Coexistence of tmexCD3-toprJ1b tigecycline resistance genes with two novel bla VIM-2-carrying and bla OXA-10-carrying transposons in a Pseudomononas asiatica plasmid

Introduction Tigecycline and carbapenems are considered the last line of defense against microbial infections. The co-occurrence of resistance genes conferring resistance to both tigecycline and carbapenems in Pseudomononas asiatica was not investigated. Methods P. asiatica A28 was isolated from hospital sewage. Antibiotic susceptibility testing showed resistance to carbapenem and tigecycline. WGS was performed to analyze the antimicrobial resistance genes and genetic characteristics. Plasmid transfer by conjugation was investigated. Plasmid fitness costs were evaluated in Pseudomonas aeruginosa transconjugants including a Galleria mellonella infection model. Results Meropenem and tigecycline resistant P. asiatica A28 carries a 199, 972 bp long plasmid PLA28.4 which harbors seven resistance genes. Sequence analysis showed that the 7113 bp transposon Tn7389 is made up of a class I integron without a 5’CS terminal and a complete tni module flanked by a pair of 25bp insertion repeats. Additionally, the Tn7493 transposon, 20.24 kp long, with a complete 38-bp Tn1403 IR and an incomplete 30-bp Tn1403 IR, is made up of partial skeleton of Tn1403, a class I integron harboring bla OXA-10, and a Tn5563a transposon. Moreover, one tnfxB3-tmexC3.2-tmexD3b-toprJ1b cluster was found in the plasmid and another one in the the chromosome. Furthermore, plasmid PLA28.4 could be conjugated to P. aeruginosa PAO1, with high fitness cost. Discussion A multidrug-resistant plasmid carrying tmexCD3-toprJ1b and two novel transposons carrying bla VIM-2 and bla OXA-10 -resistant genes was found in hospital sewage, increasing the risk of transmission of antibiotic-resistant genes. These finding highlight the necessary of controlling the development and spread of medication resistance requires continuous monitoring and management of resistant microorganisms in hospital sewage.

The genes encoding resistance determinants such as MBLs are usually found in plasmids or are associated with integrons and transposons (Mann et al., 2022). Integrons are able to capture genes that are part of gene cassettes via a site-specific recombination event and transposons contribute significantly to the transfer and transmission of antibiotic resistance (AR) in bacterial populations (Alavi et al., 2011;Mann et al., 2022). It is commonly believed that the hospital sewage provides a significant platform for the generation of new transposons and many of the novel transposons have been reported from the sewage. Acinetobacter johnsonii M19 isolated from hospital sewage carries a novel transposon Tn6618 containing carbapenem resistant gene bla OXA-23 , while Shewanella xiamenensis T17 carries the novel transposon Tn6297 encoding OXA-416 (Yousfi et al., 2017;Zong et al., 2020).
P. asiatica, a newly proposed unique species of the genus Pseudomonas, belongs to the Pseudomonas putida group, which is a potential human pathogen that can cause nosocomial illness (Tohya et al., 2019a). Moreover, the most prevalent carbapenem resistance gene in the genome of clinical isolates of P. aeruginosa is the bla VIM-2 Metal -b -lactamase (MbL) gene, which is usually present in part of the cassette repertoire of class 1 integrons/ transposons (Botelho et al., 2018). The bla VIM-2 gene has been found in P. asiatica (Brovedan et al., 2021;Tohya et al., 2021), indicating that it is an important reservoir of this gene.
Here, we describe a novel plasmid that co-harbors the tigecycline association resistance gene tmexCD3-toprJ1b, a bla VIM-2 -carrying novel transposon Tn7389, as well as bla OXA-10 -carrying novel transposon Tn7493 from a Pseudomonas asiatica strain.

Bacterial isolation and identification
Wastewater samples were collected from a large tertiary hospital in Luzhou in August 2019. The sewage samples were collected from outflow of the sewage treatment stations of hospital. The samples were collected in sterile glass bottles (200ml) at a set time each time. Sewage samples were mixed and diluted with sterile water in a ratio of 1:10 and subsequently inoculated on a MacConkey agar plate at 37°C for 18-24h in the presence of antibiotics: meropenem (0.5 mg/ L). One strain, named A28, was isolated and purified three times on Luria-Bertani (LB) broth agar medium following the repeated plate streaking method. The species was identified by detecting the 16S rRNA gene with universal primers 27F (5′-AGA GTT TGA TYM TGG CTC AG-3′) and 1492R (5′-GGY TAC CTT GTT ACG ACT T-3′), and further confirmed by WGS analysis (Smyth et al., 2020).

Antimicrobial susceptibility test
The minimal inhibitory concentrations (MICs) of A28 to antimicrobial agents were determined by broth microdilution method according to the recommendations of the CLSI 2021 breakpoints. Escherichia coli strain ATCC 25922 was used as quality control.

Conjugation assay and fitness cost of plasmid carriage
Conjugation assays were carried out using sodium azideresistant E. coli J53, rifampicin-resistant E. coli EC600 (Rif r ), and rifampicin-resistant P. aeruginosa PAO1 as recipients. Transconjugants were selected on LB agar plates containing meropenem (0.5 mg/L) and sodium azide (100 mg/L) or rifampicin (100 mg/L). The donor and recipient strains were mixed in ratios of 1:1, then cultured overnight on LB agar plates at 37°C.The resistance genes of bla VIM-2 in transconjugants were validated by PCR. A growth curve assay was used to calculate the fitness of the plasmid between P. aeruginosa transconjugants and P. aeruginosa PAO1 (Zhang et al., 2022). Overnight cultures were diluted 1:50 in LB without antibiotics and measured at OD 600 every 15 minutes for 11 hours on a Synergy H1 (Labsystems) instrument, with each sample repeated three times. Student's t-test was used for statistical analysis, with a significance threshold of 95% (P< 0.05).

Biofilm formation
The ability of the transconjugant and wild-type strain to generate biofilms was determined using crystal violet staining . The bacterial suspension was discarded and washed three times with sterile water after standing culture at 37°C for 24 hours. Crystal violet was dissolved in a 33% acetic acid solution, and its OD 595 value was determined.

Galleria mellonella killing assay
By using serial dilutions, the transconjugant PAO1-A28 and P. aeruginosa PAO1 were divided into two different amounts of bacterial suspensions ranging from 1×10 5 c.f.u. ml −1 to 1×10 6 c.f.u. ml −1 . Using a microsyringe, 10µl of the prepared bacterial suspensions were injected into the body cavity of G. mellonella through the right hind foot. The control group was injected with 10 µl PBS buffer. Ten G. mellonella were injected with bacteria in each group and placed in a Petri dish at 37°C for 72 hours. At 12-hour intervals, G. mellonella was observed to survive.

Identification of the novel transposon Tn7389
Tn7389 is a new transposon with a 7113 bp backbone and three accessory modules. A complete Tn402-like tni module showed 99.98% nucleotide sequence similarity with the genes for transposase (tniA), transposase helper proteins (tniB, tniQ) and decomposition enzymes (tniC) of Klebsiella aerogenes Tn5090 (Encoding a consistent sequence of corresponding proteins). The 5' CS of Tn7389 is an incomplete class 1 integron that lacks the 3' CS and contains the antibiotic resistance gene cassette(aacA4-bla VIM-2 ) and lacks the 3' CS ( Figure 2). Tn7389 differs from the In1701 gene cassettes found on P. aeruginosa DMC-27B, and their integrase is one base inconsistent (Jahan et al., 2020). Tn7389 has two resistance genes, bla VIM-2 and aacA4, but In1701 only has one carbapenem resistance gene, bla VIM-5 . Tn402-like transposons Tn6635 and Tn6636 harboring bla VIM-2 were also discovered in two P. asiatica strains, and these two transposons carried the same entire Tn402-like tni module, but only the tniA gene had one base mutation (G409A) compared to Tn7389 (Brovedan et al., 2021). Tn7389 has the same structure as the Tn6017 transposon found in P. aeruginosa and P. putida isolated from a Spanish hospital (Juan et al., 2010). However, the similarity of tni modules is only 86.36%. Tn7389 displayed an inconsistent arrangement of resistance genes on the gene cassettes compared to the Tn402-like transposon on the plasmid of P. asiatica LD209 (Marchiaro et al., 2014). Compared to the megaplasmid PZXPA-20-602K, Tn7389's variable region (VR) lacks the dhfrIIc gene, whereas the Tn5090-like transposon of PZXPA-20-602K has a complete type 1 integron 3' CS region with a size of more than 46 kbp .

Identification of the novel transposon Tn7493
The bla OXA-10 gene locates within a compound Tn1403-like transposon of 20.24 kp length, flanked by a complete 38-bp IR of Tn1403 and an incomplete 30-bp IR of Tn1403, and was named Tn7493 (Figure 3). Two cassettes, aacA4-bla OXA-10 , encoding resistance to aminoglycosides and oxacillinase, were found in the class 1 integron. Upstream gene cassettes were 5' CS of intI1 and IRi, flanked by tnpAR and 38-bp IR of transposon Tn1403 (Stokes et al., 2007), and tnpR, 39-bp-long IRs of Tn5563a. Downstream of aacA4bla OXA-10 was sul1-type 3' -CS, orf5-hp, and IRt, almost identical to the transposon Tn6217 reported from P. aeruginosa (Xiong et al., 2013). On the flanks of IRt were two reverse insertion sequences, IS26, with an aph(3')-I gene in the middle. The 3' CS is a truncated transposon Tn5563a that contains a mercury resistance operon (merPTR) (Szuplewska et al., 2014), without the 3' CS of Tn1403 and Tn5393.

FIGURE 3
Genetic environment of the novel Tn1403-like transposon Tn7493 in P. asiatica A28. The extents and directions of genes are shown by arrows labeled with gene names. The construction of sequence comparison was performed using BLAST (http://blast.ncbi.nlm.nih.gov). Genetic environment of the novel Tn402-like transposon Tn7389 in P. asiatica A28. The construction of sequence comparison was performed using BLAST (http://blast.ncbi.nlm.nih.gov). Green arrows, integrases of a class of integrons; Light blue arrow: Tn402 tni module; red arrows, antibiotic resistance genes; purple arrows, Tn5563a-like genes; gray arrows, hypothetical protein.
Conjugation assay, fitness cost, biofilm formation, and G. mellonella killing assay The plasmid PLA28.4 could not be transferred to the recipient cell E. coli J53/C600 by conjugation but could be transferred to P. aeruginosa PAO1. The transfer frequency of PLA28.4 was (2.039±0.077) × 10 -8 per recipient. Consequently, we evaluated the effect of acquiring resistance plasmids on biological fitness and observed significant differences in growth rate related to plasmid acquisition in P. aeruginosa PAO1 from 4h-12h (P <0.0001, Figure 5A). Biofilm formation was significantly reduced in the transconjugant strain (P<0.05) ( Figure 5B). We examined the susceptibility of G. mellonella to the transconjugant PAO1-A28 and P. aeruginosa PAO1, which were injected with 1×10 5 c.f.u. ml −1 to 1×10 6 c.f.u. ml −1 of the strains and incubated in the dark at 37°C for up to 72 h. As shown in Figures 5C, D, compared with PAO1, the transconjugant PAO1-A28 showed significantly reduced virulence against G. mellonella (P<0.05). The decreased virulence of transconjugant to G. mellonella might be due to the adaptive cost of plasmids. The genetic context of the multidrug resistant efflux pump tnfxB3-tmexC3.2-tmexD3b-toprJ1b. The extents and directions of genes are shown by arrows labeled with gene names. Black arrows, tnfxB1-tmexCD1-toprJ1-like gene clusters; pink arrows, int and int-like genes, predicted to encode site-specific integrases; blue arrows, umuC and umuD; green arrows, mobile related genes; red arrows, antibiotic resistance genes; yellow arrows, mercury resistance genes; gray arrows, hypothetical protein. Regions of homology between 96% and 100% are shaded.

Discussion
As an important reservoir of ARB and ARG, hospital sewage is an important medium for ARG to spread to other environments. In this study, a tigecycline and carbapenem-resistant culture obtained from hospital sewage belonged to P. asiatica ST15, which is a newly proposed unique species of the genus Pseudomonas, belongs to the Pseudomonas putida group (Tohya et al., 2020). Sequencing analysis revealed that it coharboring carrying a tmexCD3-toprJ1b, a novel Tn5090-like transposon Tn7389 harboring bla VIM-2 , and a Tn1403-like transposon Tn7493 harboring bla OXA-10 . Tn5090 (also known as Tn402) was discovered on IncP-7 plasmid R751 from K. aerogenes in 1994 (Rådström et al., 1994). In Tn7389, two 25-bp initial reverse repeat (IRi) and terminal reverse repeat (IRt) of Tn5090/Tn402 transposon families were located 171 bp downstream of intI1 and 116bp upstream of tniA, respectively, suggesting that the bla VIM-2 could be mobilized using the tni machinery. The integrase and recombination sites containing class 1 integrons can be inserted and removed in the form of gene cassettes at attI1 (Toleman and Walsh, 2011). Multiple Tn5090-like transposons carrying bla VIM-2 have been found in Pseudomonas in a growing number of investigations, suggesting that Tn5090-like transposons are key mobile components of VIM-2 transmission in Pseudomonas (Santos et al., 2010). The bla VIM-2 gene could be mobile via the tni mechanism, which may promote its transmission among other pathogens in the hospital sewage environment and requires closer monitoring.
Tn1403 was discovered on RPL11 plasmids from clinical P. aeruginosa isolates expressing resistance to ampicillin, streptomycin, puromycin, and chloramphenicol (Veźina and Levesque, 1991). Tn1403-like transposons have been found primarily in Pseudomonas spp. and have been shown to carry diverse types of ARGs, suggesting that they may play an important role in ARG and metal resistance gene transmission in Pseudomonas. In addition, disinfectant-sulfanilamide resistance (qacED1-sul1) genes cause bacterial resistance to chlorinecontaining disinfectants and allows bacteria to survive in disinfected water, which poses a threat to health care systems.
Although there are different variants of the MDR efflux pumps tmexCD1-toprJ1, similar structures have also been found in Aeromonas caviae, Raoultella planticola, and Klebsiella quasipneumoniae, suggesting potential horizontal transfer mechanisms among various species (Wang et al., 2021a;Dong et al., 2022;Gao et al., 2022). The transfer of tnfxB2-tmexCD1-toprJ1 has previously been found to be mobilized by site-specific integrase (Lv et al., 2020). However, it could be linked to umuCD, a neighboring mutant DNA repair system, because integrase can accelerate the excision and integration of umuCD . The proximity of umuCD to the efflux pump structure in various bacteria revealed that it might help spread tmexCD1-TopRJ1-like gene clusters.
The IncP-7 plasmid is a conjugative transfer plasmid with a narrow host range . Although most reports suggest that IncP-7 plasmids could only be transmitted in Pseudomonas (Xiong et al., 2013), pCAR1 was discovered to be transferable to Sterotrophomonas-like strains in natural water . Moreover, the IncP-7 type plasmid pNK546b in P. aeruginosa NK546 also assisted the transmission of another resistant plasmid pNK546a that could not be self-transmissible (Li et al., 2020). In this study, the IncP-7 plasmid PLA28.4 of P. asiatica could be transferred to P. aeruginosa PAO1, suggesting PLA28.4 has the capacity to transmit numerous resistance genes in hospital sewage, according to this study. Collectively, plasmid fitness cost studies found that transferring the PLA28.4 plasmid into P. aeruginosa PAO1 resulted in a lower growth rate, less biofilm generation, and lower pathogenicity, demonstrating that transmission of the PLA28.4 plasmid caused bacteria to pay a cost of adaptation.
We discovered a P. asiatica carrying a plasmid containing the tmexCD1-toprJ1-like gene cluster, and two novel transposons carrying bla VIM-2 and bla OXA-10 , respectively. Controlling the development and spread of medication resistance requires continuous monitoring and management of resistant microorganisms in hospital sewage.

Conclusion
We discovered a P. asiatica carrying a plasmid containing the tmexCD1-toprJ1-like gene cluster, and two novel transposon carrying bla VIM-2 and bla OXA-10 , respectively. Controlling the development and spread of medication resistance requires continuous monitoring and management of resistant microorganisms in hospital sewage.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/ genbank/, CP063456.1.

Author contributions
SL, WW, BYZ and QHL collected the data. FYZ and LG performed the bioinformatic analyses. QL, QC, AM-R wrote the initial draft of the manuscript. QL, RJH, LX and YSZ conceived the project, reviewed the articles and extracted the data. XX contributed to the revision of this article. All authors contributed to the article and approved the submitted version.

Funding
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