Co-transfer of IncFII/IncFIB and IncFII plasmids mediated by IS26 facilitates the transmission of mcr-8.1 and tmexCD1-toprJ1

Purpose This study aimed to characterise the whole-genome structure of two clinical Klebsiella pneumoniae strains co-harbouring mcr-8.1 and tmexCD1-toprJ1, both resistant to colistin and tigecycline. Methods K. pneumoniae strains TGC-02 (ST656) and TGC-05 (ST273) were isolated from urine samples of different patients hospitalised at separate times in 2021. Characterisation involved antimicrobial susceptibility testing (AST), conjugation assays, whole-genome sequencing (WGS), and bioinformatics analysis. Comparative genomic analysis was conducted on mcr-8.1-carrying and tmexCD1-toprJ1-carrying plasmids. Results Both K. pneumoniae isolates displayed a multidrug-resistant phenotype, exhibiting resistance or reduced susceptibility to ampicillin, ampicillin/sulbactam, cefazolin, aztreonam, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, nitrofurantoin, trimethoprim/sulfamethoxazole, apramycin, tigecycline and colistin. WGS analysis revealed that clinical strain TGC-02 carried the TmexCD1-toprJ1 gene on a 200-Kb IncFII/IncFIB-type plasmid, while mcr-8 was situated on a 146-Kb IncFII-type plasmid. In clinical strain TGC-05, TmexCD1-toprJ1 was found on a 300-Kb IncFIB/IncHI1B/IncR-type plasmid, and mcr-8 was identified on a 137-Kb IncFII/IncFIA-type plasmid. Conjugation experiments assessed the transferability of these plasmids. While transconjugants were not obtained for TGC-05 despite multiple screening with tigecycline or colistin, pTGC-02-tmex and pTGC-02-mcr8 from clinical K. pneumoniae TGC-02 demonstrated self-transferability through conjugation. Notably, the rearrangement of pTGC-02-tmex and pTGC-02-mcr8 via IS26-based homologous recombination was observed. Moreover, the conjugative and fusion plasmids of the transconjugant co-harboured the tmexCD1-toprJ1 gene cluster and mcr-8.1, potentially resulting from IS26-based homologous recombination. Conclusion The emergence of colistin- and tigecycline-resistant K. pneumoniae strains is concerning, and effective surveillance measures should be implemented to prevent further dissemination. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-024-00676-5.


Introduction
Klebsiella pneumoniae, a member of the Enterobacteriaceae family, is a significant clinical species commonly associated with nosocomial infections such as pneumonia, bloodstream infection, urinary tract infection, and soft tissue infection [1].It poses an emerging challenge for clinical settings worldwide due to the extensive use of antibiotics, leading to the emergence and rapid dissemination of multidrug-resistant K. pneumoniae, especially those resistant to last-line antibiotics such as carbapenems, colistin, and tigecycline [2].
Colistin is regarded as a last-line antibiotic, used either alone or in combination with other drugs to combat severe infections caused by carbapenem-resistant pathogens [11].The extensive use of colistin in veterinary and human medicine has given rise to colistin resistance [12].Plasmid-mediated colistin resistance (mcr) genes have extended colistin resistance horizontally among different species.The mobile colistin gene mcr-8, found on an IncFII-type conjugative plasmid in K. pneumoniae [13], has given rise to five identified variants (mcr-8.1 to mcr-8.5)[14].
This tmexCD1-toprJ1 gene cluster can be horizontally transferred along with the colistin resistance gene mcr-8 and is primarily associated with K. pneumoniae [6].In this study, we aimed to characterise the whole-genome structure of two clinical Klebsiella pneumoniae strains resistant to both colistin and tigecycline, co-harbouring mcr-8.1 and tmexCD1-toprJ1, underscoring the convergence and co-transmission risk of these resistance genes.

Patients and bacterial strains
We collected two K. pneumoniae isolates (TGC-02 and TGC-05) that exhibited resistance to both tigecycline and colistin.K. pneumoniae strain TGC-02 was isolated from a urine sample from a 79-year-old male patient diagnosed with prostatic hyperplasia.Initially, the patient sought treatment for urinary difficulties at a local hospital, which included the insertion of a urinary catheter.In October 2021, he was admitted to our hospital for further evaluation and management.However, surgery was ruled out due to underlying health issues.During his hospitalisation, he was diagnosed with a urinary tract infection and received treatment with cefuroxime sodium.Upon discharge, the patient regained the ability to urinate independently.K. pneumoniae strain TGC-05 was isolated from a urine sample obtained from a 28-year-old male patient with ureteral stones at the same hospital in August 2021.Initially, the patient was admitted to the urology department of our hospital in June 2021 due to left ureteral stones.He underwent lithotripsy, and a double-J stent was inserted, which was removed two months later.During a follow-up examination in August, the patient was diagnosed with a left epididymal cyst and left scrotal inflammation.Subsequently, the double-J stent was removed, and treatment with cefuroxime sodium was administered.The patient was discharged in good condition.The species of the isolate was determined using matrix-assisted laser desorption/ionisation timeof-flight mass spectrometry (MALDI-TOF/MS) (Bio-Mérieux, France), and was confirmed by whole-genome sequencing (WGS).

Antimicrobial susceptibility testing
The antibiotic susceptibility testing (AST) was conducted for a range of antibiotics, including aztreonam, cefepime, ceftriaxone, ceftazidime, ertapenem, imipenem, piperacillin/tazobactam, trimethoprim/sulfamethoxazole, ciprofloxacin, levofloxacin, gentamicin, amikacin, ampicillin, ampicillin-sulbactam, cefazolin, cefotetan, tobramycin, and nitrofurantoin.The VITEK-2 compact system (Bio-Mérieux, France) was employed to perform these tests.The results were interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI) breakpoints (CLSI, 2022).Additionally, the minimum inhibitory concentrations (MICs) of tigecycline and colistin were determined using the broth microdilution, and the interpretations were made following the guidelines provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2022).

Conjugation assay
The transferability of tigecycline and colistin resistance genes was determined through a filter mating assay, using clinical strains resistant to tigecycline and colistin as donors and rifampin-resistant Escherichia coli C600 as the recipient.Transconjugants carrying different resistance plasmids were screened on Mueller-Hinton (MH) plates containing various antibiotics for conjugation assays.The antibiotics were combined as follows: tigecycline (4 µg/ml) and rifampin (2.5 mg/ ml); colistin (2 µg/ml) and rifampin (2.5 mg/ml); and apramycin (30 µg/ml) and rifampin (2.5 mg/ml).Antibiotic susceptibility testing and PCR analysis were performed to confirm the transfer of plasmids carrying the TmexCD1-ToprJ1, mcr-8.1 and/or aac(3')-IV resistance genes.Specific PCR primers can be found in Additional file 1: Table S1.

Whole-genome sequencing, assembly, and annotation
Total genomic DNA was extracted from the clinical isolates and transformants using a commercial genomic DNA kit (Qiagen, Hilden, Germany).Subsequently, genomic DNA sequencing was carried out employing the Illumina HiSeq platform (Novogene Co., Ltd., Beijing, China) and a PacBio RSII sequencer (Biozeron Biological Technology Co., Ltd., Shanghai, China).The paired-end short Illumina reads and long PacBio reads were subjected to hybrid assembling using Unicycler v0.5.0 [15] in normal mode.
For comparative analsysism plasmid and genetic context comparisons were conducted using the BLAST Ring Image Generator (BRIG) [21] and Easyfig [22] tools, respectively.

Pulsed-field gel electrophoresis
S1-pulsed-field gel electrophoresis (PFGE) was employed to validate both the size and number of plasmids present in the transconjugant and clinical strains.To achieve this, bacterial whole-cell DNA from the clinical isolates and their transconjugants was embedded in agarose plugs and subjected to digestion with S1 nuclease (Takara, Tokyo, Japan).As a reference marker, Salmonella enterica serovar Braenderup H9812, digested with XbaI, was utilised.The DNA fragments were separated using the CHEF-Mapper PFGE system (Bio-Rad) under the following conditions: 14 °C, 6 V/cm, and a 120° pulse angle for 16 h, with the initial and final pulses lasting 2.16 and 63.8 s, respectively.Subsequently, the PFGE results were analysed using InfoQuest software version 4.5 (Bio-Rad Laboratories, Hercules, CA, USA).

Analysis of plasmids carrying mcr-8 and tmexCD1-toprJ1 genes within the NCBI database
Within the NCBI database (until September 2023), the complete sequences of mcr-8 and tmexCD1-toprJ1 were utilised for separate searches of complete sequences of plasmids bearing mcr-8 (coverage = 99%, identity = 100%) bearing and tmexCD1-toprJ1 (coverage and identity > 96%).Typing of these plasmids were carried out using the PlasmidFinder [17].Additionally, an analysis of IS26 distribution among these plasmids was conducted through a local Blast search.

Analysis of Mcr-8 carrying plasmid
The plasmid pTGC-02-mcr8 comprised 146,089 base pairs (bp) plasmid and possessed an IncFII-type replicon.It consisted of two main regions: an approximately 70-kb backbone region, carrying a conjugal transfer region (traABCDEGHIKLMNQTUVWX) that promoted horizontal plasmid transfer among bacteria, and an approximately 70-kb drug resistance region encompassing antibiotic resistance genes, insertion sequences (ISs), and transposons.In the conjugation assay, pTGC-02-mcr8 demonstrated self-transferability.
To assess the transferability of the tmexCD1-toprJ1bearing plasmid pTGC-05-tmex from TGC-05, conjugation assays were employed using Escherichia coli J53, Escherichia coli C600, and K. pneumoniae NTUH-K2044 as recipients but were unsuccessful in obtaining transconjugants following several attempts.Likewise, electrotransformation and chemical transformation of plasmid pTGC-05-tmex into E. coli DH5a as the recipient yielded no positive results.In silico analysis of the T4SS using the oriTfinder web tool confirmed that the oriT sequence of pTGC-05-tmex was partially deleted, which might explain the non-transferability of this plasmid.
To elucidate the mechanism of transmission and rearrangement of resistance plasmids, WGS and annotation of JTGC-02-2-Tmex were conducted.
It is hypothesized that the formation of these two novel plasmids, pJTGC-02-p1 and pJTGC-02-p2, occurred through two rounds of homologous recombination (Fig. 5).The first round of homologous recombination, based on IS26 located upstream of the tmexCD1-toprJ1 region (101,949-169,465 bp, including conjugation transfer cluster and partial MDR) on the pTGC-02-tmex plasmid and upstream of the Tet(A) region (66,965-11,5375 bp, including conjugation transfer cluster, partial MDR and a segment of backbone region) on the pTGC-02-mcr8 plasmid, resulted in the formation of a giant fusion plasmid.The second round of homologous recombination, based on an about 2 KB identical sequence composed by X polypeptide-hypothetical protein-YafZhypothetical protein (X-H-YafZ-H) located downstream of the tmexCD1-toprJ1 gene cluster on the pTGC-02tmex plasmid and downstream of the Tet(A) region on the pTGC-02-mcr8 plasmid, caused the dissociation of the fusion plasmid into two novel plasmids pJTGC-02-p1 and pJTGC-02-p2.In summary, the two rounds of homologous recombination resulted in the replacement of the Tet(A) region on the pTGC-02-mcr8 plasmid with the tmexCD1-toprJ1 region on the pTGC-02-tmex plasmid, leading to the formation of pJTGC-02-p1 co-harboring mcr-8 and tmexCD1-toprJ1.

IS26 was commonly detected on plasmids carrying mcr-8 and tmexCD1-toprJ1
A total of 58 complete sequences of mcr-8-bearing plasmids were obtained, with 56 sequences of them originating from Klebsiella species (Fig. 6a).Among the mcr-8-bearing plasmids, 28 of them were from human origin.According to plasmid typing results, 34 of these plasmids belonged to the IncFII/IncFIA type, and 6 plasmids carried at least one copy of IS26.Regarding tmexCD1-toprJ1-bearing plasmids, a total of 156 complete sequences were acquired, with 94 originating from Klebsiella species and 37 from Pseudomonas species (Fig. 6b).Among the tmexCD1-toprJ1-8-bearing plasmids, 62 of them were from human origin.Plasmid typing results indicated that 42 plasmids belonged to the IncFIB/IncHI1B type, and 127 plasmids harboured at least one copy of IS26.Among the 127 plasmids, 53 contain at least one copy of IS26 within 2 KB proximity to tmexCD1-toprJ1, potentially facilitating the spread of tmexCD1-toprJ1.Additionally, among the 156 tmexCD1-toprJ1-bearing plasmids, 9 of them co-carried tmexCD1-toprJ1 and mcr gene (Additional file 2: Table S2).

Discussion
Colistin and Tigecycline are often considered the last line of defense against life-threatening infections caused by multidrug-resistant gram-negative pathogens.A recent nationwide surveillance study in China revealed a low occurrence of tmexCD-toprJ positive clinical Klebsiella spp.(7/2795, 0.25%) [26].However, there is growing concern about the emergence of transmissible plasmids carrying tmexCD1-toprJ1, especially when they facilitate the co-transfer of tmexCD1-toprJ1 and mcr genes through the same or different plasmids [7,[27][28][29][30][31].In our study, we present findings on two clinical strains that carry both TmexCD1-toprJ1 and mcr-8 genes, aiming to uncover the mechanisms behind the accumulation of resistance.The most significant discovery in our research is the observed co-transfer of TmexCD1-toprJ1 and mcr-8 via the formation of novel hybrid plasmids mediated by IS26.
Previous research has suggested that tmexCD1-toprJ1mediated tigecycline resistance primarily originates in chickens, as evidenced by the significant disparity in the prevalence of tmexCD1-toprJ1-positive strains between animal and human sources (52.4% vs. 2.5%) [26].Alarmingly, approximately one-third of tmexCD-toprJ positive Klebsiella spp.were found to carry colistin resistance genes [26].Our study, based on public databases, indicates that around 32.7% and 39.7% of  3)-IV, as well as plasmids pTGC-02-mcr8 and pTGC-02-tmex from clinical strain TGC-02.The plasmid fusion site is indicated by the black box.c A linear comparative analysis to illustrate the formation of transconjugants tmexCD1-toprJ1-positive plasmids are of animal origin and human origin, respectively, while approximately 41.4% and 48.3% of mcr-8-positive plasmids are of animal and human origins.These seemingly contradictory findings may serve as evidence that tmexCD1-toprJ1-positive and mcr-8-positive plasmids are rapidly spreading from animals to humans.The prevailing plasmid types associated with tmexCD1-toprJ1 and mcr-8 are IncFIB/ IncHI1B and IncFII/IncFIA, respectively.Among the 156 tmexCD1-toprJ1-bearing plasmids, 9 of them co-carried both tmexCD1-toprJ1 and mcr genes.The co-occurrence of tmexCD-toprJ with mcr genes on the same plasmid poses a significant challenge for clinical management and potentially accelerates their dispersion.
IS26 is well-studied and known to create clusters of antibiotic resistance genes interspersed with directly oriented genes, a phenomenon observed in multi-resistant pathogens.It is frequently reported to generate a translocatable unit (TU) element, facilitating the transfer of a single IS26 copy along with an adjacent DNA segment to a new location [28].This process can result in the deletion or inversion of DNA segments through a replicative route, as observed in previous studies [28].Wan et al. suggested that tmexCD1-toprJ1 may integrate into pHN111RT-1 through IS26-mediated cointegration with a circular intermediate, highlighting the potential for tmexCD1-toprJ1 transmission among different plasmids and strains [9].We hypothesize that the approximately 30-kb tmexCD1-toprJ1-associated fragment could have integrated into the IncFII plasmid pTGC-02-tmex via IS26-based homologous recombination during transmission.Further research is needed to explore the formation of circular intermediates within the tmexCD1-toprJ1associated fragment.
IS26 is capable of forming cointegrates through two mechanisms: the copy-in route, where cointegrates form between DNA molecules containing IS26, and the targeted conservative route, where recombination targets one or both ends of the IS elements [32].Wang et al. reported that plasmids carrying mcr-1 could be co-transferred with plasmids containing bla NDM-1 or tmexCD1-toprJ1 through plasmid hybridization [31].Based on whole-genome sequencing (WGS) and S1-PFGE analyses, it is suggested that targeted conservative recombination of plasmids is likely mediated by IS26 elements located on plasmids carrying mcr-1, bla NDM-1 , or tmexCD1-toprJ1 [31].Cointegration via the conservative route occurs at a frequency over 50 times higher than that of copy-in cointegrate formation [32].According to our research, IS26 is common to both mcr-8 and tmexCD1-toprJ1-bearing plasmids, increasing the likelihood of cointegration events and co-transferability.Consequently, further IS26-mediated mobilization of tmexCD1-toprJ1 and mcr-8 among different plasmid types remains possible.These events may contribute to the rapid and widespread accumulation of tigecycline and colistin resistance in Enterobacteriaceae, especially in the face of antibiotic selective pressures in the future.

Conclusion
The simultaneous emergence of mobilized colistin and tigecycline resistance genes in clinical isolates is a concerning evolutionary trend.IS26 is widely distributed on mcr-8 and tmexCD1-toprJ1-bearing plasmids, which may accelerate the formation of colistin-and tigecyclineresistant strains and the creation of new hybrid plasmids.Therefore, it is imperative to enhance monitoring efforts to prevent the further spread of colistin-and tigecyclineresistant Klebsiella pneumoniae in healthcare settings.

Fig. 5 Fig. 6
Fig. 5 Hypothesized Mechanism of Plasmid Formation through Two Rounds of Homologous Recombination.The formation of pJTGC-02-p1 and pJTGC-02-p2 plasmids occurred through two rounds of homologous recombination.a The first-round homologous recombination, driven by IS26, resulted in the fusion of pTGC-02-tmex and pTGC-02-mcr8, creating a giant fusion plasmid.b The second-round homologous recombination, involving an identical sequence downstream of tmexCD1-toprJ1 and Tet(A) regions, led to the dissociation of the fusion plasmid into the two novel plasmids, pJTGC-02-p1 and pJTGC-02-p2.This process replaced the Tet(A) region with the tmexCD1-toprJ1 region, giving rise to pJTGC-02-p1, which co-harbors mcr-8 and tmexCD1-toprJ1.pTGC-02-tmex is represented in yellow, pTGC-02-mcr8 in blue, IS26 in purple double strands, X-H-YafZ-H in green double strands, and antibiotic resistance genes in red rectangles