Occurrence and characterization of plasmids carrying tmexCD1-toprJ1, bla DHA-1, and bla CTX-M-127, in clinical Klebsiella pneumoniae strains

Objective Today, the emergence of Klebsiella pneumoniae with the tmexCD1-toprJ1 gene cassette in patients has presented a significant clinical challenge. Methods To present the detailed genetic features of the tmexCD1-toprJ1 gene cassette of K. pneumoniae strain F4_plasmid pA, the whole bacterial genome was sequenced by Illumina and nanopore platforms, and mobile genetic elements related to antibiotic resistance genes were analyzed with a series of bioinformatics methods. Results K. pneumoniae strain F4 was determined to be a class A+C beta-lactamase, and was resistant to routinely used antibiotics, especially tigecycline, because of the oqxAB gene localized on the F4_chromosome and tmexCD1-toprJ1 on F4_plasmid A. After plasmid transfer assays, the F4_plasmid pA or F4_plasmid pB could be recovered with an average conjugation frequencies of 3.42×10-4 or 4.19×10-4. F4_plasmid pA carried tmexCD1-toprJ1 and bla DHA-1 accompanied by genetic intermixing of TnAs1, Tn5393, TnAs3, and In641, while F4_plasmid pB, bearing bla CTX-M-174, had structural overlap of TnAs3 and In641. Conclusions We suggested that plasmids carrying tmexCD1- toprJ1 might be strongly related to IS26-integrated loop intermediates. This study showed that due to the structural evolution of F4 and related strains, their resistances were so strong that effective antibiotics were virtually unavailable, therefore their spread and prevalence should be strictly controlled.


Introduction
In recent years, a novel plasmid-mediated resistance-noduledivision (RND) efflux pump, tmexCD1-toprJ1, has emerged and been widely characterized from patient, animal, and food samples of Klebsiella pneumoniae (Lv et al., 2020).This novel multidrug resistance plasmid gene cluster, tmexCD1-toprJ1, was first reported in 2020 from animal-derived K. pneumoniae in China, exhibiting resistance or reduced susceptibility to several classes of antibiotics, including cephalosporins, phenolics, quinolones, and tetracyclines, and conferring resistance to the last-line antibiotics tigecycline and eravacycline (Lv et al., 2020;Wang CZ et al., 2021;Ghaheh et al., 2022).
The chromosomal or plasmid-encoded RND family tmexCD1-toprJ1 expresses multidrug resistance (MDR) in Gram-negative bacteria (Li et al., 2015;Du et al., 2018), usually requiring the action of three gene products to be effective.Homologous transfer of the entire gene cluster encoding the RND-type tripartite drug efflux pump from chromosome to plasmid has been rarely reported thus far (Tauch et al., 2003;Li et al., 2015).The tripartite efflux system formed by the RND pump can directly export antibiotics outside the cell (Li et al., 2015), but only when regulatory genes (such as tnfxB, araC, or tetR) are efficiently expressed intracellularly does this lead to MDR in most clinically pathogenic bacteria carrying tmexCD1-toprJ1 (Salehi et al., 2021).
Transposons could carry multiple drug-resistant genes in different plasmids, especially, Tn3 played a crucial role in the evolution of drug-resistant plasmids in Enterobacteriaceae (Algarni et al., 2023).And Integrons could be integrated into antibiotic resistance gene cassettes with multiple IS elements or with transposons to form complex structures containing multiple resistance genes (Algarni et al., 2023), that contributed to the widespread spread of drug-resistant genes among bacteria (Algarni et al., 2022).
In this study, we compared and analyzed the MDR region of the F4 plasmids bearing tmexCD1-toprJ1, bla DHA-1 , and bla CTX-M-127 accompanied by intermingling of In641 or In553 with TnAs1, Tn5393, and/or TnAs3 with those of related plasmids, characterized the structure of the plasmids.

Materials and methods
Bacterial strains and sequencing of the 16S rRNA gene K. pneumoniae strain F4 was isolated from a sputum sample of a patient in Taizhou Municipal Hospital affiliated with Taizhou University in 2019.EC600 and Escherichia coli DH5a (TaKaRa, Dalian, China) were employed as hosts for cloning.In order to verify the strain F4 belonged as K. pneumoniae, the nearly complete 16S rRNA gene of the strain was amplified by PCR using the following primers: 5'-AGAGTTTGATYMTGGCTCAG-3' (forward) and 5'-TACCTTGTTACGACTT-3' (Y, T or C; M, A or C) (reverse).The length of the amplicon was about 1500 bp (Frank et al., 2008).The Taq enzyme was a 3:1 mixture of Fermentas Taq : Pfu ThermoFisher Scientific, Burlington, VT, USA), and the 30 ml reaction consisted of 1.5 U of enzyme.Amplification was performed using a temperature program, including initial denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C for 40 s, annealing at 50°C for 40 s, extension at 72°C for 1 min, and final extension at 72°C for 5 min.The PCR products were identified by bidirectional sequencing.

Conjugation experiments
Based on a previously reported (Chen et al., 2015), conjugation experiments were performed in lysogeny broth (LB) with the strain EC600 as the recipient and with the strain F4 as the donor strains.Donor and recipient cells in logarithmic phase (0.5 mL of each) were added to 4 mL of fresh LB, which was followed by incubation at 35°C for 18-24 h without shaking.The transconjugants were selected on trypticase soy agar (TSA) plates containing 10 mg/L of rifampicin and 0.02 mg/L of imipenem.

Plasmid-electroporation assays
Transformation experiments were undertaken using E. coli DH5a electroporated cells as recipient cells for plasmid electroporation.Conjugation frequency was calculated as the number of transconjugates per initial donor cell.To prepare electrocompetent cells, bacteria were grown to OD600 = 0.5-0.6 and precipitated by centrifugation at 4°C.Two rounds of washes and centrifugation (6,000 rpm) were performed at 4°C with 1 vol milliQ water, ending with 1/50 volume 10% glycerol.Cells were resuspended in 1/500 vol 10% glycerol and aliquoted into 50 mL samples.Aliquots were frozen on dry ice, stored at -70°C and set aside.Aliquots were mixed with less than 10 ng of DNA in a 0.2 cm cuvette (Bio-Rad, California, USA) and then electrically pulsed (2.5 kV, 25 mF and 200 W) in a MicroPulser (Bio-Rad, California, USA).Electroporated cells were added to 1 mL of LB and incubated at 37°C with shaking for antibiotic expression.After incubation, the cells were cultured on antibiotic-containing medium.When plasmids from the strain F4 were used in electroporators, it was also selected appropriately with 10 mg/L of rifampicin and 0.02 mg/L of imipenem.

Antimicrobial susceptibility test
Bacterial resistance was detected by BioMerieux VITEK2 (MICs value) and disk diffusion test (mm value) (Table 1), the results were determined in accordance with the 2022 Clinical and Laboratory Standards Institute (CLSI) Guidelines (CLSI, 2022).Twenty-two antibiotics and antibiotics + enzyme inhibitors (Table 1) were detected, and E. coli ATCC 25922 was used as the quality control strain.

Detection of classes A, B, C and D beta-lactamases Dual inhibitor diffusion synergy test
Amoxicillin/clavulanic acid (AMC, 20/10 µg) was placed in the center of the plate, and cefotaxime (CTX, 30 µg), cefepime (FEP, 30 µg), ceftriaxone (CRO, 30 µg), and cloxacillin (CXC, 200 µg) were placed around the AMC, with CTX and AMC spaced 25 mm apart and the rest spaced 20 mm apart.CXC was obtained from MW & E, UK, and the others were from Oxiod, UK.
Interpretation of test results: AMC was synergistic with CRO or CTX, indicating the sample was positive for extended-spectrum beta lactamases (ESBLs); AMC was not co-operative with CRO or CTX but was in synergy with FEP, and thus positive for ESBLs and AmpC; and CXC co-interacted with CRO or FEP, showing a positive result for AmpC enzyme.

AmpC enzyme confirmatory test
Using a modified enzyme extraction three-dimensional test, colonies to be tested were picked and incubated overnight on blood plates according to the literature (Coudron et al., 2000).To make a bacterial suspension with 0.5 McFarland turbidity, 25 µL of bacterial suspension was inoculated into 6 mL trypsin-digested soy broth, incubated overnight on a constant temperature shaker at 35°C at 200 r/min, and centrifuged at 4°C at 4000 r/min for 20 min.The supernatant was discarded, the precipitate was repeatedly freeze-thawed five times at −80°C, and 1.5 mL of 0.01 mol/L phosphate buffer (pH 7.0) was added, vortexed, and mixed.The supernatant was then centrifuged at 14,000 r/min for 2 h at 4°C, thus obtaining the enzyme extract.
Based on the standard paper diffusion method, 0.5 mL of E. coli ATCC25922 was applied to a Mueller-Hinton (MH) agar plate.Cefoxitin (FOX, 30 mg) was placed in the center of the plate, a slit b-lactams Cephamycin Fluoroqinolones Qu et al. 10.3389/fcimb.2023.1260066Frontiers in Cellular and Infection Microbiology frontiersin.orgwas cut radiologically from inside to outside at 5 mm from the edge of the paper plate with a sterile scalpel, and 40 mL of crude enzyme extract was added to the slit from inside to outside with a microsampler to avoid the enzyme solution overflowing the slit, then incubated overnight at 35°C.If an expanded long bacterial area appeared at the junction of the slit and the inhibition circle, it was judged as a positive three-dimensional test, meaning the sample was AmpC-enzyme positive.

Confirmation test for ESBLs
The differences in the diameter of the inhibition circles between ceftazidime (CAZ, 30 µg), ceftazidime/clavulanic acid (30/30 µg), and cefotaxime/clavulanic acid (30/30 µg) were determined as a positive result for ESBLs when the difference in the diameters of the inhibition circles of either group of drugs was ≥5 mm, based on the 2022 CLSI Guidelines (CLSI, 2022).

Classes B and D beta-lactamases tests
The modified carbapenem inactivation method (mCIM) and modified carbapenem inactivation + EDTA (eCIM) methods recommended by CLSI (CLSI, 2022) were performed for the detection of metallo-b-lactamases.And the increase of the zone of inhibition by ≥5 mm of eCIM vs. mCIM was interpreted as a positive result for metallo-b-lactamases (class B carbapenemase).
There was no definitive report of a class D carbapenemase phenotypic assay, which could be confirmed by a process of elimination; if the strain was not inhibited by a class A or B inhibitor, it definitely belonged to class D carbapenemase (Class D beta-lactamase).

Nucleotide sequence accession numbers
The sequences of the F4_chromosome, F4_plasmid pA, F4_plasmid pB, and F4_plasmid pC were deposited on the GenBank database with the accession numbers of CP090397.1,OM144974.1,OM144975.1,and OM144976.1,respectively (Table 2).The GenBank accession numbers of the related plasmids compared with F4_plasmid pA and F4_plasmid pB were also shown in Table 2.

Antimicrobial susceptibility test, enzymatic properties, and transferable features
The strain was confirmed to be K. pneumonia by BLAST of the 16S rRNA and genomic sequences and average nucleotide homology analysis.MICs for the drug susceptibility test of strain F4 were shown in Table 1; Results for the drug susceptibility test of BioMerieux VITEK2 without MICs using the paper diffusion method, e.g., cefoperazone/avibactam, were also listed in Table 1.Enzyme characterization was confirmed to be class A+C betalactamases.The MLST of the strain F4 sequence was analyzed as ST15 using MLST 2.0 and BacWGSTdb 2.0.
After bacterial conjugative transfers and electroporation assays, the transconjugants integrating the F4_plasmid pA or F4_plasmid pB could be recovered with an average conjugation frequencies of 3.42×10 -4 or 4.19×10 -4 , respectively.However, F4_plasmid pC failed in multiple plasmid transfer experiments.Overview of drug resistance genes for F4_chromosome, F4_plasmid pA, F4_plasmid pB, and F4_plasmid pC One F4_chromosome and three plasmids, including F4_plasmid pA, F4_plasmid pB, and F4_plasmid pC, were identified in strain F4.The F4_chromosome was approximately 5.25 Mb in length and only carried four antibiotic resistance genes, oqxB, oqxA, bla SHV-106 , and bla CTX-M-27 (Table 1, Figure S1).F4_plasmid pA is a complete plasmid with a length of about 276.5 kb, harboring 22 different categories of drug resistance genes (Table 1, Figure S2).F4_plasmid pB was also a complete plasmid with a length of about 79.7 kb, carrying nine different types of drug resistance genes (Table 1, Figure S2).The length of F4_plasmid pC was 5.721 kb, but it belonged to a plasmid fragment with only quinolone (qnrB2) and sulfonamide (sul1) resistance genes (Table 1, Figure S2).F4_plasmid pC shared identity with the back-end sequence of F4_plasmid pB and a portion of the sequence before In553 of plasmid L99-05, but in the opposite directions (Figure 1).
The nearly 13.74 kb-long segment of pCTXM27_020046 from the repA gene to the partial 3′-CS of In553 (excluding qacEdelta1 and sul1) was almost identical to the approximately 13.81 kb-long F4_plasmid pB from the 3′-CS remnant of incomplete In553 (without qacEdelta1 and sul1) to the repA gene but in opposite directions (Figure 1).Similarly, an approximately 50.40 kb-long segment of pCTXM27_020046, from the sul1 gene of In553 to IS26 located at a site of approximately 64 kb, was almost identical to an approximately 47.80 kb-long segment of F4_plasmid pB, from IS26 located at a site of about 23 kb to the sul1 gene located at site of nearly 73 kb, but in opposite directions (Figure 1).
When comparing F4_plasmid pB with Plasmid L99-05, the two plasmids were almost identical in structure and length, except for a small segment of sequence at the beginning of F4_plasmid pB that included In553, which was identical to the end of Plasmid L99-05 (Figure 1).When comparing Plasmid L99-05 with p19110124-2, the two plasmids were also nearly identical in structure and length, except that approximately a third of the final sequence length of p19110124-2 including In553 was the same as approximately a third of the length of the beginning of plasmid L99-05.F4_ plasmid pC contains five genes, two of which are the drug resistance genes qnrB4 and sul1, and showed a high degree of sequence identity with the back-end sequence of F4_plasmid pB and a segment of Plasmid L99-05 located in front of In553 (Figure 1).
Structurally, TnAs3 was divided into several parts by In553, some IS, and partial plasmid backbone genes located on F4_plasmid pB and its other closely related plasmids pCTXM27_020046, Plasmid L99-05, and p19110124-2, developing an intricate yet intersecting complex MDR region for each plasmid (Figure 1).

Genetic context of toprJ1-tmexCD1-tnfxB1 from F4_plasmid pA and closely related plasmids
By finely analyzing the MDR region in Figure 2, we discovered that IS26 was present at both the front and back of toprJ1-tmexCD1-tnfxB1.IS26-mediated translocation is the most efficient and most likely to occur when a copy of IS26 was involved (Harmer et al., 2014).

Discussion
tmexCD-toprJ-positive bacteria are usually directly involved in multidrug resistance and can carry different drug resistance genes (Dong et al., 2022).The major functional tmexCD transporters have evolved into different isoforms.tmexCD-toprJ has a highly diverse genetic environment related to various mobile elements (Dong et al., 2022).If horizontal and vertical gene transfer of tmexCD-toprJ had occurred, it would likely have led to widespread clinical dissemination (Dong et al., 2022).
So far, tmexCD-toprJ-positive bacteria have originated largely in China from 2020, and have been occasionally discovered in Vietnam and other countries, provoking warnings of global spread (Hirabayashi et al., 2021).This may be related to the IS26integrated loop intermediates described in this study (Figure 3).IS26 could be transferred not only by a transposon consisting of two IS26 and the related gene, but also by seamless transfer of the related gene to the end of the existing IS26 (Harmer and Hall, 2015;Harmer and Hall, 2016).The efficiency of the second transfer was 60 times higher than that of the first transfer when the plasmid contained IS26 (Harmer et al., 2014).Furthermore, all transfer units, such as F4_plasmid pA and similar plasmids, carried the traB gene (Figures 2, 3), which might accelerate transfer across species (Burbank and Van Horn, 2017).The spread of the tmexCD-toprJ gene cluster involves ISs (such as IS26 and IS903B), transposons (such as TnAs1, Tn5393, and TnAs3), integrons (such as In641), integration and conjugation elements (ICEs), and plasmids (Figure 2) (Sun et al., 2020;Wang Q. et al., 2021).These mobile genetic elements have contributed significantly to the development of bacterial antibiotic resistance.Incomplete TnAs1, Tn5393, and TnAs3 with integron components were found in the tmexCD1-toprJ1 gene clusters in the MDR regions of F4_plasmid pA and similar plasmids (Figure 2), and a complete or incomplete In641 appeared near the tmexCD1-toprJ1 gene clusters; we speculate that these structures of the F4_plasmid pA and similar plasmids might have accompanied the spread of the tmexCD1-toprJ1 gene cluster at an early stage and subsequently been disrupted and partitioned by other genetic mobile elements during the evolutionary process.Similarly, although F4_plasmid pB does not contain the tmexCD1-toprJ1 gene cluster, incomplete TnAs3 with In553 was also split from the MDR regions of F4_plasmid pB and similar plasmids, and complete or incomplete In553 appeared as one of the TnAs3 components in these MDR regions (Figure 3).The presence of the oqxAB gene in the F4_chromosome increases the complexity of resistance to some antibiotics.A strain F4 carrying F4_plasmid pA with TnAs1, Tn5393, TnAs3, In641, tmexCD1-toprJ1, and bla DHA-1 (Figure 2), alongside F4_plasmid pB with TnAs3, In641, and bla CTX-M-174 (Figure 1) and the F4_chromosome bearing the oqxAB gene (Figure S1), would be extremely challenging for clinical prevention and treatment.Mobilization and dissemination of bacterial plasmids carrying toprJ1-tmexCD1 and incorporating IS26 as a mediator would be more dangerous in hospitals.
Overexpression of oqxAB, the RND-type efflux pump gene on the chromosome, plays an essential role in tigecycline resistance (Sheng et al., 2014;Bialek-Davenet et al., 2015).Strain F4 was highly resistant to 22 routinely used antibiotics and antibiotic + enzyme inhibitors, including tigecycline, and this resistance was directly associated with the oqxAB gene localized on the F4_chromosome (Figure S1) and tmexCD1-toprJ1 on plasmid A (Table 1).The high resistance to blactams and b-lactams + enzyme inhibitors, especially the novel combination of cefoperazone + avibactam, was directly related to bla DHA-1 on F4_plasmid pA and bla CTX-M-174 on F4_plasmid pB (Table 1), so was cephalexin resistance (Table 1).Overall, strain F4 expressed to resistance to Aminoglycoside, b-lactams antibiotics, blactams antibiotics + enzyme inhibitors, Cephalexin, Fluoroqinolones, Tetracycline, Glycylcycline (tigecycline), Phenicol, Sulfonamide, Macrolide and Rifamycin (Table 1).The emergence of the plasmidmediated multidrug resistance gene cluster tmexCD1-toprJ1 decreases susceptibility to many clinically important antimicrobial drugs and constitutes a serious problem of multidrug resistance, which is the most troublesome issue in the human clinical setting.It's worth noting that if tnfxB function was absent, overexpression of tmexCD1-toprJ1 could enhanced resistance to tetracyclines, fluoroquinolones, cephalosporins, macrolides, and chloramphenicol (Table 1), and in the presence of upstream tnfxB, tmexCD1-toprJ1 would not been affected the resistance level (Lv et al., 2020).Structural differences in the composition of resistance genes between F4_plasmid pA, F4_plasmid pB and F4_plasmid pC were responsible for the differences in antibiotic resistance among strains F4 (Table 1).

Conclusion
It has risen to such a terrible extent that there are almost no effective antibiotics available, and its spread and prevalence should be effectively prevented for strain F4 and related strains, because of the intermingling of In641 with TnAs1, Tn5393, and TnAs3 in F4_ plasmid pA and closely related plasmids carrying tmexCD1-toprJ1 and bla DHA-1 , and the structural overlaps of In553 and TnAs3 in F4_plasmid pB and closely related plasmids bearing bla CTX-M-147 , together with the oqxAB genes located on the F4 chromosome.
FIGURE 3Schematic diagram of whether small loop intermediates could be formed by the integration of IS26s.(A) Schematic representation of a small loop intermediate that could be formed.(B) Schematic representation of a small loop intermediate that could not be formed.The diagram of F4_plasmid pA was created by the R package genoPlotR v0.8.11 software (http://genoplotr.r-forge.r-project.org/).The small loop diagrams were established using CGview v2.0.3 (https://github.com/paulstothard/cgview).

TABLE 1 Continued
*Disc diffusion method.

TABLE 2
Profiles of the K. pneumoniae plasmids studied in the paper.