The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in Klebsiella spp

  1. Natalia C Rosas
  2. Jonathan Wilksch
  3. Jake Barber
  4. Jiahui Li
  5. Yanan Wang
  6. Zhewei Sun
  7. Andrea Rocker
  8. Chaille T Webb
  9. Laura Perlaza-Jiménez
  10. Christopher J Stubenrauch
  11. Vijaykrishna Dhanasekaran
  12. Jiangning Song
  13. George Taiaroa
  14. Mark Davies
  15. Richard A Strugnell
  16. Qiyu Bao
  17. Tieli Zhou  Is a corresponding author
  18. Michael J McDonald  Is a corresponding author
  19. Trevor Lithgow  Is a corresponding author
  1. Centre to Impact AMR, Monash University, Australia
  2. Infection Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Australia
  3. School of Biological Sciences, Monash University, Australia
  4. The First Affiliated Hospital of Wenzhou Medical University, China
  5. Infection Program, Biomedicine Discovery Institute and Department of Biochemistry & Molecular Biology, Monash University, Australia
  6. Wenzhou Medical University, China
  7. School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, China
  8. Department of Microbiology and Immunology, The Peter Doherty Institute, The University of Melbourne, Australia
6 figures, 6 tables and 1 additional file

Figures

Figure 1 with 3 supplements
Experiment overview.

(A) Carbapenem-resistant Klebsiella spp. were isolated from a patient, and the genome was sequenced and assembled. The genetic cause of resistance was confirmed by re-engineering the carbapenem …

Figure 1—source data 1

Strain information for Figure 1B.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig1-data1-v1.xlsx
Figure 1—source data 2

Growth rate analysis of Escherichia coli BW25113 strains expressing the indicated open-reading frames cloned into plasmid pJP-CmR.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig1-data2-v1.xlsx
Figure 1—source data 3

Antibiotic resistance genes identified in pNAR1.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig1-data3-v1.xlsx
Figure 1—source data 4

Transmembrane transporter systems identified in pNAR1.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig1-data4-v1.xlsx
Figure 1—source data 5

β-lactamase prediction and classification using DeepBL.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig1-data5-v1.xlsx
Figure 1—source data 6

Minimum inhibitory concentration (MIC) analysis of Escherichia coli BW25113 expressing DeepBL candidates and MIC analysis of FK688 pNAR1ΔblaDHA-1 strains expressing DHA-1.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig1-data6-v1.xlsx
Figure 1—figure supplement 1
Physical map of the K. quasipneumoniae subsp. similipneumoniae FK688 chromosome.

The position of genes encoding antibiotic resistance determinants (oqxAB, blaOKP-B-21, kdeA, and fosA5) and major outer membrane porin proteins (ompK35, ompK36, ompK37, and ompK38) are indicated. …

Figure 1—figure supplement 2
The phylogeny of Klebsiella.

Maximum likelihood phylogenetic tree of 377 publicly available Klebsiella genomes shows K. pneumoniae and K. quasipneumoniae as distinct species. The inner ring indicates the country of strain …

Figure 1—figure supplement 3
Domain architecture of DeepBL candidates.

(A) The Conserved Domain Architecture Retrieval Tool (CDART; Geer et al., 2002) was used to create the graphical display of domain architectures for the protein sequences identified from DeepBL …

Figure 2 with 2 supplements
Reconstruction of mutant OmpK36 protein based structural characteristics of OmpK36 in K. quasipneumoniae subsp. similipneumoniae.

(A) PSIPRED (Buchan and Jones, 2019) secondary structure prediction of the OmpK36, using the protein sequence encoded in the K. quasipneumoniae subsp. similipneumoniae genome. The location of the 16 …

Figure 2—figure supplement 1
Gene alignment of four major porins.

Gene synteny comparison alignments of ompK35, ompK36, ompK37, and ompK38 (green arrows) and neighbouring open reading frames (ORFs) with either predicted (purple arrows) or unknown (grey arrows) …

Figure 2—figure supplement 2
Comparative sequence analysis of ompK35 and ompK36 genes of Klebsiella spp. and reference genomes.

(A) BLAST searches of sequence data held at NCBI did not identify any other Klebsiella strains carrying a tnpA insertion in the 5’ end of ompK35. However, such an insertion is seen in E. coli

Figure 3 with 2 supplements
Evolution and physical map of plasmid pNAR1.

(A) Schematic representation of the in vitro evolution experiment. After passage #3 (P3), a ceftazidime-susceptible (CAZS) mutant evolved, lacking a 17 kb region of pNAR1 that included blaDHA-1

Figure 3—source data 1

Growth rate analysis of K. quasipneumoniae strains.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Detailed physical map of the FK688 plasmid pNAR1.

The coloured lines in the outer concentric circles represent the location of predicted coding sequences in the forward and reverse DNA strands. Annotated CoDing Sequences (CDS) are shown in dark …

Figure 3—figure supplement 2
Growth rate analysis of K. quasipneumoniae strains.

Bacteria was cultured in cation-adjusted Mueller-Hinton Broth (CAMHB), and the OD600 was measured every hour for 24 hr. Error bars represent SD (n=3). The genotypes of the strains are indicated in …

Competitive fitness assay of FK688 strain variants against GFP-labelled FK688.

(A) Schematic of the competitive fitness assay experiment (Materials and methods). FACS: fluorescence-activated cell sorting. (B) The relative fitness of the engineered mutant strains relative to …

Figure 5 with 2 supplements
Genotypic and phenotypic evolution of FK688 ΔompK36 and ompK36+strains.

(A) Schematic of the evolution experiment of Lineage A (FK688:ΔompK36 pNAR1) and Lineage B (FK688:ompK36+pNAR1). 20 replicate populations (A1, A2, A3,…A20 and B1, B2, B3,…B20) for each lineage were …

Figure 5—source data 1

Fitness assay ancestral and evolved lineages A and B strains.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig5-data1-v1.xlsx
Figure 5—source data 2

Relative numbers of opaque colonies in the 20 replicate populations of FK688 ΔompK36 pNAR1 (lineage A) and ompK36+pNAR1 (lineage B) strains after 200 generations.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig5-data2-v1.xlsx
Figure 5—source data 3

FK688 OmpK36+, B3(o), and B3(t) genome modification and SNP analysis.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig5-data3-v1.xlsx
Figure 5—source data 4

Glucuronic acid measurement of ancestral and evolved strains.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig5-data4-v1.xlsx
Figure 5—source data 5

Original gel image for Figure 5.

https://cdn.elifesciences.org/articles/83107/elife-83107-fig5-data5-v1.zip
Figure 5—figure supplement 1
Comparative sequence analysis of the fim gene cluster in evolved K. quasipneumoniae strains.

Sequence comparison of FK688 ompK36+ revealed a tnpA gene from the IS4 family (blue arrow) inserted upstream of fimE in B3(o). Sequence comparisons were performed with ViPTree (Nishimura et al., 2017

Figure 5—figure supplement 2
Comparative sequence analysis of the fim gene cluster in evolved K. quasipneumoniae strains.

Schematic of the FK688 and its evolved strains A2(o) and A2(t) fim gene cluster with 100% gene similarity between the strains. Sequence comparisons were performed with ViPTree (Nishimura et al., 2017

Competitive fitness assay of FK688 strain variants in the presence of ceftazidime.

(A) The fitness of FK688 mutants measured in Lysogeny Broth (LB) media supplemented with ceftazidime across a concentration range from 0.125 to 2 µg/mL. Only strains with an intact pNAR1 plasmid, …

Tables

Table 1
Antimicrobial susceptibility profiling of K. quasipneumoniae FK688.
AntimicrobialAntimicrobialMIC (µg/mL)*
ClassDrugFK688E. coli (ATCC 25922)Breakpoints
PenicillinsAmpicillin>20488≥32
CephemsCefazolin>20482≥8
Cefotaxime10240.125≥4
Ceftazidime>20480.5≥16
CarbapenemsErtapenem640.016≥2
Imipenem80.25≥4
Meropenem40.03≥4
LipopeptidesPolymyxin B42≥4
AminoglycosidesGentamicin12≥16
Tobramycin12≥16
Kanamycin24≥64
TetracyclinesTetracycline1281≥16
FluoroquinolonesCiprofloxacin10.016≥1
  1. *

    Drug-sensitive, italics; drug-resistant, bold-text.

  2. Resistant clinical breakpoint for Enterobacterales given by CLSI, 2022.

Table 2
Antimicrobial susceptibility profiling of FK688-derived strains.
Antimicrobial ClassAntimicrobialDrugMIC (µg/mL)*
ΔompK36ompK36+
pNAR1pNAR1ΔblaDHA-1pNAR1pNAR1pNAR1ΔblaDHA-1
PenicillinsAmpicillin>2048256128>2048128
CephemsCefazolin>20483232>20484
Cefotaxime10240.50.5320.125
Ceftazidime>204810.55120.25
CarbapenemsErtapenem640.50.50.50.016
Imipenem80.250.2510.12
Meropenem40.060.1250.1250.03
LipopeptidesPolymyxin B44444
AminoglycosidesKanamycin24244
TetracyclinesTetracycline1282562128128
FluoroquinolonesCiprofloxacin10.060.0610.125
  1. *

    Drug-sensitive, italics; drug-resistant, bold-text.

Table 3
Antimicrobial susceptibility profiling FK688 ΔompK36 and ompK36+ strains and their respective evolved strains.
MIC (µg/mL)*
AntimicrobialAntimicrobialΔompK36ompK36+
ClassDrugpNAR1pNAR1 ΔblaDHA-1 A2(o)pNAR1 ΔblaDHA-1A2(t)pNAR1pNAR1 ΔblaDHA-1B3(o)pNAR1 ΔblaDHA-1 B3(t)
CephemsCefazolin>2048321>204821
Cefotaxime10240.50.25320.1250.25
Ceftazidime>20480.50.255120.250.25
CarbapenemsErtapenem640.50.0310.50.0160.031
Imipenem80.1250.12510.250.125
Meropenem40.1250.0160.120.0160.016
TetracyclinesTetracycline128128128128128128
FluoroquinolonesCiprofloxacin10.1250.2510.0310.063
AminoglycosidesKanamycin222422
Tobramycin0.511111
Gentamicin0.510.50.50.50.5
LipopeptidesPolymyxin B444484
  1. *

    Drug-sensitive, italics; drug-resistant, bold-text.

Table 4
List of plasmids used in this study.
PlasmidRelevant characteristics*Source/reference
pKD4Contains kanamycin resistance cassette (kan) flanked by FRT sites (FRT-kan-FRT); oriR6K, AmpR, KmRDatsenko and Wanner, 2000
pJET1.2/bluntBlunt-end cloning vector for insertion of DNA fragments with single deoxyadenosine overhangs; AmpRThermo Scientific
pDonor(OmpK36)pJET1.2/blunt carrying FRT-kan-FRT and K. quasipneumoniae FK688 ompK36 regions (donor plasmid for lambda Red recombination-mediated repair of ompK36 gene in FK688); AmpR, KmRThis study
pACBSRArabinose-inducible promoter; I-SceI endonuclease; lambda Red recombination genes, CmRHerring et al., 2003
pFLP-BSRpACBSR carrying fragment length polymorphism (FLP) recombinase to excise the kanamycin cassette, temp-sensitive replication; CmRRocker et al., 2020
pJP-CmRDerivative of pJP168 for anhydrotetracycline inducible protein expression. CmRRocker et al., 2020
pJP-blaDHA-1pJP-Cm containing blaDHA-1 from FK688This study
pJP-blaOKP-B-21pJP-Cm containing blaOKP-B-21 from FK688This study
pJP-blaSHKpJP-Cm containing CKCOFDID_01495 from FK688This study
pJP-blaOPHCpJP-Cm containing CKCOFDID_02113 from FK688This study
pJP-blaDAEpJP-Cm containing pbpG from FK688This study
pJP-blaTRNpJP-Cm containing CKCOFDID_04153 from FK688This study
pJP-blaABHpJP-Cm containing dhmA from FK688This study
pJP-blaDACpJP-Cm containing dacB from FK688This study
  1. *

    Amp, ampicillin; Km, kanamycin; Cm, chloramphenicol.

Table 5
List of strains used in this study.
StrainRelevant characteristics*Source or reference
K. quasipneumoniae
FK688 ΔompK36 pNAR1Wildtype, clinical isolate from a bloodstream infection case from the First Affiliated Hospital of Wenzhou Medical University, China. Expresses β-lactamase blaOKP-B-21. Deficient in ompK35 and ompK36 porin expression. Harbours a 258 kb plasmid pNAR1 (AmpR, TetR, RifR, TrpR, StpR, EryR, SdzR, CipR).Bi et al., 2017
ΔompK36 pNAR1ΔblaDHA-1FK688 with a 17 kb deletion from tnpA-sul1 in pNAR1.This study
ΔompK36 pNAR1FK688 cured of pNAR1.This study
ompK36+ pNAR1FK688 with a genetically repaired and functional ompK36 gene. Carries pNAR1.This study
ompK36+ pNAR1ΔblaDHA-1FK688 with a genetically repaired and functional ompK36 gene. It has a 17 kb deletion from tnpA-sul1 in pNAR1.This study
FK688-GFP+FK688 with a constitutively expressed green fluorescent protein (GPF). GFP gene inserted downstream of the glmS gene via pGRG-eGFP.This study
A2(o)
ΔompK36 pNAR1ΔblaDHA-1
Evolved strain from Kq1. It has a 17 kb deletion from tnpA-sul1 in pNAR1. Forms opaque colonies.This study
A2(t)
ΔompK36 pNAR1ΔblaDHA-1
Evolved strain from Kq1. It has a 17 kb deletion from tnpA-sul1 in pNAR1. Forms translucent colonies.This study
B3(o) ompK36+ pNAR1ΔblaDHA-1Evolved strain from Kq4. It has a 17 kb deletion from tnpA-sul1 in pNAR1. Forms opaque colonies.This study
B3(t) ompK36+ pNAR1ΔblaDHA-1Evolved strain from Kq4. It has a 17 kb deletion from tnpA-sul1 in pNAR1. Forms translucent colonies.This study
K. pneumoniae
B5055Hypermucoviscous phenotype. Wildtype, clinical isolate, serotype K2;O1Statens Serum Institut, Denmark
B5055 nmB5055 deletion mutant ∆wza-wzc::km (non-mucoid); KmRProf. Richard Strugnell University of Melbourne
E. coli
DH5αF endA1 hsdR17(rK, mK+) supE44 λ– thi-1 recA1 gyrA96 relA1 deoR Δ(lacZYA-argF) U169 Φ80dlacZ∆M15; NalR
E. coli DH5α was used for cloning purposes
Invitrogen
ATCC 25922CLSI control strain for antimicrobial susceptibility testingATCC
BW25113 (WT)rrnB3 ΔlacZ4787(::rrnB-3) hsdR514 Δ(araD-araB)567 Δ(rhaD-rhaB)568, rph-1Baba et al., 2006
  1. *

    Amp, ampicillin; Tet, tetracycline; Rif, rifamycin; Trp, trimethoprim; Stp, streptomycin; Ery, erythromycin; Sdz, sulfadiazine; Cip, ciprofloxacin; Nal, nalidixic acid.

Table 6
List of oligonucleotide primers used in this study.
PrimerSequence (5–3’)*Description
Construction of FK688 OmpK36+ strains
K36_insert-RgcgcgacctactacttcaacaaaaacatgtccacctatgttgactacaaaatcaacctgctgConstruction of pDonor(OmpK36) plasmid
K36_insert-Fgttgaagtagtaggtcgcgcccacgtcaacatatttcaggatgtcctggtcgcc
K36_Km-Fctaaggaggatattcatatggtcgcaagctgcataacaaa
K36_Km-Rgaagcagctccagcctacacattagaactggtaaaccaggcccag
K36_ISceI-Rtagggataacagggtaatgcccgacggtgatatccatc
K36_ISceI-Ftagggataacagggtaatgcttcggtacctctgtaacttatga
pKD4-FtgtgtaggctggagctgcttcKanamycin cassette from pKD4
pKD4-Rcatatgaatatcctccttag
Cloning of putative β-lactamases genes for anhydrotetracycline-inducible expression
blaDHA-1_For_NRgtccCCATGGtgaaaaaatcgttatctgcaac
blaDHA-1_Rev_NRcgtcAAGCTTattccagtgcactcaaa
blaOKP_F_NRtagcGAATTCatgcgttatgttcgcctgtgcc
blaOKP_R_NRgcatAAGCTTctagcgctgccagtg
blaSHK1_F_NRgttcCCATGGtgataagaaaaccactggcc
blaSHK1_R_NRatgcAAGCTTaacgcagctcgcg
blaOPHC2_F_NRctagGAATTCatgacaccagctcccttttataccctgac
blaOPHC2_R_NRacggAAGCTTtcgctgtgatcggtgtt
blaDAE1_F_NRtgcaGAATTCatgatgccgaaatttcgagtctctttgc
blaDAE1_R_NRgatcAAGCTTttaatcgttctgcgcg
blaABH1_F_NRacgtCCATGGTGaacagattatccctgatcc
blaABH1_R_NRgatcAAGCTTacaaccgatcggcg
blaDAC1_F_NRaaggCCATGGtgcgatttcccagatttatc
blaDAC1_R_NRaagcAAGCTTtagttgttctggtacaaatcc
blaTRN1_F_NRcgtaCCATGGtgactgaacgggtttattacac
blaTRN1_R_NRaatcAAGCTTacgtcagggaatagctgatc
pJPMCS_ForcctaatttttgttgacactctatcattgpJP-CmR-gene insert sequencing primers
pJPMCS_Revgccaggcaaattctgttttatcagaccg
  1. *

    Restriction endonuclease recognition sites are capitalised.

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