Resistance of Klebsiella pneumoniae Strains Carrying blaNDM–1 Gene and the Genetic Environment of blaNDM–1

Objective Regional dissemination is the major cause of the widespread prevalence of a plasmid-encoding NDM-1 enzyme. We investigated the drug resistance, joint efficiency, and gene environment of a Klebsiella pneumoniae strain carrying blaNDM–1 gene. Materials and Methods Carbapenem-non-susceptible strains were analyzed using the VITEK 2 Compact. Strains carrying blaNDM–1 were identified using polymerase chain reaction and sequencing. Antimicrobial susceptibility testing and plasmid conjugation experiments were then conducted. Strains carrying blaNDM–1 were subjected to Southern blot analysis. After the gene mapping of blaNDM–1, library construction, and sequencing, plasmids were subsequently spliced and genotyped using the software Glimmer 3.0, and then analyzed using Mauve software. Results Among 1735 carbapenem-non-susceptible strains, 54 strains of blaNDM–1-positive bacteria were identified, which consisted of 44 strains of K. pneumoniae, 8 strains of Acinetobacter baumannii and 2 strains of Escherichia coli. Strains carrying blaNDM–1 had a resistance rate of more than 50% in most antibiotics. Plasmid conjugation between strains carrying blaNDM–1 and E. coli strain J53 had a success rate of 50%. Southern blot analysis indicated that each strain had multiple plasmids containing blaNDM–1. Among the five plasmids containing blaNDM–1 in K. pneumoniae for sequencing, two plasmids with complete sequences were obtained. The findings were as follows: (i) The p11106 and p12 plasmids were highly similar to pNDM-BTR; (ii) the p11106 and p12 plasmids showed differences in the 20–30 kb region (orf00032–orf00043) from the other six plasmids; and (iii) blaNDM–1 was located at orf00037, while ble was found at orf00038. Two tnpA genes were located in the upstream region, and orf00052 (tnpA) in the 36 kb region was in the downstream sequence. Conclusion blaNDM–1-containing bacteria exhibit multidrug resistance, which rapidly spreads and is transferred through efficient plasmid conjugation; the multidrug resistance of these bacteria may be determined by analyzing their drug-resistant plasmids. The presence of ble and tnpA genes suggests a possible hypothesis that blaNDM–1 originates from A. baumannii, which is retained in K. pneumoniae over a long period by transposition of mobile elements.


INTRODUCTION
The clinical application of sulfa drugs can be traced back to the 1930s, which marked a new era of antimicrobial therapy. Once exposed to antibacterial drugs, bacteria spontaneously change their metabolic pathways or produce corresponding inactivating substances to resist antibiotics, exhibiting drug resistance. Notably, the abuse of antibiotics poses selective pressure of bacteria, conferring a survival advantage on drug-resistant bacteria. Consequently, numerous drug-resistant bacteria are spread in different pathogens.
New Delhi metallo-β-lactamase1 (NDM-1), also known as metallo-β-lactamase or metal-β-lactamase, was first isolated from a highly infectious and pathogenic multidrug-resistant strain of Klebsiella pneumoniae in 2009 (Yong et al., 2009). Cases of infection with the bla NDM-1 gene have subsequently been reported in more than 20 countries and regions worldwide, including the United Kingdom and India (Kumarasamy et al., 2010). The therapeutic efficacy of multiple antibiotic treatments for bacteria carrying bla NDM-1 is usually unsatisfactory. Therefore, bacterial strains carrying bla NDM-1 are also called superbugs. With a prolonged length of stay, bacteria-carrying bla NDM-1 have a higher probability of being isolated from stool samples. However, infection with bla NDM-1 cannot be determined from clinical symptoms and signs (Bush and Fisher, 2011). Superbugs pose a serious challenge to antibiotic therapy.
The bla NDM-1 gene is mainly distributed in plasmids and occasionally in the chromosomes of Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis (Girlich et al., 2015;Rahman et al., 2015;Shen et al., 2017). In clinical practice, bla NDM-1 plasmids from different bacterial species isolated from the same patient typically have a similar structure, suggesting the significance of plasmids in the spread of bla NDM-1 . Plasmids containing bla NDM-1 vary in size from 30-300 kb, and exist in different types, such as IncA/C, IncL/M, and IncR (Carattoli et al., 2015;Gamal et al., 2016). Specifically, IncA/C has a wide range of hosts and can exist in multiple strains, such as Enterobacteriaceae, Pseudomonas, Acinetobacter, and Vibrio cholerae, providing convenience for bla NDM-1 in different species of bacterial hosts (Wailan and Paterson, 2014).
The sequences and genetic modes of bla NDM-1 have been identified in previous studies. However, the bla NDM-1 gene environment is yet to be determined. In addition, studies mostly focus on bla NDM-1 in Acinetobacter (Bontron et al., 2016;Wang et al., 2017) and rarely on bla NDM-1 carried by K. pneumoniae. In the present study, 1735 carbapenem-nonsusceptible bacteria were collected from The First Affiliated Hospital of Nanchang University. These strains were sequenced, conjugated, and compared with the corresponding plasmids. This study aimed to analyze the differences in the bla NDM-1 gene environment and provide directions in clarifying the origin and propagation of the bla NDM-1 gene.

Sample Collection and Identification
Approval was obtained from the Medical Ethics Committee of The First Affiliated Hospital of Nanchang University and informed consent was obtained from each subject. Carbapenemnon-susceptible bacteria were then collected from The First Affiliated Hospital of Nanchang University from January 2013 to December 2016. A total of 1735 carbapenem-nonsusceptible bacteria were isolated and then identified using VITEK 2 Compact (Pioneering Diagnostics, France) at the Microbiology Laboratory at The First Affiliated Hospital of Nanchang University.

Polymerase Chain Reaction and Sequencing
Polymerase chain reaction (PCR) templates were prepared by boiling. Fresh bacteria were harvested and diluted in 500 µL ddH 2 O for 10 min in a boiling water bath. The supernatant was collected as PCR templates. Subsequently, a PCR system with a total volume of 50 µL was prepared, consisting of 25 µL of Taq Mix (Takara, Dalian), 2 µL of forward primer, 2 µL of reverse primer, 2 µL of template, and 19 µL of ddH 2 O. Subsequently, 29 cycles of PCR were conducted. The bla NDM-1 primer sequences were forward 5'-GGCGGAAGGCTCATCACGA-3' and reverse 5'-CGCAACACAGCCTGACTTTC-3'. The amplified product was 287 bp.
The PCR products were analyzed using electrophoresis with 1% agarose gel and 1 × TAE at 120 V for 25 min. Strains verified by sequencing to contain bla NDM-1 were used as markers. Electrophoresis results were obtained using an ultraviolet (UV) transilluminator. Positive PCR products were sequenced (Synbio Technology, Suzhou, China), and the sequencing results were compared using the software BLAST. Strains that were positive for PCR and matched the sequencing results were identified as those containing bla NDM-1 .

Plasmid Conjugation
The receptor strain was sodium azide-resistant E. coli strain J53. The donor and receptor strains were implanted in the Mueller-Hinton plate and cultured at 37 • C for 16-18 h. Strains in appropriate amounts were inoculated in a glass tube containing 5 mL of LB medium and then cultured at 37 • C for 16-18 h. Subsequently, 400 µL of the donor strain and 200 µL of the receptor strain were added to a glass tube containing 800 µL of LB broth medium and then cultured at 37 • C for 16-18 h. Meanwhile, the donor strain, screened in 180 µg/mL sodium azide, and the receptor strain, screened in 0.5 µg/mL of imipenem, were used as blank controls. Exactly 100 µL of the aforementioned mixture was added to the Mueller-Hinton plate and cultured at 37 • C for 16-18 h. Conjugation strains in good condition were ultimately identified using the VITEK 2 compact automatic microbial identification instrument.

Southern Blot Analysis
Southern blot analysis was conducted using 1% agarose gel with 1 × TAE and run on 120 V electrophoresis for 40 min. The gel was incubated in 0.25 mol/L HCl for 15 min, 0.5 mol/L NaOH for 20 min twice, and 0.1 mol/L phosphate buffer for 15 min twice. Membrane transfer was conducted in a 20 × saline-sodium citrate buffer. The membrane was washed in a 2 × saline-sodium citrate buffer and then dried in a baking oven at 80 • C for 2 h. PCR products carrying bla NDM-1 were labeled using the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche, United States). DIG-labeled DNA products were prepared and examined using Southern blot analysis, and images were obtained using a UV transilluminator.

Plasmid Sequencing
Five qualified plasmids containing bla NDM-1 were used to construct a sequencing library. These 5 vectors were from 4 strains, which showed high resistance to the antibiotics and successfully conjugated with E. coli J53. The data are provided as Supplementary Material and Supplementary Table S2. Briefly, 1 µg of plasmid was placed in a Covaris tube, and the DNA was separated into 400 bp fragments using Covaris S2 (Covaris, United States). Small DNA fragments were generated for library construction using the NEXTflex DNA Sequencing Kit compatible with Biomek FXp (Bio Scientific, United States). The library fragments were subjected to pairedend sequencing (2 × 150 bp) on the HiSeq2500 Sequencing System (Illumina, United States).
Clean reads after pre-processing were assembled using Velvetver.1.2.03 software. Gene prediction and annotation analyses were performed using Glimmer 3.0 software. The p11106 and p12 plasmids were compared with the plasmid sequences without bla NDM-1 of the seven species. Similarities in the plasmid sequences were depicted using Mauve software. Gene functions in different regions were annotated and analyzed.

Screening and Identification of Strains
Carrying bla NDM-1 Among the 1735 carbapenem-non-susceptible strains harvested in this experiment, 54 strains (3.1%) were bla NDM-1 -positive. These strains consisted of 44 strains of K. pneumoniae, 8 strains of A. baumannii, and 2 strains of E. coli. The bla NDM-1 gene was not found in P. aeruginosa, Enterobacter cloacae, Bacillus, Maltophilia, or Pseudomonas cepacia. All sequencing results of the 54 strains were consistent with the NCBI database 1 . The positive rates of each strain are listed in Table 1.
These 54 multidrug-resistant strains were obtained from 43 patients at The First Affiliated Hospital of Nanchang University from January 2013 to December 2016. The patients were from different cities and provinces. Temporal and regional differences suggested that the same strains could not be causing the outbreak. Patient records are shown in Supplementary Table S1.

Determination of Drug Resistance
The resistance rate of the strains carrying bla NDM-1 exceeded 50% in most antibiotics (Figure 1). The tested antibiotics exhibited nearly 100% resistance to imipenem and more than 1 http://blast.ncbi.nlm.nih.gov/Blast.cgi  Frontiers in Microbiology | www.frontiersin.org 90% resistance to meropenem and piperacillin. Meanwhile, the bla NDM-1 -positive strains showed the lowest resistance (40%) to amikacin.

Plasmid Conjugation
The plasmid conjugation experiment was conducted on all bla NDM-1 -positive strains and J53. A total of 27 strains (21, K. pneumoniae strains; 5, A. baumannii strains; and 1, E. coli strain) successfully conjugated their plasmids containing the bla NDM-1 gene ( Table 2). The success rate of plasmid conjugation was 50%.

Location of the bla NDM-1 Gene
Bla NDM-1 -positive strains that were successfully conjugated were subjected to Southern blot analysis to detect the location of bla NDM-1 . The results further demonstrated the conjugation of bla NDM-1 in E. coli strain J53 (Figure 2).

Sequencing of Plasmids
Plasmid libraries were constructed from genomic DNA. Qualified plasmid DNAs were sequenced, and the results suggested excellent qualifications for library construction and sequencing (Table 3). After removal of DNA from host genes and other plasmid DNAs, approximately 20% of the reads were extracted from target plasmid DNAs with over 2000× coverage. Subsequently, the clean reads of each plasmid were synthesized using Velvet. Large-fragment sequence assembly is presented in Table 4. Although coverage was sufficiently high (>2000×), the synthesis was not satisfactory, with the contigs between 33 and 161. Thus, artificial gap closing with the KU862632.1 strain was conducted using the Cytoscape platform, and two complete plasmid (p12 and p11106) sequences were generated. Synthesis of p243323, p32, and p7-1973 failed ( Table 4).
The size of p12 was the same as that of p11106 (58757 bp). Cytosine was located on 50396 bp of p11106, thymine was located in p12, and the remaining regions were the same. In the following gene prediction and annotation analysis, p12 was used as an example. As   depicted in Figure 3, 46 genes were reverse transcribed, and 39 were forward transcribed without chain specificity (p = 0.627). We uploaded the sequencing data to the NCBI database under the SRA (Sequence Read Archive) accession number PRJNA596354.

Comparative Analysis of the bla NDM-1 Gene Environment
Plasmid sequences from seven species were subjected to similarity analysis using Mauve software. The baseline characteristics of the selected plasmids are listed in Table 5. p11106 and p12 were compared with the aforementioned plasmids, and their similarity information is depicted in Figure 4 and Table 6.
In addition to the plasmid pNDM-BTR, differences between p11106 and p12, and the remaining seven plasmids were  mainly enriched in the 20-30 kb region. Although the plasmid pMR3-OXA181 was not absent in this region, it exhibited a considerably low homology to p11106 and p12.
The corresponding genes mapping this 10 kb region were orf00032-orf00043. Moreover, gene annotation showed that two genes (TnpA and a mobile element protein) with similar functions were located in the upstream sequences of this region. Bla NDM-1 was also located in these sequences (orf00037). IMP-4 and β-lactamase were absent in this region. The gene annotations of orf00032-orf00043 are presented in Table 7.
As shown in Table 7, (i) the p11106 and p12 plasmids were highly similar to pNDM-BTR; (ii) the p11106 and p12 plasmids showed differences in the 20-30 kb region (orf00032-orf00043) from the other six plasmids; and (iii) bla NDM-1 was located in the middle of the orf00032-orf00043 region, whereas ble was found nearby. Two tnpA genes were on the top, and orf00052 (tnpA) in the 36 kb region was in the downstream sequence.
Our experiment obtained 27 successfully conjugated J53 strains resistant to sodium azide, which is consistent with previous studies (Wailan et al., 2015;Kocsis et al., 2016). Conjugation with K. pneumoniae, A. baumannii, and E. coli as donor plasmids containing bla NDM-1 achieved a success rate of approximately 50%. Southern blot analysis confirmed that bla NDM-1 was mainly expressed in plasmids, and each drug-resistant strain carrying bla NDM-1 could contain multiple bla NDM-1 -positive plasmids. The bla NDM-1 -positive plasmids exhibited a relatively strong ability for conjugation transfer. Specifically, IncA/C showed adaptation to a wide range of hosts. It can be found in Enterobacteriaceae, Pseudomonas, Aeromonas, Vibrio cholera, and other bacterial genera, and can be transmitted within or between strains through conjugation (Wailan and Paterson, 2014;Carattoli et al., 2015;Gamal et al., 2016).
Sequencing of five plasmids obtained from the collected K. pneumoniae strains was highly qualified (coverage >2000×). Regardless, the synthesis of p243323, p32, and p7-1973 failed; only p11106 and p12 were successfully synthesized. This result could be explained by the complexity of the plasmid structure and interruption of the host genome and other plasmids during plasmid extraction. Among the sequencing reads, only 20-30% of the plasmids were identified to carry thebla NDM-1 gene, significantly increasing the difficulty of synthesis. The only difference between p11106 and p12 was the 50396 bp (orf00074 was mapped) region, where c.587T > C occurred in p11106 and thus resulted in p.196Ile > Thr. A total of 85 genes were contained in the p11106 sequence, and most of them had functions that could be identified using BLAST.
The structures of plasmids p11106 and p12 were similar, and their strains Kp.11 and Kp.12 were also similar in resistance (Supplementary Table S2). This finding suggests that resistant plasmids are a key factor in determining the drug resistance of strains.
The genetic environment of a certain gene helps reveal the origin and genetic characteristics of the gene. Poirel et al. (2011) uncovered the full-length or truncated ISAba125 sequences in the upstream region of bla NDM-1 and conserved bleomycin (ble) resistance genes in the downstream region. They hypothesized that ISAba125 sequences are carried by bla NDM-1 when it transfers from the original host, and the bla NDM-1 and ble genes come from the same original host strain . In 2012, Toleman analyzed all available bla NDM-1 -associated sequences and found that ISAba125 sequences are always present in the 100 bp upstream region of bla NDM-1 , and demonstrated that bla NDM-1 is a chimera. The chimeric process occurs in A. baumannii and is mediated by ISAba125 (Jones et al., 2014(Jones et al., , 2015Toleman et al., 2015). Our sequencing results identified certain differences in the 20-30 kb region between p11106, p12, and the compared plasmids, except for pNDM-BTR. On the basis of the aforementioned findings, we propose the following: (a) p11106 and p12 are structurally similar to pNDM-BTR, indicating that p11106 and p12 may present characteristics of pNDM-BTR (McGann et al., 2015); (b) the differences in the 20-30 kb region between p11106, p12, and the compared plasmids support the theory of gene polymorphism (Potron et al., 2011;Mata et al., 2012;Datta et al., 2017); (c) the existence of ble indicates that bla NDM-1 may originate from A. baumannii; and (d) the presence of two TnpA in the upstream region of bla NDM-1 and orf00052 (TnpA) in the 36 kb downstream region suggests that bla NDM-1 is acquired from plasmid transposition and long-term preservation (Campos et al., 2015;An et al., 2016;Khong et al., 2016).
This experiment screened drug-resistant strains carrying bla NDM-1 gene from carbapenem-non-susceptible bacteria collected from The First Affiliated Hospital of Nanchang University between January 2013 and December 2016. We assessed their drug resistance and plasmid conjugation transfer ability. Two complete plasmid sequences were obtained after sequencing analysis of five bla NDM-1 -positive plasmids isolated from K. pneumoniae and compared with bla NDM-1 -negative plasmids with known sequences. However, data on abundance were limited owing to the small sample size of drug-resistant strains as well as complexity and interferences during plasmid synthesis. In addition, the conclusion drawn from sequencing in this study is yet to be verified by molecular experiments. Gene deletion (IMP-4 and β-lactamase) occurred in p11106 and p12. The presence of a potential relationship between gene deletion and bla NDM-1 requires further study.

CONCLUSION
In conclusion, NDM-1-resistant bacteria exhibit multidrug resistance and spread to a certain extent in the hospital via efficient plasmid conjugation transfer. K. pneumoniae may be an important intermediate retention host in the dissemination process. Nosocomial bla NDM-1 -carrying bacteria are of concern.
Drug-resistant plasmids may be a key factor in determining drug resistance. The p11106 and p12 plasmids containing the bla NDM-1 gene isolated from K. pneumoniae exhibit the characteristics of the pNDM-BTR plasmid and have a polymorphic gene environment. The existence of ble in the surrounding environment of bla NDM-1 suggests that bla NDM-1 is derived from A. baumannii. Meanwhile, the presence of TnpA in both the upstream and downstream regions of bla NDM-1 indicates that bla NDM-1 may be acquired from gene transposition and persists over a long period in K. pneumoniae strains.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
Written informed consent was obtained from all participants, and the purpose and procedures of the study were explained to them. Ethical approval for this study was obtained from the Institutional Review Board of The First Affiliated Hospital of Nanchang University.

AUTHOR CONTRIBUTIONS
TX and CC designed the study, collected and analyzed the data, and drafted the manuscript. TX, CC, JW, YL, and QZ contributed to the performance of the experiment and data collection. NC and XW reviewed the study design. WZ contributed to the review of data analysis and data interpretation. All authors have approved the final version of the manuscript.