Genomic Characteristics and Phylogenetic Analyses of a Multiple Drug-Resistant Klebsiella pneumoniae Harboring Plasmid-Mediated MCR-1 Isolated from Tai’an City, China

Klebsiella pneumoniae is a clinically common opportunistic pathogen that causes pneumonia and upper respiratory tract infection in humans as well as community-and hospital-acquired infections, posing significant threats to public health. Moreover, the insertion of a plasmid carrying the mobile colistin resistance (MCR) genes brings obstacles to the clinical treatment of K. pneumoniae infection. In this study, a strain of colistin-resistant K. pneumoniae (CRKP) was isolated from sputum samples of a patient who was admitted to a tertiary hospital in Tai’an city, China, and tested for drug sensitivity. The results showed that KPTA-2108 was multidrug-resistant (MDR), being resistant to 21 of 26 selected antibiotics, such as cefazolin, amikacin, tigecycline and colistin but sensitive to carbapenems via antibiotic resistance assays. The chromosome and plasmid sequences of the isolated strain KPTA-2108 were obtained using whole-genome sequencing technology and then were analyzed deeply using bioinformatics methods. The whole-genome sequencing analysis showed that the length of KPTA-2108 was 5,306,347 bp and carried four plasmids, pMJ4-1, pMJ4-2, pMJ4-3, and pMJ4-4-MCR. The plasmid pMJ4-4-MCR contained 30,124 bp and was found to be an IncX4 type. It was the smallest plasmid in the KPTA-2108 strain and carried only one resistance gene MCR-1. Successful conjugation tests demonstrated that pMJ4-4-MCR carrying MCR-1 could be horizontally transmitted through conjugation between bacteria. In conclusion, the acquisition and genome-wide characterization of a clinical MDR strain of CRKP may provide a scientific basis for the treatment of K. pneumoniae infection and epidemiological data for the surveillance of CRKP.


Introduction
Klebsiella pneumoniae is a clinically common Gram-negative conditional pathogen that can infect humans and a wide range of domestic animals [1]. In healthy populations, K. pneumoniae colonizes mainly the gastrointestinal tract, with a small concentration in the oropharynx [2]. In immunocompromised or immunodeficient patients, K. pneumoniae can invade the blood or tissues and cause a variety of infections, including urinary tract infections, bacteremia, pneumonia, and liver abscesses [3]. In addition, K. pneumoniae is a major pathogen in ventilator-associated pneumonia (VAP) and intensive care unit (ICU)acquired pneumonia [4]. As a result, K. pneumoniae has received widespread attention.
Colistin is considered to be the last line of defense for the treatment of clinically Gram-negative bacterial infections, but the prevalence of colistin resistance has become was tested with a BD Phoenix M50 fully automated microbial drug sensitivity analyzer (BD, Phoenix, AZ, USA) and MIC values were determined according to the manufacturer's instruction. The Kirby-Bauer (KB) disc diffusion test determined the sensitivity of cefotaxime and ceftazidime/avibactam. A single colony was resuspended in a normal saline solution, and the turbidity was 0.46~0. 54. The bacterial solution was uniformly spread on a Mueller-Hinton agar medium (Autobio, Zhengzhou, China) and a drug-sensitive paper sheet (Thermo Fisher, Waltham, MA, USA) was pasted on the agar plate inoculated with the bacteria to be tested and incubated at 35 • C for 24 h. Results were observed and determined by the size of the antibacterial zone. The drug sensitivity results of the two methods were analyzed according to the 2021 CLSI M100 Executive Standard for Antimicrobial Drug Sensitivity Testing [20,21].

Conjugation Test
The broth mating method was used in the conjugation test. In this method, rifampicinresistant E. coli EC600 was used as the recipient, and the KPTA-2108 strain was used as the donor. The donor and recipient strains were inoculated in 3 mL LB broth (Binhe, Hangzhou, China) and incubated overnight at 37 • C. The donor and recipient bacteria were mixed 1:1 and incubated at 37 • C for 16 to 18 h. The mixture was then applied to LB plates with a concentration of 4 µg/mL colistin and 2.5 mg/mL rifampicin resistance and incubated at 37 • C for 24 h. Finally, single colonies were identified using an automated microbial mass spectrometry detection system Autof ms 1000, and MIC values for colistin were determined using a BD Phoenix M50 fully automated microbial drug sensitivity analyzer.

Bacterial DNA Extraction and Sequencing
PacBio sequencing and analysis were conducted by OE Biotech Co. Ltd. (Shanghai, China). In brief, genomic DNA was extracted from bacteria using the CTAB method [22] according to the manufacturer's instructions. The genomic DNA was subjected to quality control by agarose gel electrophoresis and quantified by Qubit (Invitrogen, Waltham, MA, USA). The library was constructed with the SMRTbell Template Prep Kit 1.0 (PacBio, Menlo Park, CA, USA). Single-molecule real-time (SMRT) sequencing was performed on the PacBio Sequel II platform using DNA/Polymerase Binding Kit 3.0 (New England Biolabs, Ipswich, MA, USA). Genome assembly was performed using SMRT Analysis 2.3.0 (Pacific Biosciences, Menlo Park, CA, USA).
In order to understand the epidemic characteristics of pMJ4-4-MCR, the pMJ4-4-MCR gene sequence was searched in the nt library, and 100 similar complete plasmid sequences were obtained. All similar sequences were aligned by Clustal Omega [38], and a phylogenetic tree was generated using iTOL visualization [39]. The accession numbers and names of these 100 plasmids are shown in Supplementary Table S1.
Additionally, to further clarify the drug resistance genes carried by this isolate, the chromosomal sequence of KPTA-2108 was annotated in the CARD database for resistance genes. The names of the specific antibiotic resistance genes in the chromosome of strain KPTA-2108 and their registration numbers in the CARD database are as follows: The virulence genes of KPTA-2108 were annotated using the VFDB database. Classifying virulence genes by function, we found virulence genes associated with adherence, antiphagocytosis, efflux pump, iron uptake, nutritional factor, regulation, secretion system, serum resistance, and toxin (Table S2). In the iron uptake, enterobactin (entA-F, entS, fepA-D, fepG), salmochelin (iroE, iroN), aerobactin (iutA), and yersiniabactin (ybtP) were found. They have been linked to the pathogenicity of K. pneumoniae [40].

Characterization of pMJ4-4-MCR
Plasmid type analysis showed that pMJ4-4-MCR belongs to the IncX4 type, is approximately 30 kb in length and contains only the MCR-1 resistance gene. Structural alignment with 10 plasmids carrying MCR-1 revealed that pMJ4-4-MCR had deletions (Figure 1). Although no IS26 family transposase was annotated in pMJ4-4-MCR, successful binding assays showed that the deleted genes did not influence the delivery of colistin-resistance genes in pMJ4-4-MCR.
Phylogenetic analysis of the pMJ4-4-MCR plasmid is shown in Figure 3. BLAST comparison revealed 100 similar plasmids to pMJ4-4-MCR. The pMJ4-4-MCR was aligned to 100 IncX4 plasmids carrying MCR-1, and a phylogenetic tree was constructed. Plasmids carrying MCR-1 were found to remain significantly homologous between different strains, suggesting that IncX4-type plasmids can be transmitted horizontally among different strains. The phylogenetic tree showed that the IncX4 plasmid carrying MCR-1 was more frequent in E. coli, and the IncX4 plasmids from E. coli strains showed close homology with those from other strains. It is suggested that E. coli may be served as the vector of IncX4 plasmid transmission between different bacteria, and the IncX4-type plasmid carrying MCR-1 in KPTA-2108 may have come from other Enterobacteriaceae bacteria.  Green indicates genes associated with mobile elements, red indicates genes associated with resistance, light yellow indicates genes associated with the T4SS system, orange indicates other functional genes, hypothetical proteins are marked in gray, and regions with high homology are shaded in pink.
Phylogenetic analysis of the pMJ4-4-MCR plasmid is shown in Figure 3. BLAST comparison revealed 100 similar plasmids to pMJ4-4-MCR. The pMJ4-4-MCR was aligned to 100 IncX4 plasmids carrying MCR-1, and a phylogenetic tree was constructed. Plasmids carrying MCR-1 were found to remain significantly homologous between different strains, suggesting that IncX4-type plasmids can be transmitted horizontally among different strains. The phylogenetic tree showed that the IncX4 plasmid carrying MCR-1 was more frequent in E. coli, and the IncX4 plasmids from E. coli strains showed close homology with those from other strains. It is suggested that E. coli may be served as the vector of IncX4 plasmid transmission between different bacteria, and the IncX4-type plasmid carrying MCR-1 in KPTA-2108 may have come from other Enterobacteriaceae bacteria.  The pMJ4-4-MCR (accession number: CP107047) was compared with pMFDS2258.1 (accession number: MK869757.1), pMFDS1318.1 (accession number: MK875282.1), and pMFDS1300.1 (accession number: MK875282.1) for annotation of the genes surrounding MCR-1 and the type IV secretion system (T4SS). Open reading frames are shown as arrows. Green indicates genes associated with mobile elements, red indicates genes associated with resistance, light yellow indicates genes associated with the T4SS system, orange indicates other functional genes, hypothetical proteins are marked in gray, and regions with high homology are shaded in pink.
Phylogenetic analysis of the pMJ4-4-MCR plasmid is shown in Figure 3. BLAST comparison revealed 100 similar plasmids to pMJ4-4-MCR. The pMJ4-4-MCR was aligned to 100 IncX4 plasmids carrying MCR-1, and a phylogenetic tree was constructed. Plasmids carrying MCR-1 were found to remain significantly homologous between different strains, suggesting that IncX4-type plasmids can be transmitted horizontally among different strains. The phylogenetic tree showed that the IncX4 plasmid carrying MCR-1 was more frequent in E. coli, and the IncX4 plasmids from E. coli strains showed close homology with those from other strains. It is suggested that E. coli may be served as the vector of IncX4 plasmid transmission between different bacteria, and the IncX4-type plasmid carrying MCR-1 in KPTA-2108 may have come from other Enterobacteriaceae bacteria.  the different bacterial genera carrying the IncX4 plasmid. The green fragment indicates E. coli, the yellow fragment indicates K. pneumoniae, the orange fragment indicates Salmonella spp., and the blue fragment indicates KPTA-2108.

Discussion
K. pneumoniae is a common hospital-acquired pathogenic microorganism and a serious health problem whose drug resistance has also received wide attention and research. In recent years, isolation of colistin-resistant K. pneumoniae has been conducted in different regions. For example, in 2021, Cao et al. reported the coexistence of plasmid-borne colistin resistance gene MCR-1, carbapenem resistance gene bla NDM-5 , and bla CTX-M-55 in pneumococcal ST485 isolates, and it can be transmitted in Enterobacteriaceae strains [41]. Moreover, some isolates carried multiple MCR genes, including MCR-1, MCR-3 and MCR-8 [42]. In this study, K. pneumoniae strain KPTA-2108 carrying the MCR-1 plasmid was isolated, and whole-genome sequencing of the isolate revealed that the colistin resistance gene MCR-1 was located in the Incx4-type pMJ4-4MCR plasmid. The presence of the MCR-1 on the plasmid poses a barrier to its treatment in the clinic. A combination of experimental and previous findings suggested that IncX4-type plasmids carrying MCR-1 can be transmitted horizontally between strains [43]. Some studies have reported that IncX4 plasmids were highly transmissible, with a transfer frequency 10 2 -10 5 times higher than that of the prevalent IncFII plasmids [44] and that IncX4 plasmids can be stably maintained in host bacteria, suggesting that IncX4-type plasmid may pose a greater threat than other types [45].
The MCR-1 gene has been extensively identified worldwide since it was first reported in China in 2015 [46], especially in animals and animal food. There are many large-scale livestock breeding industries in Shandong Province, and colistin has been used for many years as a growth promoter in livestock feed or in the treatment of bacterial infectious diseases such as bovine mastitis [47], leading to a higher isolation rate of pathogenic strains carrying colistin resistance genes in livestock and poultry compared to humans, and most of them were E. coli strains [48]. The present study also confirmed that E. coli strains in Enterobacteriaceae carrying the MCR-1 resistance gene occurred more frequently than Salmonella and K. pneumoniae, and they were most likely to play a mediating role in the spread of resistance to other strains. As far as we know, this is the first time that K. pneumoniae carrying MCR-1 plasmid has been isolated from a hospital in the Tai'an area, which suggested that this type of pathogenic bacteria may have already spread in the region, so further epidemiological investigation of K. pneumoniae is necessary.
In the case of colistin resistance in KPTA-2108, it was interesting to note that colistin resistance-related genes were annotated both on the plasmid and chromosome. Among them, eptA, rosA, rosB, arnA and pmrE as endogenous genes on the chromosome have been reported to be related to colistin resistance, their expression can change due to mutations in resistant bacteria [49,50]. But their contribution to colistin resistance was not investigated in depth in this study. According to the results of conjugation and the drug sensitivity tests, the MIC of the recipient E. coli EC600 was 4, which was equal to the donor KPTA-2108 (MIC = 4) [51], thus indicating that colistin resistance genes located on the plasmid may have a greater contribution than those on the chromosome. These two different resistance mechanisms were likely to confer synergistic colistin resistance to the K. pneumoniae [52]. Genes on the chromosome can affect progeny bacteria through vertical transmission, but genes on the plasmid can spread horizontally between strains of different genera. This raises the prevalence of colistin resistance in K. pneumoniae and warns us to pay extra attention to the genetic inheritance and evolution of this resistant bacterium. However, it was noteworthy that a recent article reported that MCR-1 was found on chromosomes, which indicates that the gene had been integrated and stabilized within the chromosome [53].
Recent studies suggested that K. pneumoniae had many virulence-related factors, including adherence factors, trophic factors, nutritional factors, secretion systems, and toxins [54]. Among them, increased LPS synthesis and efficiency of iron acquisition through iron carriers were the main factors of enhanced virulence in K. pneumoniae. In order to characterize virulence genes carried on the KPTA-2108, the VFDB database was used to obtain functional annotation information of genes related to virulence. Among the plasmids of KPTA-2108, the T4SS secretion system was annotated in the pMJ4-4MCR plasmid, and it could produce sex hairs (F-hairs) and transfer resistance genes between bacteria [55]. It is also the structural basis of plasmid propagation, which explains the success of the conjugation test [56].
We analyzed the relationship between mobile genetic elements on the plasmids and resistance genes (Table S4). pMJ4-1 carried an abundance of resistance genes, and transposase annotation results showed that the plasmid carried transposases IS26, ISkpn, IS903, and ISEcl1. The resistance genes bla CTX-M-15 , bla TEM-1B , and qnrS1 in pMJ4-3 were all found to be encoded in the complex transposon ISKpn19. These results illustrate the intrinsic linkage of drug-resistance genes and mobile genetic elements on plasmids [57].
Previous studies have shown that the IncX4 plasmid plays an important role in the propagation of the MCR-1 gene in Enterobacteriaceae [58]. IncX4 plasmids carrying MCR-1 showed significant similarity in the backbone [59]. In this study, pMJ4-4MCR was shown to be an IncX4-type plasmid. Functional annotation of the plasmid structure revealed that pMJ4-4MCR encodes the MCR-1 resistance gene and the T4SS secretion system gene and encodes the complete pap2 gene. Previous reports have shown that MCR-1, together with the pap2 gene, is required for reduced colistin sensitivity [60]. These genes may be responsible for the resistance and horizontal transmission that pMJ4-4MCR has [61]. By aligning pMJ4-4MCR with the most similar plasmids for gene annotation, gene deletions were found in pMJ4-4MCR, but this did not affect the horizontal spread of MCR-1.
In conclusion, this study characterized a strain of CPKP isolated for the first time in Tai'an, Shandong Province. With the help of whole-genome sequencing and functional annotation methods, the resistance genes, virulence genes, and mobile genetic elements carried by KPTA-2108 were elucidated. For the IncX4-type pMJ4-4-MCR plasmid, which can be transmitted horizontally, the prevalent characteristics and structural features of pMJ4-4-MCR were analyzed by bioinformatics, and the mechanism of horizontal transfer of pMJ4-4-MCR was attempted to be described. The results showed that KPTA-2108 is an MDR strain with chromosomes carrying an abundance of drug resistance and virulence genes, which is manifested by the multi-drug resistance and pathogenicity to humans of KPTA-2108. IncX4 types carrying MCR-1 are more frequently prevalent in E. coli. pMJ4-4-MCR in the IV secretion system was annotated, and it is closely related to the horizontal transmission of plasmids. The increased of clinically isolated CPKP warns us that the MCR gene has been rapidly spreading before we could prepare for this phenomenon, that more attention should be paid to the prevalence of MCR-1 in livestock, hospital, and community settings, and that efforts are needed to control the spread of MCR-carrying bacteria.
Author Contributions: Q.L. performed the most experiments, analyzed data and wrote the manuscript. Z.G. analyzed data and made figures. G.Z. and N.L. helped analyze the data and revised the manuscript. G.B. and M.J. designed the whole study and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement:
The study was carried out in accordance with the approved guidelines of the Ethics Committee of Tai'an City Central Hospital with written informed consent from all subjects. All the subjects gave written informed consent.

Informed Consent Statement:
Written informed consent has been obtained from the patient(s) to publish this paper.

Conflicts of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.