Characterization of Three Porcine Acinetobacter towneri Strains Co-Harboring tet(X3) and bla OXA-58

Tigecycline is the antibiotic of last resort for the treatment of extensively drug-resistant bacterial infections, mainly those of multidrug-resistant Gram-negative bacteria. The plasmid-mediated tet(X3) gene has recently been described in various pathogens that are resistant to tigecycline. We report three tigecycline-resistant Acinetobacter towneri strains isolated from porcine faeces in China, which all contained the tet(X3)-harboring plasmids. A broth microdilution method was used to examine the antimicrobial susceptibility of the isolates, and S1-Nuclease digestion pulsed-field gel electrophoresis (S1-PFGE) was used to characterize their plasmid profiles. The whole-genome sequences of the isolates were determined with the Nanopore PromethION platform. The sequence analysis indicated that the strains were A. towneri. They showed resistance to multiple antibiotics, and all the resistance genes were located on plasmids. The three tet(X3)-harboring plasmids had a similar backbone structure, and all contained bla OXA-58 with various insertion elements (IS). ISCR2 is considered an important factor in tet(X3) mobilization. In addition to ISCR2, we demonstrate that IS26 generates a circular intermediate containing the tet(X3) gene, which could increase the dissemination risk. To our knowledge, this is the first report of tet(X3)- and bla OXA-58-harboring plasmids in A. towneri. Because the IS26 is frequently found in front of tet(X3), research should be directed toward the action of IS26 in the spread of tet(X3).

The tet(X3) gene has been identified in Acinetobacter baumannii, A. schindleri, A. indicus, and Empedobacter brevis, but limited information about the mechanisms of tet(X3) transmission in bacteria is available He et al., 2020a). It has been shown that tet(X3) is transferred by plasmids or transposons in a mechanism called 'rolling-circle (RC) transposition' via ISCR2 (He et al., 2019). Moreover, plasmids carrying both tet(X3) and bla NDM-1 were detected in A. indicus in a recent study, but with no information on the plasmid containing tet(X3) together with other carbapenemase genes in A. towneri (He et al., 2020a).
In this study, we isolated plasmids carrying both tet(X3) and bla OXA-58 from A. towneri strains isolated from porcine faeces in China. We describe the whole-genome sequences of the three A. towneri isolates harboring tet(X3) and show that IS26 plays an important role in the spread of tet(X3).

Clinical Isolates and Detection of the tet(X) Genes
One hundred and sixteen anal swabs from pigs were collected on a pig farm in Guangxi Province, southern China, in 2019. The samples were stored at low temperature and transported directly to the laboratory. Three Acinetobacter strains were isolated from three independent anal swabs and cultured on Leeds Acinetobacter Agar containing 4 mg/L tigecycline. These strains were designated GX3, GX5, and GX7. We screened for the tigecycline-resistant genes tet(X3), tet(X4), and tet(X5) with PCR, as described in a previous report (Ji et al., 2020).

Antimicrobial Susceptibility Testing
The broth microdilution method was used to examine the antimicrobial susceptibility (ampicillin, amoxicillin-clavulanate, spectinomycin, tetracycline, florfenicol, sulfisoxazole, sulfamethoxazole, ceftiofur, ceftazidime, colistin, gentamicin, meropenem, and imipenem) of the isolates according to the Clinical Laboratory Standards Institute (CLSI) guidelines (CLSI, 2019), and E. coli ATCC 25922 was used as the quality control. The breakpoint criteria for tigecycline in Acinetobacter spp. was evaluated according to the EUCAST epidemiological cut-off values (https://www.mic.eucast.org/Eucast2/). S1-Nuclease Digestion Pulsed-Field Gel Electrophoresis (S1-PFGE) S1-PFGE was performed to characterize the plasmid profiles in the three strains. Salmonella H9812 was used as the size standard. Briefly, the cultured cells (optical density at 600 nm of 1.0) were harvested and suspended in Tris-EDTA buffer (pH 8.0), and then mixed with 2% gold agarose (SeaKem ® Gold Agarose, Lonza, Atlanta, GA, USA) to make plugs. The plugs were treated with S1 nuclease (TaKaRa, Dalian, China) at 37°C for 15 min, and the DNA fragments were separated with the CHEF Mapper XA system (Bio-rad, USA), as previously described (Barton et al., 1995).

Whole Genome Sequencing
Whole-genome sequencing (WGS) of the three A. towneri isolates was performed with the Nanopore PromethION platform (Biomarker Technologies, Beijing, China). The sequences were assembled with Canu v1.5. Pilon v1.22 was used to improve the draft genome assemblies by correcting bases.

Genome Analysis
The bioinformatics analysis of these sequences was conducted at the CGE server (https://cge.cbs.dtu.dk/services/), including multi-locus sequence typing (MLST) to identify the (STs), and ResFinder to detect drug-resistance genes. The WGS annotations were designed with the Rapid Annotations using Subsystem Technology (RAST) annotation pipeline (version 2.0) (http:// rast.nmpdr.org/). A phylogenetic tree based on plasmid replication initiator protein genes was constructed with the MEGA version 7.0 software using the neighbour-joining method (Kumar et al., 2016). The sequence alignment was generated with Easyfig version 2.1 (Sullivan et al., 2011).

Strain Identification and Antimicrobial Susceptibility Testing
A 16S rDNA PCR detection and sequencing analysis suggested that these strains belonged to the genus Acinetobacter. The rpoB gene was then used to identify the Acinetobacter isolates at the species level. This analysis showed that strain GX3 was identical to GX5, and when compared with Acinetobacter spp. from the GenBank database, it shared a high degree of nucleotide similarity (99.85%) with strain 19110F47 isolated from the faeces of swine in China (CP046045.1). The rpoB gene of strain GX7 shared greatest homology with that of strain 205 (97.21% identical at the nucleotide level) isolated from swine faeces in China (CP048014.1). S1-PFGE was used to identify the plasmids in the isolates, and indicated that the GX5 and GX7 isolates both contained one large plasmid of~180-kb in size. Strain GX3 contained two plasmids, approximately 50-kb and 150-kb in size. Electroporation and conjugation experiments showed that the tet(X3)-harboring plasmids in the three isolates could not transfer to E. coli DH5a, E. coli C600, E. coli 26R 793, or A. baumannii 5AB. This could be related to the host specificity of the plasmids.
All of these strains were resistant to ampicillin, amoxicillinclavulanate, spectinomycin, tetracycline, florfenicol, sulfisoxazole, sulfamethoxazole, ceftiofur, ceftazidime, imipenem, and tigecycline, but were susceptible to gentamicin, meropenem and colistin. The strains also showed different minimal inhibitory concentration to enrofloxacin and ofloxacin ( Table S1). The tet(X3) gene was identified in all three isolates.
We analyzed the three tet(X3)-harboring plasmids from strains GX3, GX5, and GX7. All of them included several classes of resistance genes. As well as tet(X3) encoding tigecycline resistance, they contained bla OXA-58 encoding blactam resistance, sul2 encoding sulphonamide resistance, floR encoding chloramphenicol resistance, aph(3')-Ia encoding aminoglycoside resistance, and mph(E) and msr(E) encoding macrolide resistance. However, dfrA20 encoding trimethoprim resistance was only identified in pGX3 and pGX5 (Table 1). Notably, all of the resistance genes were located on plasmids in these strains ( Figure 1A).
A sequence analysis showed that the plasmids encoded the same replication protein. However, they did not belong to any known replicon type. A structural analysis of the three tet(X3)harboring plasmids showed that they shared a high degree of nucleotide similarity (≥91%), suggesting that they may be derived from the same ancestral MDR plasmid backbone ( Figure 1A).
An analysis of their genetic environments showed that the tet (X3) genes were frequently located between the integrase (xerD) and resolvase genes. This implies that xerD-tet(X3)-resolvase may play an important role in the process of gene transfer. Furthermore, the tet(X3) gene may be transferred by ISCR2, which located downstream of xerD-tet(X3)-resolvase in all the plasmids, except p94-2-tetX3. It is interesting to note that the p34AB and p80-1-2 contain complete copies of ISCR2 on each side of xerD-tet(X3)-resolvase ( Figure 1B). Moreover, a truncated transposase (DISCR2) ( Figure S2) was located upstream from xerD-tet(X3) or IS26-xerD-tet(X3) in all plasmids (except p94-2-tetX3), which implies that the complete ISCR2 may have existed previously. ISCR2 belongs to the IS91 family, which is transposed by an RC transposition mechanism that differs from those of other IS elements (Toleman and Walsh, 2010). It has been demonstrated that a 4609-bp circular intermediate occurs in p34AB (He et al., 2020b). IS91 was previously considered a rare element in the transfer of resistance genes, but it has been increasingly detected in recent studies (Bello-Loṕez et al., 2019).

Analysis of the Genetic Context of bla OXA-58
We also identified the carbapenemase gene, bla OXA-58 , in these isolates. The bla OXA-58 gene was all present in the tet(X3)harboring plasmids, pGX3, pGX5, and pGX7 in our study. Further examination of the bla OXA-58 region revealed that a truncated ISAba3 element was located upstream of bla OXA-58 and a complete ISAba3 element on downstream in all plasmids ( Figure 2C). This is consistent with a previous report that the bla OXA-58 gene was transferred by ISAba3 (Matos et al., 2019). It is notable that the isolates were resistant to imipenem (MIC, 8-16 mg/L), but they were susceptible to meropenem (MIC, 0.12-2 mg/L) ( Table S1). It may be due to bla OXA-58 hydrolyzes carbapenems weakly and often expresses poorly. Moreover, the insertion of an upstream ISAba3 can enhance expression of the bla OXA-58 , but it is truncated in these strains (Hamidian and Nigro, 2019). Overall, our findings support the notion that the co-occurrence of bla OXA-58 and tet(X3) strengthens the risk of the dissemination of these plasmids.

CONCLUSION
To the best of our knowledge, this is the first report of the cooccurrence of tet(X3) and bla OXA-58 in plasmids identified from A. towneri. What is remarkable here is that, although A. towneri is an environmental bacterium, it was detected in the faeces of swine. pGX3 has a novel plasmid backbone containing MDR genes, which are similar to the backbone structures of pGX5 and pGX7, suggesting that they may be derived from the same ancestral MDR plasmid backbone. Like ISCR2, IS26 has been proved that it can generate a circular intermediate to transfer tet (X3), providing antibiotic resistance genes with a highly mobile genetic vehicle, although the mechanisms involved require further research.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories.

AUTHOR CONTRIBUTIONS
JM, JW, and ZY conceived and designed the study. JF, YL, BY, and XW acquired the data. JM, RL, LB, and TH drafted the manuscript. JW critically revised the manuscript. All authors contributed to the article and approved the submitted version. . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.