Identification of Genetic Alterations Associated with Acquired Colistin Resistance in Klebsiella pneumoniae Isogenic Strains by Whole-Genome Sequencing
Abstract
:1. Introduction
2. Results
2.1. Antimicrobial Susceptibility
2.2. Mutations in Colistin-Resistant Isolates
2.3. Orthologs Involved in Colistin Resistance
2.4. Change of mRNA Levels in Candidate Genes Associated with Colistin Resistance
2.5. Confirmation of the Role of KP1_0061 and KP1_3620 Gene in Colistin Resistance
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains
4.2. Pulsed-Field Gel Electrophoresis (PFGE)
4.3. In Vitro Antimicrobial Susceptibility Testing
4.4. Genome Sequencing and Confirmation of Genetic Variants
4.5. Identification of Orthologous Genes
4.6. mRNA Expression Analysis
4.7. Complementation Assay
4.8. Colistin Susceptibility Testing
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Isolate | ST a | Minimum Inhibitory Concentration(mg/L) b | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
COL | PB | AMP | AZ | CTX | CAZ | CIP | GEN | IMI | P/T | SXT | ||
B0608-134 | 730 | 1 | 2 | 64 | ≤0.06 | ≤0.06 | 1 | ≤0.06 | 1 | 0.125 | 16/4 | 0.06/1.18 |
B0608-134R | 256 | >64 | 64 | ≤0.06 | ≤0.06 | 1 | ≤0.06 | 2 | 0.25 | 16/4 | 0.06/1.18 | |
B0704-039 | 11 | 0.5 | 2 | >64 | >64 | >64 | >64 | >64 | >64 | 1 | >64/4 | 4/76 |
B0704-039R1 | >8192 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | 1 | >64/4 | 4/76 | |
B0704-039R2 | >128 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | 0.5 | >64/4 | 4/76 | |
08-B063 | 23 | 0.5 | 2 | 64 | ≤0.06 | ≤0.06 | 0.5 | ≤0.06 | 0.5 | 0.125 | 8/4 | 0.12/2.37 |
08-B063R | 512 | >64 | >64 | ≤0.06 | ≤0.06 | 0.5 | 0.125 | 0.5 | 0.125 | 16/4 | 0.5/9.5 | |
B0701-068 | 152 | 0.5 | 2 | 64 | ≤0.06 | ≤0.06 | 1 | ≤0.06 | 1 | 0.25 | 16/4 | 0.06/1.18 |
B0701-068R | 1024 | >64 | >64 | ≤0.06 | ≤0.06 | 1 | ≤0.06 | 1 | 0.25 | 16/4 | 0.125/2.37 |
Set | Gene Name | Function | Nucleotide Alteration | Amino Acid Alteration | Fold Change of mRNA Expression |
---|---|---|---|---|---|
B0608-134 versus B0608-134R | Intergenic region between KPN_02065 and KPN_02066 | KPN_02065, putative transport protein KPN_02066, putative response regulator consisting of a CheY-like receiver domain and a winged-helix DNA-binding domain | Insertion of Insertion sequence (IS)5-like element | - | 208.99 ± 48.27-fold increase for KPN_02065 |
4.48 ± 0.1-fold increase for KPN_02066 | |||||
B0704-039 versus B0704-039R1 | Intergenic region between KP1_3468 and KP1_3469 | KP1_3468; conserved hypothetical protein KP1_3469; putative outer membrane | Insertion of IS5-like element | - | 2.51 ± 0.49-fold increase for KP1_3468 |
1.03 ± 0.11 fold increase for KP1_3469 | |||||
B0704-039 versus B0704-039R2 | KP1_3468 | Hypothetical protein | T26A, C108G, A128T | L9Q, S36R, K43I | 7.91 ± 0.31-fold increase |
mgrB | PhoPQ regulatory protein | Insertion of IS5-like element | Frameshift | - | |
B08-063 versus B08-063R | phoQ | Sensor protein PhoQ | A803C | Y268S | 7.49 ± 0.52-fold increase |
12-bp deletion at 341st nucleotide | Deletion of 5 amino acids at position 114 | ||||
KP1_3620 | Hypothetical protein | 1 bp deletion at 51st nucleotide | Premature stop at position 14 | - | |
KP1_0061 | Repressor of galETK operon | T104G | V35G | 2.11 ± 0.35-fold increase | |
B0701-068 versus B0701-068R | KP1_3468 | Hypothetical protein | Insertion of IS5-like element | Frameshift | 0.9 ± 0.14-fold increase |
KPN_02067 | Sensor histidine protein kinase (RstA regulator) | G593T | G198V | 1.36 ± 0.05-fold increase | |
mgrB | PhoPQ regulatory protein | Insertion of IS5-like element | Frameshift | - |
Isolates | Colistin MICs(mg/L) |
---|---|
08-B063 | 0.5 |
08-B063/pBAD33 | 0.5 |
08-B063/pKP1_0061R | 0.5 |
08-B063/pKP1_3620R | 0.125 |
Primer | Sequence (5′→ 3′) | Amplicon Size (bp) | Reference |
---|---|---|---|
Primers for sequencing | |||
134-target1-F | TGAATGCACAAGGTAAAGCCAGG | 500 | This study |
134-target1-R | CCGTCTTCGGCTCTTAAGGTTTT | ||
039-target1-F | GACACAGCCAGCGATGCCAG | 416 | This study |
039-target1-R | TTGTATGATCCATGGCGTGA | ||
063-target1,2-F | ATGCAGGTAATAACCAATCT | 751 | This study |
063-target1,2-R | ATCCCGGAAAACCTGAATAT | ||
063-target3-F | CCATAATAAAAAAAATAATC | 362 | This study |
063-target3-R | GCCCCTCCCCACACATCTTT | ||
063-target4-F | ACGGCCGAGCGCCAGAAACG | 720 | This study |
063-target4-R | TCGCATCCGCATCCGCCAGG | ||
068-target1-F | TATGATGCACACCTGTCGGG | 780 | This study |
068-target1-R | CACTGTGGAATAACACCCCA | ||
068-target2-F | GGCACTGAATAATGCGTAAG | 480 | This study |
068-target2-R | GCCTGAAACAAATCTCTCAG | ||
mgrB-ext-F | CAGCCAGCGATGCCAGATTT | 380 | [5] |
mgrB-ext-R | CCTGGCGTGATTTTGACACGA | ||
Primers for qRT-PCR | |||
RT-KPN_02065-F | CCCCGGGGTGCTATCTCAC | 135 | This study |
RT-KPN_02065-R | CCGCCCAGGATGGCATTTAT | ||
RT-KPN_02066-F | TCGCTGCCATACTGACAGGCT | 148 | This study |
RT-KPN_02066-R | TTCCAGCCATCGTACACGGG | ||
RT-KP1_3468-F | AAAAATTACGGTGGGTTTTACTGA | 109 | This study |
RT-KP1_3468-R | ATGCCGCTGAAAAACTGAAC | ||
RT-KP1_3469-F | TGGATTGGCGTATTATTGAGC | 122 | This study |
RT-KP1_3469-R | TGAACATAAAGTGCGGTGCT | ||
RT-phoQ-F | TGCCAGGGAAGCGGACTAC | 100 | This study |
RT-phoQ-R | GCGGCGGATCAGTGATAAAC | ||
RT-KP1_0061-F | TTATCACCGACTGCAACACC | 138 | This study |
RT-KP1_0061-R | CGACATCGGTCTCAATGATG | ||
RT-KPN-02067-F | GGGAAACCTTAGTGCCAGAGC | 111 | This study |
RT-KPN-02067-R | TGCGCATCCAGCGTCTGTAA | ||
RT-rpoB-F | CGCGTATGTCCGATCGAAA | 100 | This study |
RT-rpoB-F | GCGTCTCAAGGAAGCCATATTC |
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Choi, M.; Ko, K.S. Identification of Genetic Alterations Associated with Acquired Colistin Resistance in Klebsiella pneumoniae Isogenic Strains by Whole-Genome Sequencing. Antibiotics 2020, 9, 374. https://doi.org/10.3390/antibiotics9070374
Choi M, Ko KS. Identification of Genetic Alterations Associated with Acquired Colistin Resistance in Klebsiella pneumoniae Isogenic Strains by Whole-Genome Sequencing. Antibiotics. 2020; 9(7):374. https://doi.org/10.3390/antibiotics9070374
Chicago/Turabian StyleChoi, Myeongjin, and Kwan Soo Ko. 2020. "Identification of Genetic Alterations Associated with Acquired Colistin Resistance in Klebsiella pneumoniae Isogenic Strains by Whole-Genome Sequencing" Antibiotics 9, no. 7: 374. https://doi.org/10.3390/antibiotics9070374