Characterization of IncHI1B Plasmids Encoding Efflux Pump TmexCD2-ToprJ2 in Carbapenem-Resistant Klebsiella variicola, Klebsiella quasipneumoniae, and Klebsiella michiganensis Strains

Tigecycline serves as one of the last-resort antibiotics to treat severe infections caused by carbapenem-resistant Enterobacterales. Recently, a novel plasmid-mediated resistance-nodulation-division (RND)-type efflux pump gene cluster, TmexCD1-ToprJ1, and its variants, TmexCD2-ToprJ2 and TmexCD3-ToprJ3, encoding tetracyclines and tigecycline resistance, were revealed. In this study, we reported three TmexCD2-ToprJ2-harboring Klebsiella species strains, collected from two teaching tertiary hospitals in China, including one K. quasipneumoniae, one K. variicola, and one K. michiganensis. The three strains were characterized by antimicrobial susceptibility testing (AST), conjugation assay, WGS, and bioinformatics analysis. AST showed that K. variicola and K. quasipneumoniae strains were resistant to tigecycline with MIC values of 4μg/ml, whereas the K. michiganensis was susceptible to tigecycline with an MIC value of 1μg/ml. The TmexCD2-ToprJ2 clusters were located on three similar IncHI1B plasmids, of which two co-harbored the metallo-β-lactamase gene blaNDM-1. Conjugation experiments showed that all three plasmids were capable of self-transfer via conjugation. Our results showed, for the first time, that this novel plasmid-mediated tigecycline resistance mechanism TmexCD2-ToprJ2 has spread into different Klebsiella species, and clinical susceptibility testing may fail to detect. The co-occurrence of blaNDM-1 and TmexCD2-ToprJ2 in the same plasmid is of particular public health concern as the convergence of “mosaic” plasmids can confer both tigecycline and carbapenem resistance. Its further spread into other clinical high-risk Klebsiella clones will likely exacerbate the antimicrobial resistance crisis. A close monitoring of the dissemination of TmexCD-ToprJ encoding resistance should be considered.

Tigecycline serves as one of the last-resort antibiotics to treat severe infections caused by carbapenem-resistant Enterobacterales. Recently, a novel plasmid-mediated resistancenodulation-division (RND)-type efflux pump gene cluster, TmexCD1-ToprJ1, and its variants, TmexCD2-ToprJ2 and TmexCD3-ToprJ3, encoding tetracyclines and tigecycline resistance, were revealed. In this study, we reported three TmexCD2-ToprJ2-harboring Klebsiella species strains, collected from two teaching tertiary hospitals in China, including one K. quasipneumoniae, one K. variicola, and one K. michiganensis. The three strains were characterized by antimicrobial susceptibility testing (AST), conjugation assay, WGS, and bioinformatics analysis. AST showed that K. variicola and K. quasipneumoniae strains were resistant to tigecycline with MIC values of 4 μg/ml, whereas the K. michiganensis was susceptible to tigecycline with an MIC value of 1 μg/ml. The TmexCD2-ToprJ2 clusters were located on three similar IncHI1B plasmids, of which two co-harbored the metallo-β-lactamase gene bla  . Conjugation experiments showed that all three plasmids were capable of self-transfer via conjugation. Our results showed, for the first time, that this novel plasmid-mediated tigecycline resistance mechanism TmexCD2-ToprJ2 has spread into different Klebsiella species, and clinical susceptibility testing may fail to detect. The co-occurrence of bla NDM-1 and TmexCD2-ToprJ2 in the same plasmid is of particular public health concern as the convergence of "mosaic" plasmids can confer both tigecycline and carbapenem resistance. Its further spread into other clinical high-risk Klebsiella clones will likely exacerbate the antimicrobial resistance crisis. A close monitoring of the dissemination of TmexCD-ToprJ encoding resistance should be considered.

INTRODUCTION
Tigecycline is one of the last-resort antibiotics used to treat severe infections caused by carbapenem-resistant Enterobacterales (Cheng et al., 2020). However, increasing studies reported the emergence of tigecycline resistance in clinical settings, which is frequently caused by the overexpression of non-specific active efflux pumps [tet(A) and tet(K)] or mutations within the drugbinding site in the ribosome [tet(M); Grossman, 2016;Linkevicius et al., 2016]. In Klebsiella pneumoniae, tigecycline resistance is also frequently associated with the overexpression of ramA, which can directly regulate multidrug resistance efflux pumps AcrAB and OqxAB (Ruzin et al., 2005(Ruzin et al., , 2008. These tigecycline resistances are primarily mediated by chromosome-encoding mechanisms thus could not be easily transferred horizontally. The newly emerging mobile tigecycline resistance mechanism is of particular public health concern (He et al., 2019;Lv et al., 2020). The enzymatic modification gene variants [tet(X)] are highly transferable between species (Linkevicius et al., 2016;He et al., 2019;Sun et al., 2019).
Recently, a novel plasmid-mediated resistance-nodulationdivision (RND)-type efflux pump gene cluster, TmexCD1-ToprJ1, has been identified in K. pneumoniae . This gene cluster was first identified in animal isolates but soon later was also found in clinical isolates (Sun et al., 2020). TmexCD1-ToprJ1 was initially reported in China, but it has now been found in clinical K. pneumoniae isolates outside of China (Hirabayashi et al., 2021), suggesting this resistance has started to spread into other global regions. Two homologous variants, TmexCD2-ToprJ2 (Wang et al., 2021a) and TmexCD3-ToprJ3 (Wang et al., 2021b), have also been identified in Raoultella ornithinolytica and Proteus mirabilis, displaying similar tigecycline resistance profiles. Worrisomely, the mobile tigecycline gene clusters have also been found in clinical carbapenemresistant K. pneumoniae strains (Chiu et al., 2017). Here, we reported the identifications of three clinical TmexCD2-ToprJ2-encoding Klebsiella strains, including two carbapenemresistant strains co-harboring bla VIM-8/NDM-5 or bla NDM-1 .

Bacterial Strains
Klebsiella variicola strain JNQH579 was recovered from a sputum sample of a 64-year male patient in intensive care units (ICUs) at a tertiary hospital in Jinan City, Shandong Province, in March 2021, who had been hospitalized for 17 days due to severe pneumoniae and renal failure. The patient had received tigecycline treatment for 7 days at a dosage of 100 mg (IV) q12h before the isolation of the strain. Based on the antibiotic susceptibility testing results, antibiotic therapy was switched to aztreonam 0.5 g (IV) q8h in combination with tigecycline. The respiratory symptoms improved after antibiotic treatment and the patient continued to be hospitalized for 3 months due to cardiovascular and renal disease. Klebsiella quasipneumoniae strain JNQH473 was recovered from a urine sample of a 1-month-old infant with sepsis at a tertiary hospital in Xiamen City, Fujian Province, in August 2019, who was hospitalized at the department of pediatrics for 7 days. During her hospitalization, the neonate received multiple antimicrobial treatments, including cefotaxime, vancomycin, and ceftazidime. The patient was fully recovered after antimicrobial and supportive treatment and was discharged on hospital day 13. Klebsiella michiganensis strain JNQH491 was recovered from a blood culture of a 48-year male patient at the department of oncology, who had received chemotherapy due to nasopharyngeal carcinoma in the same hospital as JNQH473. The patient was discharged home after a cycle of chemotherapy on hospital day 11. The overall strain features of the three strains are listed in Table 1. All the patients reported no recent travel abroad.

Antibiotic Susceptibility Testing
Antibiotic susceptibility testing (AST) was performed using the VITEK 2 (bioMérieux, Nürtingen, Germany) system. Minimum inhibitory concentrations (MICs) for tigecycline were performed by broth microdilution according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. ATCC 25922 (Escherichia coli) and ATCC 27853 (Pseudomonas aeruginosa) were used as quality control strains for susceptibility testing. All the tests were performed in duplicate in different days. The breakpoints were interpreted according to CLSI guidelines except for tigecycline, of which the EUCAST epidemiological cutoff value >2 μg/ml (for K. pneumoniae) was used. 1

Genomic Analysis
The in silico multilocus sequence typing (MLST) was carried out using MLST v. 2.19.0, 3 and antibiotic resistance/plasmid replicon gene detections were carried out using ABRicate v. 0.9.9 4 using CARD (Jia et al., 2017) and PlasmidFinder (Carattoli et al., 2014) database, respectively. The intrinsic variants of oqxAB, chromosomal ampH and fosA, which confer resistance to quinolones, β-lactams and fosfomycin, respectively, in Enterobacterales rather than Klebsiella species, are therefore not reported (Henderson et al., 1997;Lam et al., 2021). Kleborate v. 1.0.0 (Lam et al., 2021) was used for Klebsiella K locus and O locus typing. The comparative analysis of TmexCD2-ToprJ2 harboring plasmids was done by BLASTn and illustrated using CGView Server (Grant and Stothard, 2008). Easyfig (Sullivan et al., 2011) was used to visualize the genetic context comparisons. Selected plasmids were compared with Mauve 2.3.1 (Darling et al., 2010), followed by visualization using genoPlotR (Guy et al., 2010). Plasmid distance trees were generated using Mashtree (Katz et al., 2019). Alignment between three TmexCD-ToprJ variants was done by Clustal Omega (Madeira et al., 2019). ISFinder 5 was used to identify ISs. Tn number was identified using Tn Number Registry (Roberts et al., 2008). In order to examine the distribution and relationships of TmexCD2-ToprJ2 and its variants (TmexCD1-ToprJ1, TmexCD2-ToprJ2), plasmid sequences were downloaded from NCBI 6 and compared.

Conjugation Experiment
Conjugation experiments were performed by plate mating using E. coli J53 (AziR) as the recipient as described in our previous study (Hao et al., 2021). Briefly, overnight cultures of the donor strains and the recipient strain E. coli J53 were mixed (1:1) and applied onto an LB agar plate, followed by overnight culture at 37°C. Transconjugants were selected on Mueller-Hinton (MH) agar containing sodium azide (200 μg/ml) and tigecycline (0.5 μg/ml).

Susceptibility of Three Klebsiella Isolates
The MICs for JNQH473, JNQH491, and JNQH579 were shown in Table 2. The MICs of tigecycline in JNQH473 and JNQH579 were both 4 μg/ml. However, JNQH491 was susceptible to tigecycline with a MIC value of 1 μg/ml. These strains were resistant to almost all tested β-lactam antibiotics except that JNQH491 and JNQH579 were susceptible to aztreonam. All the strains were susceptible to amikacin with an MIC value of less than 2 μg/ml.

Transfer of TmexCD2-ToprJ2 Harboring Plasmids via Conjugation
The TmexCD2-ToprJ2 harboring IncHI1B plasmids were successfully transferred into E. coli J53 from three JNQH strains. Further PCRs confirmed that other plasmids in the donor strains were not co-transferred along with the TmexCD2-ToprJ2 harboring IncHI1B plasmids into the recipient E. coli J53 strain (Supplementary Figure S2). The conjugation frequency was 10 −6 , 10 −6 , and 10 −5 for JNQH473, JNQH491, and JNQH579 per recipient cell, respectively. The MICs for the E.coli J53 transconjugants are shown in Table 2. The MICs of tigecycline against E. coli J53 transconjugants were 1 μg/ml, which were 8-fold higher than that of the E. coli J53 itself. In addition, the transfer of bla NDM-1 together with the TmexCD2-ToprJ2 genes for JNQH491 and JNQH579 in the E. coli J53 transconjugants conferred resistance to all tested β-lactams (ampicillin, amoxicillin-clavulanate, piperacillin/tazobactam, cefoxitin, ceftriaxone, cefepime, imipenem, and ertapenem) except for aztreonam. All transconjugants exhibited resistance to quinolones (ciprofloxacin and levofloxacin, ≥4 μg/ml), which was likely due to the combinational effects of both quinolone resistance determinant qnrS1 and the TmexCD2-ToprJ2. Further, E.coli J53 transconjugants for JNQH491 and JNQH579 exhibited resistance to gentamycin, while it was susceptible for JNQH473. Aac(3)-IId is likely the main source of the disparity on the basis that aac(3)-IId was co-harbored with TmexCD2-ToprJ2 in the same plasmids for JNQH473 and JNQH491 strains, while it was absent on the pJNQH579-2 plasmid.

DISCUSSION
TmexCD1-ToprJ1 is the first reported plasmid-encoded RND efflux pump, conferring resistance to multiple drugs including tigecycline . More recently, a variant of TmexCD2-ToprJ2 was identified in a clinical R. ornithinolytica strain NC189, which demonstrated similar tigecycline resistance as the TmexCD1-ToprJ1 (Wang et al., 2021a). In comparison with TmexCD1-ToprJ1, most of which were detected in K. pneumoniae (Figure 2), TmexCD2-ToprJ2 was identified among a variety of Enterobacteriaceae species, including R. ornithinolytica, C. freundii, Aeromonas hydrophila, and K. pneumoniae (Wang et al., 2021a). Our study demonstrated that the TmexCD2-ToprJ2 had spread into carbapenem-resistant K. pneumoniae species complex (KpSC), which is an emerging pathogenic species and frequently detected clinically (Wyres et al., 2020). The highly conserved synteny and structure of the TmexCD2-ToprJ2 harboring plasmids suggested the likelihood of horizontal transfer of a highly similar plasmid between different Klebsiella species. We also found the co-existence of TmexCD2-ToprJ2 and bla NDM-1 genes in pJNQH491-2 and pJNQH579-2, confirmed by the resistance profile of the transconjugants. The co-transfer of bla NDM-1 with TmexCD2-ToprJ2 for JNQH491 and JNQH579 in the E. coli  ,aadA16,qnrS1,mphE,msrE,catB3,catII.2,sul1,dfrA27,bla OXA-1 ,bla NDM-1 J53 conjugants conferred resistance to tigecycline and all tested β-lactams except aztreonam. In addition, analysis of plasmids from the database from NCBI revealed frequent co-occurrence of TmexCD2-ToprJ2 and carbapenem-resistant genes in the same plasmid. Our finding is of particular public health concern as the convergence of "mosaic" plasmids can confer both tigecycline and carbapenem resistance, thus leading to a serious challenge to the treatment of bacterial infections. It has been demonstrated in a previous study that TmexCD2-ToprJ2 functions as an efflux pump system by the efflux inhibition experiments (Wang et al., 2021a). TmexCD2-ToprJ2 exhibits a broad substrate spectrum toward tetracyclines, eravacycline, tigecycline (8-fold MIC increase), ciprofloxacin (4-fold MIC increase), and slightly decreased susceptibility (2-fold MIC increase) to cefotaxime and cefepime (Wang et al., 2021a). Our finding is consistent with previous study that TmexCD2-ToprJ2 gene cluster caused 8-fold increase in the tigecycline MICs in the E. coli transconjugants (Wang et al., 2021a). However, strain K. michiganensis JNQH491 was susceptible to tigecycline with a MIC value of 1 μg/ml irrespective of the presence of TmexCD2-ToprJ2 gene. We speculated that the differences in plasmid copy numbers or transcription of promoter sequence in this K. michiganensis strain might contribute to the different susceptibility profiles observed in strains from this study (Shaheen et al., 2011). Further studies are needed to evaluate how much TmexCD2-ToprJ2 will affect the therapeutic FIGURE 1 | Comparative structural analysis of pJNQH473-3, pJNQH491-2, pJNQH579-2, and two TmexCD2-ToprJ2 harboring plasmids NZ_CP063149.1 and NZ_CP063748.1. Open reading frames (ORFs) of pJNQH579-2 are shown as the outermost ring, with plasmid replicons, insertion sequences (IS), and antimicrobial resistance genes highlighted. effects of tigecycline in vivo, including clinical isolates with low MIC values.
An increasing number of studies have supported that the TmexCD-ToprJ-like efflux pump system may originate from the chromosome of Pseudomonas species, as their structures are closely related to the chromosomal MexCD-OprJ system in P. aeruginosa Sun et al., 2020;Wang et al., 2021a,b). These findings suggested TmexCD-ToprJ-like clusters FIGURE 2 | Linear comparisons of TmexCD2-ToprJ2-bearing genetic contexts in pJNQH473-3 and that in pJNQH579-2, pC2315-2-NDM, and plasmid pKOX_ R1(NC018107.1). Light blue shading indicates shared regions of homology, while green shading indicates inversely displayed regions of homology. Colored arrows indicate ORFs. The red arrows indicate the antibiotic resistance genes. The yellow arrows indicate IS. The nucleotide sequence of the umuC gene (green arrow) representing the insertion site is shown above the gene. The 16,846 bp putative "hp1-hp2-tnpA-tnfxB2-IS881-TmexCD2-ToprJ2" transposon located on pJNQH473-3 and 34,827 bp larger putative transposon on pJNQH579-2 are marked with bilateral black arrows.
Frontiers in Microbiology | www.frontiersin.org 8 October 2021 | Volume 12 | Article 759208 might be originated from chromosomal genes in Pseudomonas species, through horizontal transfer into Enterobacteriaceae species. The MexCD-OprJ family proteins act as efflux pumps, conferring intrinsic resistance to tetracycline, chloramphenicol, and norfloxacin in P. aeruginosa (Poole et al., 1993). The rapid expansion of the TmexCD-ToprJ cluster has been attributed to various mobile genetic elements, such as ICEs, transposons (e.g., Tn5393), or IS element (e.g., IS26; Sun et al., 2020;Wan et al., 2020;Wang et al., 2021b;Yang et al., 2021). In our study, a genetic structure (hp1-hp2-tnpA-tnfxB2-IS881-TmexCD2-ToprJ2) constitutes a putative transposon system. Further, a larger putative transposon comprised of 34,827 bp harboring TmexCD2-ToprJ2 was also inserted into the umuC-like gene of pJNQH579-2. As such, the umuC gene appears to be a "hotspot" for TmexCD2-ToprJ2 integration in IncHIB plasmids, while the molecular mechanism underlying the site-specific integration deserves further studies.

CONCLUSION
Overall, we report the first time of three IncHI1B type plasmids encoding efflux pump TmexCD2-ToprJ2 in carbapenem-resistant K. variicola, K. michiganensis, and K. quasipneumoniae species. The sequence analysis identified a putative transposon element for TmexCD2-ToprJ2 transmission, with the genetic structure of "hp1-hp2-tnpA-tnfxB2-IS881-TmexCD2-ToprJ2." Of note, the co-existence of bla NDM-1 and TmexCD2-ToprJ2 is of particular public health concern. The emergence of such Klebsiella strains underscores FIGURE 3 | Comparison of linear plasmid maps of four TmexCD2-ToprJ2-bearing plasmids and the related plasmid pKOX-R1. The genomes were compared with Mauve and the elements were designated as Mauve blocks. Red shaded regions between plasmids indicates shared regions of homology, while blue shading indicates inversely displayed regions of homology. The tree was created using Mashtree.
the importance of clinical awareness of this pathotype and the need for continued monitoring of TmexCD-ToprJ family resistance genes in China and around the world.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the First Affiliated Hospital of Shandong First Medical University. Written informed consent to participate in this study was provided by the participants' legal guardian/ next of kin. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
MH and WM conceived the study and designed the experimental procedures. BZ and XD collected the strains. YW, BZ, ML, JM, XL, and FC performed the experiments. MH, JG, and SL analyzed the data. WM, YH, and YW contributed to reagents and materials. MH and LC wrote the manuscript. All authors contributed to the article and approved the submitted version.