Emergence of a clinical Klebsiella pneumoniae harboring an acrAB-tolC in chromosome and carrying the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 in a plasmid

Objective The emergence of clinical Klebsiella pneumoniae strains harboring acrAB-tolC genes in the chromosome, along with the presence of two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 genes on a plasmid, has presented a significant clinical challenge. Methods In order to study the detailed genetic features of K. pneumoniae strain SC35, both the bacterial chromosome and plasmids were sequenced using Illumina and nanopore platforms. Furthermore, bioinformatics methods were employed to analyze the mobile genetic elements associated with antibiotic resistance genes. Results K. pneumoniae strain SC35 was found to possess a class A beta-lactamase and demonstrated resistance to all tested antibiotics. This resistance was attributed to the presence of efflux pump genes, specifically acrAB-tolC, on the SC35 chromosome. Additionally, the SC35 plasmid p1 carried the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65, as well as bla TEM-1 with rmtB, which shared overlapping structures with mobile genetic elements as In413, Tn3, and TnAs3. Through plasmid transfer assays, it was determined that the SC35 plasmid p1 could be successfully transferred with an average conjugation frequency of 6.85 × 10-4. Conclusion The structure of the SC35 plasmid p1 appears to have evolved in correlation with other plasmids such as pKPC2_130119, pDD01754–2, and F4_plasmid pA. The infectious strain SC35 exhibits no susceptibility to tested antibioticst, thus effective measures should be taken to prevent the spread and epidemic of this strain.


Introduction
The increasing prevalence and global spread of multidrug-resistant bacteria, including in regions such as South/North America, Europe, Africa, the Middle East, South-East Asia, and Oceania, posed a significant threat to human health worldwide (Hamidian and Nigro, 2019).Among these bacteria, carbapenem-resistant Klebsiella pneumoniae had emerged as a major cause of infections globally (World Health Organization (WHO), 2014).Plasmids played a crucial role in the dissemination of drug-resistant genes, such as bla KPC-2 (Zhai et al., 2021).Various types of plasmids had been found to carry bla KPC , with the IncFIIk plasmid being the most frequently detected and widely reported (Villa et al., 2010;Pitout et al., 2015;Li et al., 2021).The production of carbapenemase enzymes, including KPC, NDM, and OXA-48, was one of the major resistance mechanisms in K. pneumoniae (Pitout et al., 2015).
The presence of CTX-M type extended-spectrum betalactamases (ESBLs) was another significant factor contributing to bacterial resistance to cephalosporins.These ESBLs had been widely detected in European countries, as well as in Asia and South America (Cantón and Coque, 2006).Mobile genetic factors played an important role in the emergence of multidrug-resistant bacteria (Yu et al., 2024).There were notable variations in the distribution of CTX-M types across different countries and continents (Yu et al., 2024).In Asia, CTX-M-14 and CTX-M-55 were the predominant genotypes, while CTX-M-1 was more prevalent in Europe (Yu et al., 2024), and higher frequency of IncI-I(a) plasmids were found in Eurasian and Asian transmission clusters (Yu et al., 2024).The transfer of CTX-M-14 was mainly mediated by ISEcpl, while IS26 played a significant role in the transfer of CTX-M-65.Additionally, bla TEM and bla CTX-M were frequently found together within the same transposable unit (Yu et al., 2024).Studies had reported concurrent involvement of bla KPC-2 , bla CTX-M-65 , and bla SHV-12 on the same plasmid (Xiang et al., 2016;Liu et al., 2018), indicating a close relationship and a highly similar genetic backbone among these resistance genes (Liu et al., 2018).Sequencing results had demonstrated that IS26 mediated the duplication of the IS26-fosA3-bla CTX-M-65 plasmid insertion element in certain strains (Wang et al., 2023).
Bacterial resistance to antimicrobial drugs could occur through four main mechanisms: (1) Inactivation of the drugs through degradation or chemical modification, (2) Mutations in the targets of the drugs, resulting in reduced binding or efficacy, (3) Changing in membrane permeability, and (4) Active efflux of drugs, reducing intracellular concentrations (Reygaert, 2018).Mechanisms (1) and (2) generally conferred resistance to specific or similar antibiotics, such as KPC or CTX-M-65, which could be present in the same strain, leading to resistance to multiple drugs within their respective antibiotic families (Yamasaki et al., 2023).On the other hand, mechanisms (3) and (4) could increase resistance to multiple structurally unrelated antibiotics (Reygaert, 2018).For example, a single multidrug efflux pump, such as tmexCD1-toprJ1 (Qu et al., 2023) or acrAB-tolC, had the ability to transport multiple structurally unrelated substrates, and its high expression could result in bacterial resistance to various antibiotic families (Yamasaki et al., 2023).Therefore, the presence or acquisition of resistance genes, along with cellular drug efflux pumps or channels, significantly enhanced bacterial multidrug resistance, making clinical treatment more challenging and complex, and posing a greater risk to infected patients.
This study aims to characterize the multidrug resistance genes present on the chromosome harboring acrAB-tolC and a plasmid carrying two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 .The research will focus on annotating the structure of resistance genes on the plasmid, comparing the structural relationships between similar plasmids, and exploring possible evolutionary processes.The study will provide detailed data that can be used for the treatment of highly multidrug-resistant strains with similar resistance mechanisms.

Bacterial strains and sequencing of the 16S rRNA gene
A 57-year-old man was admitted to the intensive care unit with a closed craniocerebral injury resulting in intracranial and pulmonary infection, but empiric antimicrobial treatment with tigecycline plus polymyxin was ineffective.Subsequently, K. pneumoniae SC35, a carbapenem-insensitive strain, was isolated from the patient's sputum at Taizhou Municipal Hospital affiliated with Taizhou University in 2021, and for this reason, this selected strain was further investigated in depth in this paper.
For species verification, the almost complete 16S rRNA gene was amplified using PCR.The forward primer used was 5'-AGAGTTTGATYMTGGGGCTCAG', and the reverse primer used was 5'-TACCTTGTTACGACTT-3' (where Y represents T or C, and M represents A or C).The length of the amplicon obtained was approximately 1,500 bp (Frank et al., 2008).The PCR amplification was performed using a mixture of Fermentas Taq and Pfu enzymes (in a 3:1 ratio) from ThermoFisher Scientific.The reaction mixture contained 30 ml of the enzyme mixture, and the PCR conditions included an initial denaturation step at 94°C for 3 min, followed by denaturation at 94°C for 40 s, annealing at 50°C for 40 s, extension at 72°C for 1 min, and a final extension step at 72°C for 5 min.A total of 30 cycles were performed.To characterize the PCR products, bidirectional sequencing was carried out.This sequencing method allowed for sequencing in both the forward and reverse directions, providing more accurate and reliable results.

Conjugation experiments
To perform conjugation, the EC600 receptor strain and SC35 donor strain were mixed together in lysozyme broth (LB), which served as the growth medium.Logarithmic phase cells of both the donor and recipient strains (0.5 mL each) were taken and cultured in 4 mL of fresh LB at 35°C for 18-24 hours without shaking.This incubation period allowed for the transfer of genetic material from the donor to the recipient strain.After incubation, Tryptic Soy Agar (TSA) plate containing selective antibiotics were used to screen for transconjugants.Specifically, the TSA plates were supplemented with 10 mg/L rifampicin and 0.02 mg/L imipenem.These antibiotic concentrations were effective in inhibiting the growth of the donor and recipient strains but allowed the growth of transconjugants that had acquired the resistance genes from the donor strain through conjugation.The transconjugants, which were the result of successful conjugation and acquisition of the resistance genes, could be identified and isolated on the TSA plates supplemented with the selective antibiotics.

Plasmid electroporation tests
For plasmid electroporation, E. coli DH5a cells were used as the recipient cells for transformation experiments.The conjugation frequency was calculated by determining the number of transduced conjugates per initial donor cell.To prepare the electroactive cells, the bacteria were grown to an optical density (OD600) of 0.5-0.6.The cells were then precipitated by centrifugation at 4°C and washed twice with 1 volume of milliQ water, followed by centrifugation at 6,000 rpm at 4°C.Finally, the cells were centrifuged with 1/50 volume of 10% glycerol.This washing process helped in removing any residual media and preparing the cells for electroporation.The prepared electroactive cells were aliquoted and stored at -70°C for backup.For electroporation, aliquots of the electroactive cells were mixed with less than 10 ng of DNA in a 0.2 cm cuvette.The electroporation was performed using a MicroPulser apparatus (Bio-Rad, California, USA) at specific settings, such as 2.5 kV, 25 mOhm, and 200 W.This electrical pulse facilitates the uptake of the plasmid DNA by the recipient cells.After electroporation, the cells were immediately transferred into 1 mL of LB medium and incubated at 37°C with shaking.To ensure the expression of the antibiotic resistance encoded by the plasmids, the cells were incubated in a culture medium containing antibiotics.In the case of strain SC35, the plasmids were properly screened using TSA plates supplemented with 10 mg/L rifampicin and 0.02 mg/L imipenem, which allowed for the selection and growth of cells carrying the plasmids.

Antimicrobial susceptibility testing
In the bacterial resistance assay, the BioMerieux VITEK2 system was used to determine the Minimum Inhibitory Concentration (MIC) values of various antibiotics (Table 1).Additionally, the disk diffusion test was performed to measure the diameter (mm) of the zones of inhibition, indicating bacterial resistance.The results obtained were confirmed using the 2022 Clinical and Laboratory Standards Institute (CLSI) guidelines (Clinical and Laboratory Standards Institute (CLSI), 2022).A total of 33 antibiotics, including antibiotics combined with enzyme inhibitors, were tested.The MIC values obtained from the VITEK2 system and the zones of inhibition from the disk diffusion test were recorded in Table 1.To ensure the reliability of the assay, E. coli ATCC 25922 was selected as the quality control strain.The MIC values and zone diameter measurements were accurately determined bacterial resistance, and the results obtained from the assay were compared to the breakpoints and interpretive criteria.

Confirmation of beta-lactamases Confirmation test for ESBLs
According to the CLSI Guidelines 2022, the presence of extended-spectrum beta-lactamases (ESBLs) was determined based on the difference in the diameter of the inhibition zone for specific groups of drugs.Specifically, positive ESBL production was confirmed if the difference in the circle of inhibition diameter was equal to or greater than 5 mm for any of the following drug groups: ceftazidime (CAZ, 30 µg) and ceftazidime/clavulanic acid (30/30 µg), ceftazidime (CTX, 30 µg) and ceftizoxime/clavulanic acid (30/ 30 µg).This criterion was used to identify bacteria that produced ESBLs, which were enzymes capable of hydrolyzing and inactivating a broad range of beta-lactam antibiotics, including cephalosporins and monobactams.

Determination for classes B and D beta-lactamases
According to the CLSI Guidelines 2022 (Clinical and Laboratory Standards Institute (CLSI), 2022), the modified carbapenem inactivation method (mCIM) and the modified carbapenem inactivation method + EDTA (eCIM) were used to determine the presence of metal-b-lactamases, specifically class B carbapenemases.In the mCIM, a carbapenem antibiotic was combined with a EDTA to inhibit the activity of metallo-b-lactamases (MBLs).If there was no MBL present, the carbapenem antibiotic was able to inhibit the bacterial growth, resulting in a zone of inhibition.If the addition of EDTA increased the zone of inhibition by 5 mm or more compared to the mCIM, it was considered positive for metal-b-lactamases (class B carbapenemases).
On the other hand, the identification of the class D carbapenemase phenotype, known as class D blactamases, currently lacked a definitive method.However, it could be inferred by a measure of exclusion.If the strain did not exhibit inhibition by class A or B inhibitors, it was inferred to possess a class D carbapenemase.

Antimicrobial susceptibility testing, enzymatic characterization and transferable properties
The strain SC35 was identified as K. pneumoniae through BLAST analysis of its 16S rRNA and genome sequences, as well as through nucleotide homology analysis.Table 1 provided the MIC values and circle of inhibition diameters of the strain SC35 in the drug susceptibility testing conducted using BioMerieux VITEK2 and the paper diffusion method.The strain SC35 exhibited resistance to all tested antibiotics (Table 1).Enzyme characterization revealed that the strain SC35 produced class A beta-lactamase.The MLST sequence was characterized as sequence type (ST) 11 using MLST 2.0 and BacWGSTdb 2.0.Through bacterial conjugation transfer and electroporation experiments, the integrated SC35 plasmid p1 was successfully recovered, with an average conjugation frequency of 6.85 × 10 -4 .
Investigation and characterization of drug-resistance genes on the SC35 chromosome and SC35 plasmids In the strain SC35, a chromosome and four plasmids were identified, including the SC35 plasmid p1, SC35 plasmid p2, SC35 plasmid p3, and SC35 plasmid p4.On the SC35 chromosome, a total of 40 different genes associated with drug resistance were found.These genes included drug-resistant nodule disintegration (RND) antibiotic efflux pumps (e.g., acrAB-tolC), major facilitator superfamily (MFS) antibiotic efflux pumps (e.g., kpnGH), and ATPbinding cassette (ABC) antibiotic efflux pumps (e.g., msbA) (Figure 1A).For example, it had been documented that upregulation of the expression of the AcrAB-TolC was the major cause of marA-mediated MDR (Li and Nikaido, 2004), whereas AcrAB-TolC overexpression significantly improved the response of marA-mediated MDR to all antibiotics (Ruiz and Levy, 2010).The characterization of drug-resistance genes on the SC35 chromosome could be found in Table 3.
Comparison of structures for the SC35 plasmid p1 with closely related plasmids, pKPC2_ 130119, pKPC2_115069, pKPC2_020003, and pKPC2_095649, Involving the core structures for bla KPC-2 and bla CTX-M-65 from Chengdu, China The SC35 plasmid p1 contained three multidrug-resistant (MDR) regions, namely MDR1, MDR2, and MDR3.The MDR1 region had a sequence length of 22.762 kbp, ranging from 3.383 kbp to 26.144 kbp.It consisted of the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 , which were double repeats of the genetic array of IS26-ISKpn6-bla KPC-2 -ISKpn27-tnpR-IS26-hp-bla CTX-M-65 -IS903-btuB-IS26 (Figure 2).The MDR2 region had a sequence length of 5.321 kbp, ranging from 68.642 kbp to 73.962 kbp, and contained the genetic array IS26-hp-rmtB-bla TEM-1 -tnpR-hp-hp-IS26.The MDR3 region had a sequence length of 18.968 kbp, ranging from 104.056 kbp to 123.023 kbp, and included In413 as well as the antibiotic resistance genes mphA, sul2, aph(3')-Ib, aph(6)-Id and tetA (Figure 2).The structure of SC35 plasmid p1 was characterized by the overlap and intersection of mobile genetic elements as Tn3, TnAs3, and In413 (Figure 2).Comparison of the SC35 plasmid p1 with pKPC2_130119 showed that the MDR1 region of SC35 plasmid p1 shared excellent    identities with the MDRs of both pKPC2_130119 and pKPC2_115069 (Figure 2).The MDR2 region of SC35 plasmid p1 was inversely identified with the posterior regions of the MDR of pKPC2_130119, ranging from 26.918 kbp to 31.518 kbp (Figure 2).The MDR3 region of SC35 plasmid p1 had no identities with the MDRs of pKPC2_130119 and pKPC2_115069 (Figure 2).Similarly, the MDR1 region of SC35 plasmid p1 was identical to the MDRs of pKPC2_ 115069 and pKPC2_095649, respectively, only in a genetic array of IS26-ISKpn6-bla KPC-2 -ISKpn27-tnpR-IS26-hp-bla CTX-M- 65 -IS903-btuB-IS26 (Figure 2).The MDR2 region of SC35 plasmid p1 was inversely identified with the posterior regions of the MDRs of pKPC2_115069 and pKPC2_095649, ranging from 15.850 kbp to 21.170 kbp (Figure 2).The MDR3 region of SC35 plasmid p1 had no identities with the MDRs of pKPC2_115069 and pKPC2_095649 (Figure 2).The MDR1 region of SC35 plasmid p1 had inversely extreme identity with the MDR of pKPC2_020003,  ranging from 82.857 kbp to 96.028 kbp (Figure 2).The MDR2 region of SC35 plasmid p1 was inversely identified with a portion of the MDR of pKPC2_020003, ranging from 78.241 kbp to 83.561 kbp.The MDR3 region of SC35 plasmid p1 also had no identity with the MDR of pKPC2_020003 (Figure 2).

Discussion
The acrAB-tolC resistance-nodulation-cell division (RND) antibiotic efflux pump, composed of the extracellular cytoplasmic fusion protein AcrA, the drug proton transporter AcrB, and the outer membrane channel protein TolC, was responsible for bacterial resistance to various agents including fluoroquinolones, tetracyclines, cephalosporins, glycylcyclines, penams, rifamycins, phenicol antibiotics, disinfectants, and antiseptics (Li et al., 2015) (Table 3).The expression of the marA gene, which regulated the transcription of the acrAB system, significantly increased the activity of the efflux pump by more than 2.8-fold and induced resistance to a wide range of antibiotics (Shi et al., 2022).In the SC35 strain, the presence of acrAB-tolC on the chromosome led to a high level of resistance to various antibiotics including fluoroquinolones, tetracyclines, cephalosporins, cephalomycins, meropenem, and glycylcyclines (tigecycline) (Table 1).
In addition to the RND efflux pump, the SC35 chromosome also contained MFS antibiotic efflux pump and ABC antibiotic efflux pump (Table 3).These efflux pumps contributed to the resistance phenotype of the SC35 strain.Furthermore, the presence of various non-efflux pump resistance genes on both the SC35 chromosome and SC35 plasmid p1, along with the overlapping and interaction of mobile genetic elements as In413, Tn3, and TnAs3 on the SC35 plasmid p1 (Figures 1-3), further contributed to the emergence of a highly multidrug-resistant strain SC35 (Table 1).The combination of these resistance mechanisms resulted in the almost complete resistance observed in the strain SC35.
In China, ST11 had been the most prevalent carbapenem-resistant strain of K. pneumoniae, and the majority of these ST11-KPC-2 strains were considered highly threatening clones.Infections caused by these prevalent strains were often difficult to treat, as they exhibited limited response to available treatment options (Zhang et al., 2017;Wang et al., 2018;Zhang et al., 2018).The SC35 strain, belonging to ST11, produced carbapenemase and possessed antibiotic resistance genes solely on the SC35 plasmid p1.Through sequencing analysis, it was discovered that the SC35 plasmid p1 carried the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 in the MDR1 region, as well as bla TEM-1 with rmtB in the MDR2 region, and other resistance genes that overlapped with mobile genetic elements as In413 and TnAs3 in the MDR3 region (Figures 2, 3).The combination of resistance genes with various functions on both the chromosome and plasmids was what contributed to the challenging situation of having limited treatment options for the SC35 strain.
By comparing the SC35 plasmid p1 with closely related plasmids from Chengdu, China (pKPC2_130119, pKPC2_115069, pKPC2_020003, and pKPC2_095649), it was found that the SC35 plasmid p1 shared the highest similarity with pKPC2_130119 in the MDR1 and MDR2 regions.Both plasmids contained the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 , and bla TEM-1 with rmtB (forward or reversed).In contrast, the other plasmids only had one tandem core structure for bla KPC-2 and bla CTX-M-65 , and bla TEM-1 with rmtB (forward or reversed).None of these plasmids, except the SC35 plasmid p1, had an MDR3 region (Figure 2).Therefore, The SC35 plasmid p1 showed a high level of similarity to the plasmid from Chengdu, China, with the only difference being the absence of an MDR3 region.Similarly, when comparing the SC35 plasmid p1 with other related plasmids (p21072329_1, pXHKP6-1, pBSI014-KPC2, and pDD01754-2) that had the bla KPC-2 core structure arranged separately from the bla CTX-M-65 core structure, it was observed that the bla CTX-M-65 core structure of SC35 plasmid p1 was more similar to that of pDD01754-2.On the other hand, the arrangement of the bla KPC- 2 core structure in pDD01754-2 was identical to that of the SC35 plasmid p1 (Figure 4A).This finding was consistent with the previous discovery of F4_plasmid pA, which shared perfect identity with the SC35 plasmid p1 (Ma et al., 2023).Based on these observations, it was hypothesized that pDD01754-2, F4_plasmid pA, and SC35 plasmid p1 might be evolutionarily linked (Figure 4B).

Conclusion
The presence of similarities in the structure of SC35 plasmid p1 with plasmids such as pKPC2_130119, pDD01754-2, and F4_plasmid pA suggests that these plasmids are interconnected in their evolutionary processes.The strain SC35, which carries multiple efflux pumps on its chromosome (such as acrAB-tolC) and harbors the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 , and bla TEM-1 with rmtB on a plasmid with overlapping structures of mobile genetic elements as In413, Tn3, and TnAs3, poses a significant threat.Effective preventive measures should be taken to stop the spread and prevalence of such antibiotic-resistant bacteria.

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

TABLE 3
Characterization of drug-resistance genes located on SC35 chromosome.