drug resistant (MDR) bacteria, is of particular concern in the least studied bacterial species.

Methods: In 2023, strain M2 was isolated from the sludge sample at a commercial bovine farm in Hebei province, China, using a MacConkey plate containing meropenem. PCR amplification and Sanger sequencing verified it co-carrying bla NDM and tet(X) genes. It was classified within the Acinetobacter genus by MALDI-TOF-MS and 16S rDNA analyses. Whole-genome sequencing (WGS) was performed on the Oxford Nanopore platform, with species-level identification via ANI and dDDH. Antimicrobial susceptibility testing was performed against 20 antibiotics. Conjugation assays employed the filter-mating method using E. coli J53 and Salmonella LGJ2 as recipients.

Results: This strain was confirmed as a novel species of Acinetobacter genus, showing resistance to meropenem, ampicillin, ceftazidime, cefepime, gentamicin, kanamycin, fosfomycin, imipenem, ertapenem, and tetracycline. Despite carrying tet(X3), it remained susceptible to tigecycline, omadacycline, and doxycycline. The genome carried 11 ARG types, multiple metal resistance genes (MRGs), and virulence factor (VF) genes. The bla NDM-1 was located in a skeleton, ISAba125-bla NDM-1-ble MBL-trpF, which was carried by an ISAba14-mediated rolling-circle-like structure in pM2-2-NDM-1 (rep_cluster_481). Integrative and conjugative element (ICE) and multiple pdif modules (driven by the XerCD site-specific recombination (XerCD SSR) system), which were associated with the mobilization of resistance determinants, were identified in this plasmid. Chromosomal tet(X3) was mediated by ISVsa3, forming a skeleton, ISVsa3-XerD-tet (X3)-res-ISVsa3.

Discussion: The co-occurrence of bla NDM and tet(X) in a novel species of the Acinetobacter genus hints that substantial undiscovered bacteria co-carrying high-risk ARGs are concealing in the agroecological system, which should cause particular concern.

Keywords: Acinetobacter; antibiotic resistance genes (ARGs); blaNDM; multi-drug resistance; pdif module; tet(X).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Structural features of the elements in the chromosome and plasmids. This figure shows the backgrounds of bla NDM and tet(X), ARGs, MRGs, genes encoding VFs, putative ICE region (oriT, T4SS, T4CP, relaxase), and pdif modules. In pM2-2-NDM-1, the red lines mark the C/D and D/C of the pdif modules.
Figure 2
Figure 2
Comparison analysis of the pdif module island. The C/D and D/C sites of each pdif module are shown in Table 3 .
Figure 3
Figure 3
Plasmid analysis. (A): Circular comparison between pM2-2-NDM-1 and the similar plasmids from the NCBI database. (B): Comparison analysis of the bla NDM-1 skeletons from pM2-2-NDM-1 and pDT01139C.
Figure 4
Figure 4
Phylogenetic tree. Bootstrap values were shown on the branch of this tree as circles. Branch lengths were displayed as numbers on each branch of this tree.
Figure 5
Figure 5
Comparison analysis. (A-1): The comparison analysis between the rolling-circle-like structure and the similar segments of plasmids from the NCBI database. (A-2): The comparison analysis between the rolling-circle-like structure and Tn7382. (B): The comparison analysis of the tet(X3) skeleton.
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