Comparative genomic analysis of Klebsiella pneumonia (LCT-KP214) and a mutant strain (LCT-KP289) obtained after spaceflight
- Yinghua Guo†1,
- Yinhu Li†2,
- Longxiang Su†3,
- De Chang1, 5,
- Wenbin Liu2,
- Tong Wang2,
- Yanting Yuan2,
- Xiangqun Fang1,
- Junfeng Wang1,
- Tianzhi Li1,
- Chengxiang Fang4,
- Wenkui Dai2Email author and
- Changting Liu1Email author
© Guo et al.; licensee BioMed Central Ltd. 2014
Received: 5 October 2013
Accepted: 7 July 2014
Published: 12 July 2014
With the development of space science, it is important to analyze the relationship between the space environment and genome variations that might cause phenotypic changes in microbes. Klebsiella pneumoniae is commonly found on the human body and is resistant to multiple drugs. To study space-environment-induced genome variations and drug resistance changes, K. pneumoniae was carried into outer space by the Shenzhou VIII spacecraft.
The K. pneumoniae strain LCT-KP289 was selected after spaceflight based on its phenotypic differences compared to the ground-control strain. Analysis of genomic structural variations revealed one inversion, 25 deletions, fifty-nine insertions, two translocations and six translocations with inversions. In addition, 155 and 400 unique genes were observed in LCT-KP214 and LCT-KP289, respectively, including the gene encoding dihydroxyacetone kinase, which generates the ATP and NADH required for microbial growth. Furthermore, a large number of mutant genes were related to transport and metabolism. Phylogenetic analysis revealed that most genes in these two strains had a dN/dS value greater than 1, indicating that the strain diversity increased after spaceflight. Analysis of drug-resistance phenotypes revealed that the K. pneumoniae strain LCT-KP289 was resistant to sulfamethoxazole, whereas the control strain, LCT-KP214, was not; both strains were resistant to benzylpenicillin, ampicillin, lincomycin, vancomycin, chloramphenicol and streptomycin. The sulfamethoxazole resistance may be associated with sequences in Scaffold7 in LCT-KP289, which were not observed in LCT-K214; this scaffold contained the gene sul1. In the strain LCT-KP289, we also observed a drug-resistance integron containing emrE (confers multidrug resistance) and ant (confers resistance to spectinomycin, streptomycin, tobramycin, kanamycin, sisomicin, dibekacin, and gentamicin). The gene ampC (confers resistance to penicillin, cephalosporin-ii and cephalosporin-i) was present near the integron. In addition, 30 and 26 drug-resistance genes were observed in LCT-KP289 and LCT-KP214, respectively.
Comparison of a K. pneumoniae strain obtained after spaceflight with the ground-control strain revealed genome variations and phenotypic changes and elucidated the genomic basis of the acquired drug resistance. These data pave the way for future studies on the effects of spaceflight.
KeywordsKlebsiella pneumoniae Comparative genomic analysis Virulence gene Resistance gene
With the rapid development of space technology, space capsules are frequently launched to explore the universe; therefore, it is important to understand space biology. Space environmental physics has elucidated the existence of properties including electromagnetic radiation, microgravity, high vacuum and strong magnetic field in the space environment; however, it is important to understand the impact of these factors on organisms. Paul et al. focused on plants, which are significant components of biological systems, and discussed the adaption and growth tropism of plants in the microgravity environment in a space shuttle . Gridhani et al. examined proton-induced perturbations in gene expression, cell cycle and cell division as well as the differences between the effects of protons and high-energy proton radiation [2, 3]. Gao et al. observed that bacterial metabolism was significantly altered in the space environment ; furthermore, exposure to the space environment might cause genetic damage . Tixador et al. studied the growth and antibiotic resistance of Escherichia coli during the mission of the space shuttle Discovery . However, mutations caused by the space environment have not been examined at the genomic level. Klebsiella pneumoniae is an important Gram-negative, opportunistic pathogen that causes severe diseases such as septicemia, pneumonia, urinary tract infections, and soft-tissue infections . Many clinical strains of K. pneumoniae are highly resistant to antibiotics, which poses a major threat to global public health. Over the past decade, the physiology, biochemistry, and regulation of K. pneumoniae pathways have been extensively studied [8–11]. However, the effect of spaceflight on K. pneumoniae has not been examined at the genomic level. K. pneumoniae is well-suited for such studies because of its characteristics.
In 2011, the Shenzhou VIII spacecraft carried K. pneumoniae strains into outer space for approximately 17 days (398 hours). The control strain was cultured at the same temperature in an incubator on earth. After spaceflight, the antibiotic resistance and pathogenicity of the strains were examined. Based on these analyses, the LCT-KP289 strain obtained after spaceflight was selected and compared to the control strain LCT-KP214. The genomes of LCT-KP289 and LCT-KP214 were sequenced to compare their genomic variations. These analyses revealed genes potentially related to drug resistance, and analysis of the putative drug-resistance genes revealed variations in the homologous genes in the two strains. Studies on these candidate resistance genes will be important to improve understanding of the drug resistance of K. pneumoniae.
Genomic features of the strains LCT-KP214 and LCT-KP289
Sequence assembly data for LCT-KP214 and LCT-KP289
Average gene length
Genome coding percentage
Statistics of plasmid alignment results in the two strains
Klebsiella pneumoniae plasmid pKPN_CZ
Escherichia coli plasmid NR1
Klebsiella pneumoniae plasmid pNDM10469
Klebsiella pneumoniae plasmid pKPN_CZ
Klebsiella pneumoniae plasmid pNDM-KN
Escherichia coli plasmid NR1
Escherichia coli IncN plasmid N3
Bacillus megaterium QM B1551 plasmid pBM400
Enterococcus faecium plasmid pM7M2
Enterococcus faecium plasmid p5753cA
Detection of genomic structural variations and functional enrichment of variant genes
In addition to the large-scale genome variations, 770 SNPs were identified (3,133 raw SNPs; 2,363 SNPs containing N bases, nearly N bases or with low sequencing depth were filtered). To analyze the genes affected by SNPs, we selected SNPs located in CDS or intergenic regions no further than 300 bp from the nearest gene. We observed that 334 and 330 genes were affected by SNPs in LCT-KP289 and LCT-KP214, respectively. Furthermore, 99 InDels were also identified (133 raw InDels; 34 InDels containing Ns or with low sequencing depth were filtered). InDel-affected genes were identified using the method described for SNPs; 299 and 304 genes were affected by InDels in LCT-KP289 and LCT-KP214, respectively (not all InDels were located in CDS regions in both strains). Based on the GO annotations, 48 and 49 functional categories were enriched in the genes containing SNPs and InDels, respectively, in the strain LCT-KP289. Most of the affected genes encoded molecular functions related to transmembrane transporters (Additional file 1: Table S1). Based on KEGG annotations, the unique gene KP214_00701 in LCT-KP214 was assigned the function “Glycerolipid metabolism (ko00561)” [Figure 2C]; this gene contained 12 SNPs and 5 InDels. KP214_00701 encodes dihydroxyacetone kinase (dha), which is required to generate ATP and NADH for microbial growth . However, this gene was not functional in LCT-KP289 because of a nonsense mutation (GAA- > TAA). This mutation in LCT-KP289 might have been caused by the severe environmental changes during spaceflight.
Analysis of phylogenetic relationships
Statistics of the annotation results for heterozygotic SNPs
Start codon nonsyn
Stop codon nonsyn
Start codon syn
Stop codon syn
SNP within CDS
Characterization of drug-resistance and related genes
Based on the assembly results, the genome of LCT-KP289 was larger than that of LCT-KP214; genome alignment and prediction of genome elements revealed that the extra sequences in LCT-KP289 were from tandem repeat fragments and interspersed repeated sequences. Analysis of genome variation revealed that LCT-KP289 contained more unique genes than LCT-KP214. GO functional annotation revealed that the unique genes of LCT-KP289 were enriched in 9 functional items, including oxidation-reduction, biofilm formation, and arginine catabolism. Therefore, the genomic changes in LCT-KP289 were related to environmental adaptation. We analyzed the evolutionary rate and phylogenetic relationships of LCT-KP289. The base frequencies of the core genomes revealed a greater number of heterozygotic SNPs in LCT-KP289 compared to LCT-KP214. The phylogenetic trees at the genome and gene levels indicated that LCT-KP289 had a greater base-substitution rate. The dN/dS analysis revealed that most genes in LCT-KP289 had dN/dS values greater than 1. Together, these data indicated that special environmental factors can stimulate base substitutions in bacteria and that environmental selection pressure might influence the direction of bacterial evolution .
To identify the genes that were most affected by the environment, the variant genes in LCT-KP214 and LCT-KP289 were annotated using the COG, KEGG and GO databases. These annotations revealed that some functional categories were enriched in LCT-KP289 compared to LCT-KP214; these categories were related to bacterial metabolism such as “Lipid transport and metabolism”, “Inorganic ion transport and metabolism” and “Secondary metabolites biosynthesis, transport and catabolism”. However, using KEGG annotation, LCT-KP289 lacked the dihydroxyacetone kinase gene, which is important for the generation of ATP and NADH, because of a premature stop codon . Therefore, we propose that, in LCT-KP289, mutations occurred more frequently in genes related to environmental adaptation.
K. pneumoniae is highly pathogenic and encodes multiple types of drug resistance genes. In this study, we analyzed the drug-resistance phenotypes of LCT-KP214 and LCT-KP289. Both strains were resistant to several drugs, except sulfamethoxazole, and LCT-KP289 was more resistant to this drug than was LCT-KP214. Based on genome alignment and ARDB annotation, we observed that LCT-KP289 contained an extra copy of the sul1 gene in scaffold7. sul1 encodes an alternative form of dihydrofolate synthetase, which cannot be inhibited by the drug . Therefore, environmental selection pressure might cause genome variations, leading to an additional copy of the sul1 gene in LCT-KP289 and subsequent enhanced drug resistance.
Furthermore, we observed gene elements related to antibiotic resistance in K. pneumoniae. Integron determinants, including gene cassettes and several antibiotic-resistance genes (e.g., emrE, ant and sul1), were observed in both strains. Antibiotics can be divided into four classes based on their mechanism of action: i) repression of bacterial cell-wall synthesis, ii) destruction of the cell membrane structure, iii) repression of protein synthesis, and iv) repression of DNA synthesis. emrE encodes an efflux pump that confers multidrug resistance . The protein encoded by the gene ant potentially modifies aminoglycosides by adenylation, conferring resistance to protein synthesis inhibitors such as spectinomycin, streptomycin, tobramycin, kanamycin, and gentamicin . Because integrons are mobile genetic elements, the antibiotic genes contained in integrons can be transferred among different strains. Based on plasmid annotation, we observed that the integrons in LCT-KP289 and LCT-KP214 might originate from Escherichia coli plasmids. We observed the gene ampC, which encodes beta-lactamase, flanking the integrons. This enzyme can inactivate cell-wall synthesis inhibitors such aspenicillin and cephalosporin . Furthermore, the Tn21 resolvase was observed near the integrons, which might enhance the mobility of the antibiotic-resistance genes. Other antibiotic-resistance genes, including those related to polymyxin and bacitracin, were dispersed in the genome of K. pneumoniae.
In this study, comparative genomics was used to analyze the strain LCT-KP289, which was selected after spaceflight, and the ground-control strain LCT-KP214 to identify the relationship between the unique space environment and genome variation; furthermore, the potential rate of evolution was studied using phylogenetic analysis. We observed that the space environment affected the evolutionary rates of environment-related genes. Phenotypic analysis revealed that LCT-KP289 had increased resistance to sulfonamides, potentially due to an increased copy number of the gene sul1. Furthermore, the observation of integrons in K. pneumoniae provides insight into the mechanism of multidrug resistance. The HMP data have shown that the bacteria were multidrug resistant and highly pathogenic. Therefore, this study paves the way for future studies on the effect of spaceflight on drug resistance and pathogenicity.
Bacterial strains and growth conditions
The K. pneumoniae strain (CGMCC 1.1736) used in this study was obtained from the Chinese General Microbiological Culture Collection Center and was carried in the Shenzhou VIII unmanned spacecraft for more than 17 days (398 hours). This strain is NCTC 5056, which was clinically isolated from patients diagnosed with pneumonia. The strains were cultured in LB, which contained tryptone (10 g/liter), yeast extract (5 g/liter), NaCl (10 g/liter), and agar powder (15 g/liter). The pH of the culture medium was adjusted to 7.0-7.2. The culture broth was identical to the culture medium but lacked Bacto Agar. The Shenzhou VIII unmanned spacecraft was launched by a Long March 2 F rocket at 5:58 on November 1, 2011 (GMT + 8). It arrived at the Tiangong-1 space station (approximately 340 km apogee distance) and successfully docked with the space station at 17:28(GMT + 8). After docking, the Shenzhou VIII spacecraft and Tiangong-1 remained connected for 12 days at an approximate apogee distance of 350 km. The Shenzhou VIII spacecraft successfully separated from the Tiangong-1 space station and docked again on November 14. Subsequently, the Shenzhou VIII spacecraft remained with the Tiangong-1 space station for three more days. The return capsule of the Shenzhou VIII spacecraft completed the experimental task and left the Tiangong-1 space station on November 17. It landed in the Inner Mongolia region at 20:38 on November 17 (GMT + 8). Microbiological samples were quickly removed and transported to Beijing by military helicopters. The samples arrived at the laboratory of the Chinese PLA General Hospital at 7:17 on November 18 (GMT + 8). As a control, the same K. pneumoniae strain was maintained in a laboratory incubator on earth under the same temperature conditions as the cabin of the Shenzhou VIII spacecraft. The temperature conditions were adjusted according to the spacecraft conditions. During spaceflight, the temperature fluctuated between 16-21°C. Because the warehouse was not equipped with a radiation-measuring device, we were unable to obtain relevant data. After the Shenzhou VIII spacecraft landed, the bacterial colonies were randomly selected from plates coated with K. pneumoniae. Phenotypic analyses, including disk diffusion tests and growth curves, were performed to compare the spaceflight clones and the ground control strain (Additional file 3: Figure S1). The ground control strain was named LCT-KP214. One clone that was obtained after spaceflight and was significantly different from LCT-KP214 was named LCT-KP289.
Growth curve and antibiotic-resistance analyses
The strains were grown on LB liquid medium for 18 h at 37°C. Approximately 20-μl suspensions were inoculated into microtiter plates (honeycomb plates) containing 350 μl LB broth and detected by Bioscreen C (Lab Systems, Helsinki, Finland) at 37°C with continuous shaking. The growth of each sample was monitored by measuring the optical density at 600 nm (OD600) at three time points. A well containing 370 μl LB was included as a negative control. The growth curve of each strain was generated based on the OD600 measurements. The K. pneumoniae strains were transferred from the plate culture system to 1.5-ml centrifuge tubes containing 1 ml of physiological saline, and the concentrations of the bacterial suspension were diluted to 107 ~ 108 bacteria per ml. Culture plates were then coated with 100 μl of bacterial suspension of each strain. Tablets of filter paper were moistened with 17 different antibiotics, including benzylpenicillin, ampicillin, cefazolin, ceftazidime, ceftriaxone sodium, azithromycin, ciprofloxacin, lincomycin, vancomycin, the pediatric compound sulfamethoxazole, chloramphenicol, cefoperazone sodium, amikacin, streptomycin, minocycline, meropenem, and piperacillin. The tablets were then placed on the surface of the culture plates. Each plate contained three types of antibiotics, and two tablets were used for each antibiotic. The plates were then incubated at 37°C for 18 ~ 24 h, and the diameters of the inhibition zones were measured and recorded. The diameter of the tablets was 6 mm.
DNA sample preparation and sequencing
K. pneumoniae genomes were sequenced using an Illumina Hiseq2000 with a multiplexed protocol. Paired-end reads of 90 bp each were generated from 500-bp and 6-kb random sequencing libraries for the control strain LCT-PK214 and the strain obtained after spaceflight, LCT-PK289. We filtered the raw data in four steps: removing reads with 5 bp of Ns, removing reads with 20 bp of low-quality (≤Q20) bases, removing adapter contamination, and removing duplicate reads. Finally, 100X and 50X filtered paired-end reads were obtained for the 500-bp and 6-kb libraries, respectively. The assembly was performed using the SOAPde novo algorithm [26, 27] (http://soap.genomics.org.cn/soapdenovo.html, version: 2.04). Local assembly and gap closure were performed on paired-end reads located in gaps. For highly complex regions, PCR gap closure was performed to obtain sequences without outer gaps. Finally, the SOAPaligner/soap2 software was used for error correction [28, 29] (http://soap.genomics.org.cn/soapaligner.html, version 2.21). The reads were mapped to the sequence, mapping information was recoded, and single-base and local proofreading were performed to analyze the assembly results.
Analysis of genomic components and identification of variations
The sequence of the query strain LCT-PK289 was compared to the reference sequence LCT-PK214 using Mummer  (http://mummer.sourceforge.net, version 3.22) and LASTZ [31, 32] (http://www.bx.psu.edu/miller_lab/dist/README.lastz-1.02.00, Version: 1.01.50). We used Mummer for chain stander and start site selection, and LASTZ was used for detailed alignment. Next, the syntenic regions and structural variations, including deletions, insertions, inversions and translocations, were identified in the alignment blocks . SNPs were identified by measuring the distances between mismatched sites in syntenic regions. SNPs located in sequence gaps, repeat regions, or scaffold ends were discarded. To validate the results of the non-redundant candidate SNPs in the genomes, the high-quality, paired-end reads were first mapped to the corresponding genomes using SOAPaligner [28, 29] (http://soap.genomics.org.cn, version 2.21). Next, the numbers of the most abundant (n1) and second-most abundant (n2) nucleotides at each SNP position in each strain (counted according to the number of reads in each strain supporting this nucleotide) were examined. High quality SNPs were defined as SNPs that satisfied the criteria n1 + n2 ≥ 10 and n1/n2 ≥ 5 and for which the quality score of each mapped base was >20. Raw small InDels shorter than 50 bp were predicted from the regions recognized as gaps in the alignment of syntenic regions. InDels containing more than one mismatch 5 bp upstream or downstream were eliminated. Read validation was then performed on the remaining InDels, and the InDels for which ≥3 query reads mapped to the InDel-removed sequence of the subject were retained.
Function enrichment of variant genes
We analyzed the relationships between all gene function variations (genes at SV regions or those containing SNPs or InDels) using different gene/protein databases (COG/GO/KEGG). This allowed us to compute the numbers of proteins for each corresponding COG/GO/KEGG term. We determined the GOG/GO/KEGG enrichment terms for the variant genes using a hypergeometric test [34, 35] and calculated the P-value. P ≤ 0.05 was considered a significant enrichment of the GOG/GO/KEGG term for the variant gene/protein. We determined the main biological function of differential proteins using function enrichment analysis.
We converted the protein sequence alignment results into multiple amino acid sequences in the CDS regions and aligned multiple sequences in the clustered gene family using the Muscle software (http://www.drive5.com/muscle, v3.8.31). Finally, we generated the gene family tree by analyzing the multiple-sequence alignment results based on Muscle using the Bayes method with the MrBayes software (MrBayes v3.1.2). The dN/dS ratios were calculated using the CODONML software (in paml version 4.4, January 2010) with the GY-HKY model .
K. pneumoniae strains LCT-KP214 and LCT-KP289 genome sequences have been deposited in GenBank under accession numbers AJHE00000000 and ATRO00000000, respectively.
Single nucleotide polymorphism
Non-redundant protein database
Cluster of orthologous groups of proteins
Kyoto encyclopedia of genes and genomes
Antibiotic resistance genes database
This work was supported by the National Basic Research Program of China (973 program) No. 2014CB744400, the Program of Manned Spaceflight No. 040203, Key Program of Medical Research in the Military ‘12th 5-year Plan’, No. BWS12J046, Key Pre-Research Foundation of Military Equipment of China No. 9140A26040312JB10078, and Beijing Novel Program, No.Z121107002512128.
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