The KP15-652 strain was isolated by urine culture from a patient with a trauma to the urinary track in Quzhou, China on October 20, 2015. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS, BioMeírieux, France) was used to identify the isolate to the species level and further confirmed using genome sequencing data.

Minimum inhibitory concentrations (MICs) of the KP15-652 strain for cefepime, cefotaxime, imipenem, meropenem, amikacin, levofloxacin, ciprofloxacin, fosfomycin, aztreonam, colistin and tigecycline were determined by broth microdilution. The results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) 2021 guidelines. Escherichia coli ATCC 25922 served as the quality control strain.

Genomic DNA of the KP15-652 strain was extracted using a Qiagen Minikit (Qiagen, Hilden, Germany) based on the manufacturer’s recommendations. Whole-genome sequencing was performed using both the Illumina NovaSeq platform (Illumina, San Diego, CA, USA) and long-read PacBio RS II platform (Pacific Biosciences, Menlo Park, CA, USA). De novo hybrid assembly of the Illumina and PacBio reads was performed using Unicycler v0.4.8 (Wick et al., 2017). Annotation was conducted using National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/) (Tatusova et al., 2016). Antimicrobial resistance genes were identified using ABRicate v0.8.13 (https://github.com/tseemann/abricate) based on the ResFinder database (http://genomicepidemiology.org/) (Zankari et al., 2012). Bacterial virulence factors were identified using the Virulence Factor Database (VFDB, http://www.mgc.ac.cn/VFs/) (Liu et al., 2022). Capsular typing was performed using Kaptive (Wyres et al., 2020a). Plasmid replicons were analyzed with PlasmidFinder v2.1 (Carattoli et al., 2014). Multilocus sequence typing (MLST) was analyzed using the Center for Genomic Epidemiology (CGE) database (https://cge.cbs.dtu.dk/services/MLST/). Insertion sequence (IS) elements were investigated through ISFinder (https://www-is.biotoul.fr/) (Siguier et al., 2006). Genetic contexts were compared with Easyfig (Sullivan et al., 2011). The results of genetic environment and plasmid circular map comparisons were manually visualized in Adobe Illustrator 2020 (https://www.adobe.com/products/illustrator.html).

Transformation and conjugation assay were performed to determine whether the plasmids carrying mcr-1.1, mcr-8.2 or tmexCD1-toprJ1 gene cluster are transferable. Plasmid DNA was extracted from clinical isolates using a Qiagen Plasmid MidiKit (Qiagen, Hilden, Germany) and then electrotransformed into E. coli DH5α. Transformants were selected on Mueller-Hinton (MH) agar plates containing colistin (2 mg/L) or tigecycline (2 mg/L), together with rifampin (700 mg/L). Conjugation experiments using E. coli J53 (sodium azide resistant) as the receptor strain were performed using the film mating method (Yang et al., 2021). Conjugants were screened on MH plates containing colistin (2 mg/L) or tigecycline (2 mg/L), together with sodium azide (200 mg/L). The identity of successful transformants or conjugants was confirmed via polymerase chain reaction (PCR) for several marker genes, such as resistance genes, and MALDI-TOF MS.

Plasmid curing assays were performed using sodium dodecyl sulfate (SDS, 10% w/v, pH 7.4) (El-Mansi et al., 2000). In brief, one colony was selected and grown in 2 mL MH broth overnight culture at 37°C. Then, 100 μL of overnight culture solution was transferred to fresh MH broth containing 10% SDS and incubated at 37°C, followed by spreading on MH agar plates without antibiotics. Plasmid cured colonies were confirmed by PCR and S1 nuclease pulsed field gel electrophoresis (S1-PFGE).

The K. pneumoniae strain, named KP15-652, was isolated from urine culture and presented multidrug resistance. MIC data revealed that KP15-652 is resistant to colistin (> 2048 mg/L), tigecycline (16 mg/L), amikacin (> 256 mg/L), levofloxacin (128 mg/L), and ciprofloxacin (> 32 mg/L) but is susceptible to cefepime (0.5 mg/L), cefotaxime (2 mg/L), imipenem (2 mg/L), meropenem (0.064 mg/L), fosfomycin (8 mg/L) and aztreonam (0.5 mg/L). ( Figure 1 ) The multidrug resistance and the high-level resistance against colistin prompted us to explore molecular resistance determinants and their transferability based on genomic analysis and phenotype confirmation.

The complete genome of isolate KP15-652 was acquired by hybrid assembly combining long- and short-reads sequencing data. The size of the circularized chromosome is 5,265,433 bp ( Table 1 ), and up to seven plasmids were identified. MLST analysis showed that the KP15-652 strain belongs to ST656 (gapA-infB-mdh-pgi-phoE-rpoB-tonB: 4-4-1-1-7-4-4). The O locus and K locus of KP15-652 are predicted to be OCL101 and KL177, with 94.88% and 99.37% nucleotide identity, respectively.

The KP15-652 genome was positive for a series of antibiotic resistance determinants ( Table 1 , Figure 1 ), conferring potential resistance to β-lactams (bla _SHV-187, blaTEM-1B and bla _DHA-1), aminoglycosides (strAB, aph(3’)-Ia, aph(4)-Ia, aac(3)-IV, aadA1, aadA2 and armA), macrolide (mph(A), msr(E) and mph(E)), sulfonamides (sul1 and sul3), quinolones (qnrB4), phenicol (cmlA1), colistin (mcr-1.1 and mcr-8.2), and tetracycline or tigecycline (tet(A), tet(M) and tmexCD1-toprJ1).

Most of the resistance genes are located on plasmids, especially on the top 4 large plasmids named p652-1, p652-2, p652-3 and p652-4, which are 275,345 bp, 192,753 bp, 52,998 bp and 43,761 bp in size, respectively. The other three plasmids harbored by KP15-652 are relatively small, of no more than 4 kb and carrying no any known resistance genes. Based on replicon analysis, three of four resistant plasmids belong to hybrid incompatibility groups, including IncFIB/IncHI1B for p652-1, IncFIB/IncFII for p652-2 and IncR/IncN for p652-3. The remaining resistant plasmid is from the IncX1 group. The other three small plasmids are rolling-circle replication models belonging to Col440I, ColRNAI, and ColpVC. Plasmid p652-1 harbors a series of resistance genes encoding aminoglycoside-modification enzymes, methylase ArmA, cephalosporinase DHA-1, quinolone resistance determinant QnrB4 and tigecycline efflux pump system tmexCD1-toprJ1. The second large plasmid, p652-2, harbors aadA2, sul1, mph(A) and the β-lactamase gene bla _TEM-1B. mcr-8.2 is located on plasmid p652-3, accompanied by the wild-type tigecycline efflux pump tet(A). Notably, there is another copy of tet(A) on the chromosome that shows an A276S mutation, which probably elevates the level of tigecycline resistance. mcr-1.1 is carried on IncX1 plasmid p652-4 along with another tigecycline efflux pump: tet(M) ( Table 1 , Figure 1 ).

Chromosome is also a repository for resistance determinants, such as the β-lactamase gene bla _SHV-187 and tigecycline efflux pump tet(A) mutant mentioned above. Moreover, mutations in the gyrA (S83I) and parC (S80I) genes that confer resistance to fluoroquinolones were detected. Concerning mutations for colistin resistance, mutations in CrrB (L204V, V237I) were identified on the chromosome ( Table 1 , Figure 1 ).

Due to the concern of resistance to last-resort antibiotics, such as colistin and tigecycline, we performed transformation/conjugation tests and plasmid curing assays to explore the transferability and contribution of corresponding resistance determinants.

Both the transformant and conjugant we acquired under colistin selection presented two distinct phenotypes when we extracted plasmids from a clinical isolate for transformation or used a clinical isolate as a donor for the conjugation assay. One of the phenotypes in each assay involved elevated colistin resistance to more than 64 times compared to the respective recipient due to acquisition of the mcr-1.1-harboring plasmid p652-4. The transformation assay particularly selected another phenotype under colistin, which was confirmed as having acquired two plasmids, tmexCD1-toprJ1-harboring p652-1 and mcr-8.2-harboring p652-3, simultaneously. This transformant showed more than 64 times increased colistin resistance and 16 times higher tigecycline resistance. In addition, another conjugant was captured with co-transfer of plasmids p652-1 and p652-4, resulting in both higher colistin and tigecycline resistance in a similar manner ( Figure 1 ).

When tigecycline was used as a selection marker, only a p652-1-harboring conjugant was acquired, with a much higher tigecycline resistance level (MIC increased 256 times). Unfortunately, several attempts for transformation under tigecycline failed. We also attempted the plasmid curing assay using a clinical isolate and successfully acquired a clone that lost the plasmid p652-2 and the mcr-1.1-harboring plasmid p652-4. The corresponding colistin resistance level of the plasmid-curing clone was reduced by more than 16 times. For many other resistance determinants carried on these four plasmids, plasmid acquisition or loss accompanying the change in resistance level in various derivate clones was mostly coincident with the predicted phenotype of antimicrobial agents mediated by the resistance determinants ( Figure 1 ).

The mcr-1.1 gene in KP15-652 is located on the approximately 44-kb plasmid named p652-4, with 44.57% GC content and belonging to IncX1. Structurally, the genetic environment of ISKpn26-mcr-1.1-pap2-ISApl1 was observed ( Figure 2A ). In addition to mcr-1.1, the tigecycline efflux pump gene tet(M) was identified on p652-4, elevating the level of tigecycline resistance. Moreover, plasmid p652-4 was found to carry type IV secretion system-related genes due to a complete vir operon, which is identical to several known plasmids, such as p869Rt_IncX1 (CP080086) and pD72-IncX1 (CP035315) ( Figure 2B ). Hence, the sole plasmid p652-4 has the potential to be transferred via conjugation.

The mcr-8.2 gene is located on the multireplicon plasmid p652-3 with IncR/IncN-typed replicons. The genetic structure of the mcr-8.2 surrounding region of the p652-3 plasmid is ISEcl1-mcr-8.2-orf-ISKpn26 ( Figure 3A ) and 100% identical to pMCR8_095845 (CP031883), pD120-1 (CP034679) and pMCR8_020135 (CP037964) ( Figure 3B ). Another tigecycline efflux pump gene, tet(A), was identified on the p652-3 plasmid, and the p652-3, pMCR8_095845, pD120-1 and pMCR8_020135 plasmids carry one or more IS903B genetic elements. No transferability system has been found for p652-3, with unsuccessful transfer of a single plasmid in the conjugation assay.

Plasmid p652-1 contains two multidrug resistance (MDR) regions, and the tmexCD1-toprJ1 gene cluster is located in MDR region 1 ( Figure 4 ). Many mobile genetic elements were also identified in MDR region 1, including four copies of IS26, a single copy of IS903B, tnp440 and a partial Tn21 ( Figure 4B ). A circular intermediate form of the tmexCD1-toprJ1 gene cluster was observed, comprising two translocatable unit (TU) elements bracketed by two IS26 elements. Importantly, a complex class 1 integron is located in the same region, consisting of a 5’ conserved segment (5’ CS) and 3’ CS ( Figure 4B ). Furthermore, various resistance genes, including aadA2, cmlA1, aadA1, qacH and sul3, were detected, and a segment of the transposase Tn21 was identified downstream of intl1. MDR region 2 also harbors various genes conferring resistance to different kinds of antimicrobial agents, including strA, strB, qnrB4, bla _DHA-1, sul1, armA, msr(E) and mph(E) ( Figure 4C ).

The 275,345 bp plasmid p652-1 might be a co-integration plasmid derived from the progenitor IncHI1B-type plasmid and IncFIB-type plasmid. It is possible that the tmexCD1-toprJ1 gene cluster is located on the IncHI1B-type plasmid, and the fusion process for its derivation may have involved IS26-mediated replicative transposition, whereby one single copy of IS26 on the IncFIB-type plasmid targeted the other copy of IS26 on the IncHI1B-type plasmid to form a fusion plasmid ( Figure 5A ). This event generated the 8-bp target site duplication (TSD) CAATGACA ( Figure 5B ). However, one IS26 copy seemed to have undergone the inversion event according to the direction of IS26 copies and the 8-bp TSD position.

We further conducted a comparison with the plasmid pHNAH81-1 (MK347425), a representative plasmid that harbors an RND efflux pump conferring resistance to tigecycline. The results showed only 6% coverage, with 99.92% identity between the p652-1 and pHNAH81-1 plasmids. Specifically, the tmexCD1-toprJ1 gene cluster shows 99.98% identity and 100% coverage with that of the pHNAH81-1 plasmid. Moreover, one single copy of IS26 was identified upstream of tmexCD1-toprJ1 in p652-1. Nevertheless, no IS26 genetic element was found on the pHNAH81-1 plasmid ( Figure 5C ).