Identification of a novel MDR plasmid co-harbouring the carbapenem resistance gene bla_VIM-2 and tigecycline resistance gene cluster tmexCD1-toprJ1 in a Pseudomonas stutzeri isolate

Tigecycline and carbapenems are a few of the last-resort antimicrobial agents used to treat serious infections caused by MDR bacteria.^1–3 However, the emergence of mobile tigecycline and carbapenem resistance genes reduces their clinical efficacy.

In September 2021, Pseudomonas stutzeri T75 was isolated from a porcine faecal sample in Liaoning province, China using brain heart infusion (BHI) agar plates supplemented with meropenem (1 mg/L) and tigecycline (4 mg/L). Strain T75 was identified to species level by 16S rRNA sequencing. Broth microdilution tests according to CLSI revealed that P. stutzeri T75 was resistant to meropenem, tigecycline, gentamicin, ciprofloxacin, amoxicillin, ceftazidime and cefepime, but susceptible to colistin. Carbapenemase genes were screened for by a multiplex PCR assay⁴ and the results showed that P. stutzeri T75 was positive for the bla_VIM-2 gene. Of the mobile tigecycline resistance genes, including tet(A), tet(X) variants and tmexCD1-toprJ1,⁵ the tmexCD1-toprJ1 gene cluster was detected.

WGS was performed using both Illumina HiSeq and Oxford Nanopore MinION platforms followed by hybrid assembly using Unicycler v 0.4.3.⁶ The genome was annotated automatically using RAST (https://rast.nmpdr.org/) followed by manual correction. The acquired antimicrobial resistance genes (ARGs) and plasmid replicon types were identified with the CGE services (www.genomicepidemiology.org/services/). The plasmid relaxase typing was performed using MOB-typer software.⁷ Plasmid comparisons were visualized using Easyfig v 2.2.5.⁸

Isolate P. stutzeri T75 consisted of circular chromosomal DNA (4 357 264 bp) and one plasmid (194 547 bp), designated pT75-VIM. The complete genome sequence of P. stutzeri T75 was deposited in the GenBank database under accession numbers CP113226 and CP113227. Plasmid pT75-VIM contained 240 predicted ORFs with an average G + C content of 56.35%. Plasmid pT75-VIM harboured multiple ARGs including tmexCD1-toprJ1, bla_VIM-2, bla_PER-1, dfrB1, qnrVC6, aadA1 and two copies of sul1 as well as two copies of aacA4 (Figure 1a). Sequence analysis showed that an 831 bp repB gene encoding a plasmid replication initiator protein, which belonged to Pfam01051 corresponding to the Rep_3 family, was present on plasmid pT75-VIM. However, plasmid pT75-VIM could not be classified as belonging to any known incompatibility group by PlasmidFinder. MOB-typer analysis revealed that plasmid pT75-VIM was assigned to the relaxase MOB_H family. This suggests that pT75-VIM was a novel replicon type belonging to the MOB_H group. A Blastn search showed that the repB gene of pT75-VIM displayed significant nucleotide sequence identity (>97%) to that of six plasmids in GenBank (three from P. stutzeri and three from Pseudomonas aeruginosa) (Table S1, available as Supplementary data at JAC Online). All six plasmids were also non-typeable by PlasmidFinder and should belong to this novel replicon type similar to plasmid pT75-VIM. Comparative analysis showed that three of the six plasmids shared a conserved plasmid backbone with pT75-VIM, including genes for replication (repB), partition (parB and parA) and transfer (tra genes) (Figure S1). However, the genes involved in transfer were absent in the other three plasmids. According to the information available in GenBank, these novel-type plasmids were isolated from China, USA, Spain, India and South Korea (Table S1), which indicates that they are circulating in different countries and may serve as vehicles for the dissemination of ARGs. To test the transferability of plasmid pT75-VIM, a conjugation assay was performed using P. aeruginosa PAO1 (rifampicin resistant) as the recipient. However, transconjugants carrying plasmid pT75-VIM were not obtained under laboratory conditions despite repeated attempts.

Figure 1.

(a) Circular representation of the plasmid pT75-VIM. The circles show (from outside to inside): predicted coding sequences, GC skew, GC content and scale in kb. ORFs of different functions are presented in various colours. (b) Genetic environment of the bla_VIM-2 gene and the tmexCD1-toprJ1 gene cluster. The positions and transcriptional directions of the ORFs are denoted with arrows. Genes are differentiated by colours. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

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Genetic environment analysis showed that the bla_VIM-2 gene was located in a Tn402-like structure (tniA-tniB-tniQ-tniR-bla_VIM-2-aacA4-dfrB1b-intI1), which was bounded by 25 bp inverted repeats (IR_i and IR_t) (Figure 1b). In addition, 5 bp imperfect direct repeats (CTGGC/CTCGG) were observed immediately upstream and downstream of this Tn402-like structure. Blastn searches indicated that this Tn402-like structure harbouring bla_VIM-2 has previously been detected in other Pseudomonas spp., such as Pseudomonas putida, Pseudomonas monteilii and P. aeruginosa (Figure 1b), which implies that this Tn402-like structure may act as a mobile element for transmission of bla_VIM-2 in Pseudomonas spp. Another important resistance gene cluster tmexCD1-toprJ1 was found in a new genetic structure (ΔISPa86-hp-hp-tmexCD1-toprJ1-hp-hp-intI1-ISPst3) (Figure 1b). Blastn searches showed that no similar genetic structure containing tmexCD1-toprJ1 is currently deposited in the GenBank database. In this novel genetic context for tmexCD1-toprJ1, two different mobile elements were identified in the flanking region of tmexCD1-toprJ1, including ISPst3 and ΔISPa86 (Figure 1b), which might play a role in the translocation and transmission of tmexCD1-toprJ1. To the best of our knowledge, this is the first report of tmexCD1-toprJ1 in P. stutzeri. Combined with previous reports,^9,10 it is further suggested that tmexCD1-toprJ1 probably originated from Pseudomonas spp.

In conclusion, we identified a novel-type plasmid co-harbouring bla_VIM-2 and tmexCD1-toprJ1 in a porcine P. stutzeri. Furthermore, attention should be paid to the potential risks of transfer of the plasmid-borne bla_VIM-2 and tmexCD1-toprJ1 from P. stutzeri to other Pseudomonas spp., such as P. aeruginosa. Therefore, surveillance studies are urgently warranted to investigate the prevalence of this novel plasmid type in clinical isolates.

Acknowledgements

We thank Prof. Dongsheng Zhou (Beijing Institute of Microbiology and Epidemiology) for providing rifampicin-resistant P. aeruginosa PAO1.

Funding

This work was supported by the National Key Research and Development Program of China (grant no. 2022YFF0710505), Central Public-interest Scientific Institution Basal Research Fund (1610302022001) and the German Federal Ministry of Education and Research (BMBF) under project number 01KI2009D as part of the Research Network Zoonotic Infectious Diseases.

Transparency declarations

The authors have no conflict of interest to declare.

Supplementary data

Figure S1 and Table S1 are available as Supplementary data at JAC Online.

References

Author notes