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Abstract

Objectives

In this study, we developed an IS26-based CRISPR/Cas9 system as a proof-of-concept study to explore the potential of a re-engineered bacterial translocatable unit (TU) for curing and immunizing against the replication genes and antimicrobial resistance genes.

Methods

A series of pIS26-CRISPR/Cas9 suicide plasmids were constructed, and specific guide RNAs were designed to target the replication gene of IncX4, IncI2 and IncHI2 plasmids, and the antibiotic resistance genes mcr-1, blaKPC-2 and blaNDM-5. Through conjugation and induction, the transposition efficiency and plasmid-curing efficiency in each recipient were tested. In addition, we examined the efficiency of the IS26-CRISPR/Cas9 system of cell immunity against the acquisition of the exogenous resistant plasmids by introducing this system into antimicrobial-susceptible hosts.

Results

This study aimed to eliminate the replication genes and antimicrobial resistance genes using pIS26-CRISPR/Cas9. Three plasmids with different replicon types, including IncX4, IncI2 and IncHI2 in three isolates, two pUC19-derived plasmids, pUC19-mcr-1 and pUC19-IS26mcr-1, in two lab strains, and two plasmids bearing blaKPC-2 and blaNDM-5 in two isolates were all successfully eliminated. Moreover, the IS26-based CRISPR/Cas9 system that remained in the plasmid-cured strains could efficiently serve as an immune system against the acquisition of the exogenous resistant plasmids.

Conclusions

The IS26-based CRISPR/Cas9 system can be used to efficiently sensitize clinical Escherichia coli isolates to antibiotics in vitro. The single-guide RNAs targeted resistance genes or replication genes of specific incompatible plasmids that harboured resistance genes, providing a novel means to naturally select bacteria that cannot uptake and disseminate such genes.

Introduction

Antibiotic-resistant (AR) bacteria are one major cause of healthcare-associated infections in the world, bringing millions of deaths and tremendous economic losses. Among the ways of spreading, horizontal gene transfer (HGT) is the main contributor to the rapid dispersion of antibiotic resistance genes (ARGs).1 Plasmids play an important role in facilitating horizontal genetic exchange and therefore promoting the acquisition of resistance genes. Plasmids often acquire the resistance from pre-existing resistance genes from the bacterial resistome pool.2 The acquisition occurs through the concerted activities of various mobile genetic elements (MGEs) that are able to move within or between DNA molecules, including ISs, transposons and gene cassettes/integrons, especially when antimicrobial selection pressure exists. These mobile elements collectively play a significant role in the spreading of ARGs.2 MGEs responsible for ARG dispersion have been characterized in various organisms. ISs are the simplest MGEs that typically carry one or more (sometimes two) transposase (tnp) genes and are widespread in all domains of life.3 ISs are discrete DNA segments, which are able to move themselves to new locations in the same or different DNA molecules within a single cell, sometimes along with the neighbouring genes.4 IS26, an 820 bp DNA segment, was first discovered over three decades ago, encoding a transposase (Tnp26) of 234 amino acids.5 It has been deemed to be a critical element in the dissemination of ARGs among Gram-negative bacteria, as it is associated with genes conferring resistance to different classes of antibiotics, including carbapenems.6 ARGs flanked by IS26s resemble class I or composite transposons. An investigation from Harmer and co-workers6 revealed that IS26 was able to mobilize resistance genes within bacteria by a single copy of IS26 forming a ‘translocatable unit’ (TU) or by two copies of IS26 forming a class I transposon, Tn(I). The IS26-mediated transfer usually recognizes other pre-existing IS26s to form a cointegrate.6,7

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci have evolved to confer sequence-directed immunity against exogenous genes in most archaea and many bacteria.8–11 The system was later developed into a promising gene-editing tool for many applications, including in eukaryotes and prokaryotes.12–17 Some previous studies endorsed the potential of the reprogrammed Cas nuclease to prevent the spreading of plasmid-carrying resistance by targeting and cutting ARGs in the bacterial plasmids.18–24 As a plasmid-curing measure, the CRISPR/Cas9-based system demonstrated merits in super-easy handling, which requires only the co-expression of a Cas protein and a customized single-guide RNA (sgRNA).

Recently, several natural transposon-associated CRISPR-Cas systems have been described. A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems. The CRISPR-Cas defence systems have been recruited multiple times in nature to guide RNA-directed transposition by Tn7-like elements and have enabled genetic analysis of diverse bacteria with Tn7-mediated Mobile-CRISPRi.25–27 Compared with the Tn7-like transposons, which involve transposition-associated proteins TnsA, TnsB and TnsC, the IS26-mediated transposition only requires the single IS26-encoded transposase, which can seem like an ‘all in one’ transposon. Given the high activity of IS26-mediated transposition in various Gram-negative pathogens, we sought to develop an IS26-based transposon-CRISPR/Cas9 (IS26 Mobile-CRISPR/Cas9) system to cure ARGs and to immunize against the acquisition of ARGs in Escherichia coli hosts, as a proof of concept of re-engineering endogenous transposon in combating antimicrobial resistance. Our results indicated that the IS26 Mobile-CRISPR/Cas9 system cannot only eliminate the resistant plasmids in E. coli hosts but also provide them with the immunity to acquire the incorporation of the exogenous plasmid.

Materials and methods

Ethics

Ethical approval not applicable.

Bacterial strains and growth conditions

The strains used in this study are listed in Table 1. The primers used in this study can be found in Table S1, available as Supplementary data at JAC Online. E. coli strains were cultured at 37°C in LB medium. Medium was supplemented with diaminopimelic acid (DAP, 0.3 mM) to culture the donor strain E. coli WM3064 in a conjugation assay. When needed, antibiotics were added at the following concentrations: colistin (2 mg/L, abbreviated to CS2), chloramphenicol (25 mg/L, abbreviated to C25), sodium tellurite (25 mg/L, abbreviated to ST25) and meropenem (2 mg/L, abbreviated to MEM2) in LB liquid medium or LB agar plates.

Table1.
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Strains used in this study

StrainDescriptionReference
E. coli 001Clinical isolate carrying an IncX4 plasmid pEc001, harbouring mcr-1 gene and one single copy of IS2639
E. coli 002Clinical isolate carrying an IncI2 plasmid pEc002, harbouring mcr-1 geneThis study
E. coli 003Clinical isolate carrying an IncHI2 plasmid pEc003, harbouring mcr-1 geneThis study
ΔE. coli 001Tn: IS26-CRISPR/Cas9 integrated into the E. coli 001 host, tpmR, sgRNA: IncX4This study
ΔE. coli 002Tn: IS26-CRISPR/Cas9 integrated into the E. coli 002 host, tpmR, sgRNA: IncI2This study
ΔE. coli 003Tn: IS26-CRISPR/Cas9 integrated into the E. coli 003 host, tpmR, sgRNA: IncHI2This study
E. coli 004E. coli DH5α, ampR, mcr-1R, pUC19-mcr-1This study
E. coli 005E. coli DH5α, ampR, mcr-1R, pUC19-IS26mcr-1This study
ΔE. coli 004Tn: IS26-CRISPR/Cas9 integrated into the E. coli 004 host, tpmR, sgRNA: mcr-1This study
ΔE. coli 005Tn: IS26-CRISPR/Cas9 integrated into the E. coli 005 host, tpmR, sgRNA: mcr-1This study
ΔΔE. coli 001Tn: IS26-CRISPR/Cas9 integrated into the E. coli 005 host, tpmR, sgRNA: mcr-1This study
E. coli 006Clinical isolate carrying a plasmid pEc006, harbouring blaKPC-2 geneThis study
E. coli 007Clinical isolate carrying a plasmid pEc007, harbouring blaNDM-5 geneThis study
ΔE. coli 006Tn: IS26-CRISPR/Cas9 integrated into the E. coli 006 host, tpmR, sgRNA: blaKPC-2This study
ΔE. coli 007Tn: IS26-CRISPR/Cas9 integrated into the E. coli 007 host, tpmR, sgRNA: blaNDM-5This study
E. coli WM3064RP4 (tra) in the chromosome, dependent on DAP for growth40
E. coli WM3064: pEc001E. coli WM3064 carrying pEc001This study
E. coli WM3064: pEc002E. coli WM3064 carrying pEc002This study
E. coli WM3064: pEc003E. coli WM3064 carrying pEc003This study
E. coli WM3064: pEc006E. coli WM3064 carrying pEc006This study
E. coli WM3064: pEc007E. coli WM3064 carrying pEc007This study
StrainDescriptionReference
E. coli 001Clinical isolate carrying an IncX4 plasmid pEc001, harbouring mcr-1 gene and one single copy of IS2639
E. coli 002Clinical isolate carrying an IncI2 plasmid pEc002, harbouring mcr-1 geneThis study
E. coli 003Clinical isolate carrying an IncHI2 plasmid pEc003, harbouring mcr-1 geneThis study
ΔE. coli 001Tn: IS26-CRISPR/Cas9 integrated into the E. coli 001 host, tpmR, sgRNA: IncX4This study
ΔE. coli 002Tn: IS26-CRISPR/Cas9 integrated into the E. coli 002 host, tpmR, sgRNA: IncI2This study
ΔE. coli 003Tn: IS26-CRISPR/Cas9 integrated into the E. coli 003 host, tpmR, sgRNA: IncHI2This study
E. coli 004E. coli DH5α, ampR, mcr-1R, pUC19-mcr-1This study
E. coli 005E. coli DH5α, ampR, mcr-1R, pUC19-IS26mcr-1This study
ΔE. coli 004Tn: IS26-CRISPR/Cas9 integrated into the E. coli 004 host, tpmR, sgRNA: mcr-1This study
ΔE. coli 005Tn: IS26-CRISPR/Cas9 integrated into the E. coli 005 host, tpmR, sgRNA: mcr-1This study
ΔΔE. coli 001Tn: IS26-CRISPR/Cas9 integrated into the E. coli 005 host, tpmR, sgRNA: mcr-1This study
E. coli 006Clinical isolate carrying a plasmid pEc006, harbouring blaKPC-2 geneThis study
E. coli 007Clinical isolate carrying a plasmid pEc007, harbouring blaNDM-5 geneThis study
ΔE. coli 006Tn: IS26-CRISPR/Cas9 integrated into the E. coli 006 host, tpmR, sgRNA: blaKPC-2This study
ΔE. coli 007Tn: IS26-CRISPR/Cas9 integrated into the E. coli 007 host, tpmR, sgRNA: blaNDM-5This study
E. coli WM3064RP4 (tra) in the chromosome, dependent on DAP for growth40
E. coli WM3064: pEc001E. coli WM3064 carrying pEc001This study
E. coli WM3064: pEc002E. coli WM3064 carrying pEc002This study
E. coli WM3064: pEc003E. coli WM3064 carrying pEc003This study
E. coli WM3064: pEc006E. coli WM3064 carrying pEc006This study
E. coli WM3064: pEc007E. coli WM3064 carrying pEc007This study

mcr-1R, colistin resistant; tpmR, tellurite resistant; ampR, ampicillin resistant.

Table1.
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Strains used in this study

StrainDescriptionReference
E. coli 001Clinical isolate carrying an IncX4 plasmid pEc001, harbouring mcr-1 gene and one single copy of IS2639
E. coli 002Clinical isolate carrying an IncI2 plasmid pEc002, harbouring mcr-1 geneThis study
E. coli 003Clinical isolate carrying an IncHI2 plasmid pEc003, harbouring mcr-1 geneThis study
ΔE. coli 001Tn: IS26-CRISPR/Cas9 integrated into the E. coli 001 host, tpmR, sgRNA: IncX4This study
ΔE. coli 002Tn: IS26-CRISPR/Cas9 integrated into the E. coli 002 host, tpmR, sgRNA: IncI2This study
ΔE. coli 003Tn: IS26-CRISPR/Cas9 integrated into the E. coli 003 host, tpmR, sgRNA: IncHI2This study
E. coli 004E. coli DH5α, ampR, mcr-1R, pUC19-mcr-1This study
E. coli 005E. coli DH5α, ampR, mcr-1R, pUC19-IS26mcr-1This study
ΔE. coli 004Tn: IS26-CRISPR/Cas9 integrated into the E. coli 004 host, tpmR, sgRNA: mcr-1This study
ΔE. coli 005Tn: IS26-CRISPR/Cas9 integrated into the E. coli 005 host, tpmR, sgRNA: mcr-1This study
ΔΔE. coli 001Tn: IS26-CRISPR/Cas9 integrated into the E. coli 005 host, tpmR, sgRNA: mcr-1This study
E. coli 006Clinical isolate carrying a plasmid pEc006, harbouring blaKPC-2 geneThis study
E. coli 007Clinical isolate carrying a plasmid pEc007, harbouring blaNDM-5 geneThis study
ΔE. coli 006Tn: IS26-CRISPR/Cas9 integrated into the E. coli 006 host, tpmR, sgRNA: blaKPC-2This study
ΔE. coli 007Tn: IS26-CRISPR/Cas9 integrated into the E. coli 007 host, tpmR, sgRNA: blaNDM-5This study
E. coli WM3064RP4 (tra) in the chromosome, dependent on DAP for growth40
E. coli WM3064: pEc001E. coli WM3064 carrying pEc001This study
E. coli WM3064: pEc002E. coli WM3064 carrying pEc002This study
E. coli WM3064: pEc003E. coli WM3064 carrying pEc003This study
E. coli WM3064: pEc006E. coli WM3064 carrying pEc006This study
E. coli WM3064: pEc007E. coli WM3064 carrying pEc007This study
StrainDescriptionReference
E. coli 001Clinical isolate carrying an IncX4 plasmid pEc001, harbouring mcr-1 gene and one single copy of IS2639
E. coli 002Clinical isolate carrying an IncI2 plasmid pEc002, harbouring mcr-1 geneThis study
E. coli 003Clinical isolate carrying an IncHI2 plasmid pEc003, harbouring mcr-1 geneThis study
ΔE. coli 001Tn: IS26-CRISPR/Cas9 integrated into the E. coli 001 host, tpmR, sgRNA: IncX4This study
ΔE. coli 002Tn: IS26-CRISPR/Cas9 integrated into the E. coli 002 host, tpmR, sgRNA: IncI2This study
ΔE. coli 003Tn: IS26-CRISPR/Cas9 integrated into the E. coli 003 host, tpmR, sgRNA: IncHI2This study
E. coli 004E. coli DH5α, ampR, mcr-1R, pUC19-mcr-1This study
E. coli 005E. coli DH5α, ampR, mcr-1R, pUC19-IS26mcr-1This study
ΔE. coli 004Tn: IS26-CRISPR/Cas9 integrated into the E. coli 004 host, tpmR, sgRNA: mcr-1This study
ΔE. coli 005Tn: IS26-CRISPR/Cas9 integrated into the E. coli 005 host, tpmR, sgRNA: mcr-1This study
ΔΔE. coli 001Tn: IS26-CRISPR/Cas9 integrated into the E. coli 005 host, tpmR, sgRNA: mcr-1This study
E. coli 006Clinical isolate carrying a plasmid pEc006, harbouring blaKPC-2 geneThis study
E. coli 007Clinical isolate carrying a plasmid pEc007, harbouring blaNDM-5 geneThis study
ΔE. coli 006Tn: IS26-CRISPR/Cas9 integrated into the E. coli 006 host, tpmR, sgRNA: blaKPC-2This study
ΔE. coli 007Tn: IS26-CRISPR/Cas9 integrated into the E. coli 007 host, tpmR, sgRNA: blaNDM-5This study
E. coli WM3064RP4 (tra) in the chromosome, dependent on DAP for growth40
E. coli WM3064: pEc001E. coli WM3064 carrying pEc001This study
E. coli WM3064: pEc002E. coli WM3064 carrying pEc002This study
E. coli WM3064: pEc003E. coli WM3064 carrying pEc003This study
E. coli WM3064: pEc006E. coli WM3064 carrying pEc006This study
E. coli WM3064: pEc007E. coli WM3064 carrying pEc007This study

mcr-1R, colistin resistant; tpmR, tellurite resistant; ampR, ampicillin resistant.

Construction of the pIS26-CRISPR/Cas9 and pUC19-derivative plasmids

The conjugative suicide plasmid pIS26-CRISPR/Cas9 consisted of the CRISPR/Cas9 cassette flanked by IS26, which possessed the RP4oriT fragment and the R6K replication origin, and the pUC19-derivative plasmids were constructed using standard molecular cloning techniques. Greater details of the plasmid construction are provided in Appendix 1 of the Supplementary data. The plasmid construction steps are shown in Figure S1.

Conjugation assays

E. coli WM306428,29 (a pir+ strain that is dependent on DAP for growth) carrying pIS26-CRISPR/Cas9 was used as a donor in mating assays. The donor strain was grown overnight (∼12 h) at 37°C in LB supplemented with 300 μM DAP. Recipient strains were cultivated until the plateau stage in LB broth with 300 μM DAP at 37°C. For each donor and recipient strain, 100 μL was added to 700 μL of LB and mixed by pipetting. The mixture of donor and recipient strains was pelleted by centrifugation for 2 min at 7000 g, then resuspended in 50 μL of LB after washing with PBS. The resuspension was transferred onto cellulose membranes (MF-Millipore HAWG01300) placed on a pre-warmed LB agar plate supplemented with 300 μM DAP and incubated at 37°C for 12 h. Membranes were then transferred to microcentrifuge tubes containing 200 μL of PBS and vortexed to liberate the cells. Transconjugants were then identified as the grown colonies on the selective agar plates containing colistin and sodium tellurite in the absence of DAP (to exclude donor E. coli WM3064). The conjugation assays were performed in duplicate on two different days. A schematic diagram of the experiment is shown in Figure 1a.

The plasmid-curing workflow using the pIS26-CRISPR/Cas9 system. (a) A suicide plasmid containing the IS26-formed transposon carrying CRISPR/Cas9 was transferred to recipient bacteria by bi-parental mating. Donor cell E. coli WM3064 contained a chromosomal copy of the RP4 transfer machinery used to mobilize the suicide plasmids. Once inside the recipient cell, Tn: IS26-CRISPR/Cas9 was integrated into the plasmid or chromosome in the recipient strain. Selection on antibiotic plates lacking DAP eliminated the E. coli WM3064 donors and retained recipients with an integrated IS26-CRISPR/Cas9 library. (b) Cells were recovered and transferred into the medium with ST25 and 0.2 mg/L aTc to allow the cleavage of target plasmids (black) by CAS9 (orange), guided by the gRNA expression from the integrated plasmid itself (black). The culture was plated on ST25 LB agar and followed by PCR to identify the plasmid loss. The PCR-negative single colonies were also streaked onto CS2 or MEM2 antibiotic plates to identify the phenotype.
Figure 1.

The plasmid-curing workflow using the pIS26-CRISPR/Cas9 system. (a) A suicide plasmid containing the IS26-formed transposon carrying CRISPR/Cas9 was transferred to recipient bacteria by bi-parental mating. Donor cell E. coli WM3064 contained a chromosomal copy of the RP4 transfer machinery used to mobilize the suicide plasmids. Once inside the recipient cell, Tn: IS26-CRISPR/Cas9 was integrated into the plasmid or chromosome in the recipient strain. Selection on antibiotic plates lacking DAP eliminated the E. coli WM3064 donors and retained recipients with an integrated IS26-CRISPR/Cas9 library. (b) Cells were recovered and transferred into the medium with ST25 and 0.2 mg/L aTc to allow the cleavage of target plasmids (black) by CAS9 (orange), guided by the gRNA expression from the integrated plasmid itself (black). The culture was plated on ST25 LB agar and followed by PCR to identify the plasmid loss. The PCR-negative single colonies were also streaked onto CS2 or MEM2 antibiotic plates to identify the phenotype.

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Plasmid-curing conditions

The pIS26-CRISPR/Cas9 plasmid with different target sgRNA was generated as described above and introduced into the chosen strain by conjugation, followed by selection on agar containing ST25 + CS2 or ST25 + MEM2. Single colony transformants were selected and checked for the presence of the Tn: IS26-CRISPR/Cas9 by PCR using primers LHCas9testF and LHCas9testR (targeting the tpm and Cas9 gene on Tn: IS26-CRISPR/Cas9). Positive colonies were selected and inoculated into 5 mL of LB containing ST25 and 0.2 mg/L anhydrotetracycline (aTc). After incubation, the culture was diluted and simultaneously plated on LB agar containing ST25. In addition, ∼48 colonies grown on ST25 plates were subjected to PCR detection to confirm the loss of targeted genes or plasmids. The curing efficiency was calculated based on the PCR detection results. The experiments were conducted on two different days by two different operators; a schematic diagram of the experiment is shown in Figure 1b.

sgRNA design and cloning

The 20 nt base-pairing region (N20) of a sgRNA was designed according to an online web server (https://eu.idtdna.com/site/order/designtool/index/CRISPRCUSTOM). The sgRNA along with the tpm gene fragment, flanked by AvrII and PacI restriction sites, along with the N20 (Table 2), were amplified with a specific forward primer: AGTCCTAGGTATAATACTAGT-N20-GTTTTAGAGCTAGAAATAGCAAG (AvrII restriction site is underlined) and a reverse primer CCATGCATGTCGACTCTAGATTAATTAAG (PacI restriction site is underlined), using pIS26-CRISPR/Cas9 plasmid as the template. The PCR product was subsequently inserted into the AvrII- and PacI-digested pIS26-CRISPR/Cas9 plasmid, generating the final pIS26-CRISPR/Cas9 plasmids with targeted sgRNA. The cloning of the new targeted sgRNA into the pIS26-CRISPR/Cas9 procedure is shown in Figure S2.

Table 2.
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Selected gRNAs and the target antibiotic gene or replication gene

StrainsgRNAN20 sequence (5′ to 3′)TargetCuring efficiency (%)a
E. coli 001mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 004mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 005mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 001IncX4AGACTCAAATTCATTGAATCReplication gene of IncX4 plasmid100 ± 0
E. coli 002IncI2TCACTGGTCTGAACTATCACReplication gene of IncI2 plasmid100 ± 0
E. coli 003IncHI2TCTGTTAGAGAGAAAGAATCReplication gene of IncHI2 plasmid100 ± 0
E. coli 006blaKPC-2AACTGTAAGTTACCGCGCTGARG blaKPC-2100 ± 0
E. coli 007blaNDM-5AACGGTTTGATCGTCAGGGAARG blaNDM-5100 ± 0
StrainsgRNAN20 sequence (5′ to 3′)TargetCuring efficiency (%)a
E. coli 001mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 004mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 005mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 001IncX4AGACTCAAATTCATTGAATCReplication gene of IncX4 plasmid100 ± 0
E. coli 002IncI2TCACTGGTCTGAACTATCACReplication gene of IncI2 plasmid100 ± 0
E. coli 003IncHI2TCTGTTAGAGAGAAAGAATCReplication gene of IncHI2 plasmid100 ± 0
E. coli 006blaKPC-2AACTGTAAGTTACCGCGCTGARG blaKPC-2100 ± 0
E. coli 007blaNDM-5AACGGTTTGATCGTCAGGGAARG blaNDM-5100 ± 0
a

The values shown are the mean ± SD of three independent experiments.

Table 2.
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Selected gRNAs and the target antibiotic gene or replication gene

StrainsgRNAN20 sequence (5′ to 3′)TargetCuring efficiency (%)a
E. coli 001mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 004mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 005mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 001IncX4AGACTCAAATTCATTGAATCReplication gene of IncX4 plasmid100 ± 0
E. coli 002IncI2TCACTGGTCTGAACTATCACReplication gene of IncI2 plasmid100 ± 0
E. coli 003IncHI2TCTGTTAGAGAGAAAGAATCReplication gene of IncHI2 plasmid100 ± 0
E. coli 006blaKPC-2AACTGTAAGTTACCGCGCTGARG blaKPC-2100 ± 0
E. coli 007blaNDM-5AACGGTTTGATCGTCAGGGAARG blaNDM-5100 ± 0
StrainsgRNAN20 sequence (5′ to 3′)TargetCuring efficiency (%)a
E. coli 001mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 004mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 005mcr-1GCGGCATTCGTTATAAGGATARG mcr-1100 ± 0
E. coli 001IncX4AGACTCAAATTCATTGAATCReplication gene of IncX4 plasmid100 ± 0
E. coli 002IncI2TCACTGGTCTGAACTATCACReplication gene of IncI2 plasmid100 ± 0
E. coli 003IncHI2TCTGTTAGAGAGAAAGAATCReplication gene of IncHI2 plasmid100 ± 0
E. coli 006blaKPC-2AACTGTAAGTTACCGCGCTGARG blaKPC-2100 ± 0
E. coli 007blaNDM-5AACGGTTTGATCGTCAGGGAARG blaNDM-5100 ± 0
a

The values shown are the mean ± SD of three independent experiments.

IS26-CRISPR-Cas9 system limiting plasmid acquisition

In this section, each donor and recipient strain was grown to OD600 = 0.2–0.3 in 2 mL of LB broth with 300 μM DAP without antibiotics at 37°C and the donors and recipients were then washed three times with LB broth followed by resuspension in 2 mL of LB broth with 300 μM DAP without antibiotics. The donor and recipient bacteria were mixed 1:1 with 0.2 mg/L aTc or without aTc, and the resuspension was placed in the incubator for 4 h. The resuspension was then transferred onto cellulose membranes (MF-Millipore HAWG01300). It was then put on a pre-warmed LB agar plate supplemented with 300 μM DAP and incubated at 37°C for 8 h. Cells were spread onto selective agar supplemented with 0.2 mg/L aTc or without aTc to select the transconjugants. The conjugation efficiency was calculated as the number of conjugants per recipient. The experiments were conducted on two different days by two different operators.

Antimicrobial susceptibility testing (AST)

AST of two antibiotics (colistin and meropenem) for the parent strains and the plasmid-cured strains was performed by agar dilution and interpreted according to CLSI guidelines.30 The testing was performed in duplicate on two different days. The quality control strain E. coli ATCC 25922 was used in the testing.

Data availability

The nucleotide sequence of pIS26-CRISPR/Cas9 has been deposited in GenBank under accession number MW512918.

Results

Construction of pIS26-CRISPR/Cas9 plasmid-curing system

According to the transposition characteristics of IS26 (Figure 2a and b), during translocation the IS26-formed TU or IS26 Tn usually recognize another IS26 as a target and show significantly higher frequency (>60-fold) of forming a cointegrate when the target plasmid contains IS26.6,31 We designed a plasmid, pIS26-CRISPR/Cas9 (Figure 2c), containing an IS26 composite transposon embedded in a CRISPR-Cas9 system, (Figure 2d), which contained a replication R6K γ origin that requires the π protein (encoded by the pir gene), ensuring the vector cannot replicate in recipient cells. The Cas9 gene is driven by a Pteto promoter containing a TetR operator site induced by aTc. The expression of sgRNA is activated by the constitutive promoter pJ23119, while a tellurite resistance gene tpm was used as a selection marker since most MDR strains remain susceptible to tellurite.32 The Cas9 and tpm gene, along with sgRNA, was flanked by two IS26s in the same orientation.

Design and characterization of IS26-CRISPR/Cas9 and outcomes of transposition by IS26. (a) (i) a TU, one copy of IS26 and an adjacent region, into a recipient that lacks IS26; (ii) the TU targets an existing copy of IS26 (∼60× higher frequency than a recipient that lacks IS26). Both reactions create the same type of cointegrate.2 (b) A ‘composite transposon’-like structure is flanked by target site duplications (TSDs, grey lollipops) created during replicative transposition or preserved (if previously present) during conservative transposition. Intermolecular conservative transposition into a region that already contains two copies of IS26 flanking a resistance gene can give an array of TU.2,6,31,41 (c) Map of pIS26-CRISPR/Cas9 plasmid; the yellow oval represents the R6K replication, the grey rectangle represents the RP4 junction transfer origin, the blue rectangle represents IS26 in the same direction, the two IS26s holding pieces of DNA together to form Tns. The black arrows represent the tpm gene, which was used as a selective marker in clinical strains. (d) Tn: IS26-CRISPR/Cas9, flanked by IS26 from pIS26-CRISPR/Cas9, were ‘Mobile-CRISPR,’ combining modularity, stable genomic integration and ease of transfer to bacteria by conjugation.
Figure 2.

Design and characterization of IS26-CRISPR/Cas9 and outcomes of transposition by IS26. (a) (i) a TU, one copy of IS26 and an adjacent region, into a recipient that lacks IS26; (ii) the TU targets an existing copy of IS26 (∼60× higher frequency than a recipient that lacks IS26). Both reactions create the same type of cointegrate.2 (b) A ‘composite transposon’-like structure is flanked by target site duplications (TSDs, grey lollipops) created during replicative transposition or preserved (if previously present) during conservative transposition. Intermolecular conservative transposition into a region that already contains two copies of IS26 flanking a resistance gene can give an array of TU.2,6,31,41 (c) Map of pIS26-CRISPR/Cas9 plasmid; the yellow oval represents the R6K replication, the grey rectangle represents the RP4 junction transfer origin, the blue rectangle represents IS26 in the same direction, the two IS26s holding pieces of DNA together to form Tns. The black arrows represent the tpm gene, which was used as a selective marker in clinical strains. (d) Tn: IS26-CRISPR/Cas9, flanked by IS26 from pIS26-CRISPR/Cas9, were ‘Mobile-CRISPR,’ combining modularity, stable genomic integration and ease of transfer to bacteria by conjugation.

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Elimination of plasmids by IS26-CRISPR/Cas9 system

The transposable capacity of the constructed plasmid pIS26-CRISPR/Cas9 was firstly assessed in a clinical strain, E. coli 001, and two lab strains, E. coli 004 and E. coli 005. They all harboured the mcr-1 gene. E. coli 001 was a clinical strain isolated from Guangdong Province in South China in 2013 and carries an IncX4 plasmid (accession number KX711706), while E. coli 004 and E. coli 005 were E. coli DH5α harbouring the pUC19-derivative plasmids pUC19-mcr-1 and pUC19-IS26mcr-1, respectively. Our results showed that the pIS26-CRISPR/Cas9 plasmid targeting mcr-1 was able to transpose into bacteria with or without a pre-existing copy of IS26 (Figure 3a). After transposition, a single colony of each strain was selected and incubated in a medium supplemented with ST25 + 0.2 mg/L aTc21 and the curing efficiency was determined at different incubation times. The results showed that over 80% of mcr-1-carrying bacteria lost colistin resistance in the three strains after 12 h with 0.2 mg/L aTc treatment, while the curing frequency reached 100% at 24 h (Figure 3b). The three plasmid-cured strains were named ΔΔE. coli 001, ΔE. coli 004 and ΔE. coli 005. The validation of the genotype and phenotype of each plasmid cured strain is shown in Figure S3.

(a) E. coli WM 3064 carrying a pIS26-CRISPR/Cas9 plasmid targeting the mcr-1 gene conjugated with E. coli 001, E. coli 004 and E. coli 005 and the transposition efficiency was calculated. (b) Time course characterization of the Tn: IS26-CRISPR/Cas9 plasmid-curing system. Plasmid curing of pEc001, pUC19-mcr-1 and pUC19-IS26mcr-1 containing the mcr-1 gene. Plating on antibiotic LB agar plates was performed at 4, 8, 12 and 24 h after induction. The genotype of each plasmid-cured strain was confirmed by PCR using specific primers and the AR phenotype was confirmed using selective plates. The curing efficiency was calculated as PCR negative per 50 single colonies. Error bars represent the results from at least three biological replicates. After plasmid curing, Tn: IS26-CRISPR/Cas9 remained in the bacterial host as an immune system, and could block plasmid acquisition. (c) The Tn: IS26-CRISPR/Cas9 targeting the mcr-1 gene remained in E. coli 004 and E. coli 005 (resulting strains were ΔE. coli 004 and ΔE. coli 005). The resulting strain ΔE. coli 004 was used as a recipient and conjugated with E. coli WM3064: pEc001, E. coli WM3064: pEc002 and E. coli WM3064: pEc003, then the conjugation efficiency was calculated. (d) After plasmid curing, the resulting strains ΔE. coli 001, ΔE. coli 002 and ΔE. coli 003 with integrated Tn: IS26-CRISPR/Cas9 targeting replication genes IncX4, IncI2 and IncHI2, respectively, were also used as recipients conjugated with E. coli WM3064: pEc001, E. coli WM3064: pEc002, E. coli WM3064: pEc003, E. coli WM3064: pEc006 and E. coli WM3064: pEc007. The conjugation efficiency was calculated. (e) After plasmid curing, the resulting strains ΔE. coli 006 and ΔE. coli 007 with integrated Tn: IS26-CRISPR/Cas9 targeting blaKPC-2 and blaNDM-5 genes were also used as recipients conjugated with E. coli WM3064: pEc001 and E. coli WM3064: pEc002. Error bars represent conjugation results from at least three biological replicates and P < 0.01 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA, USA). Mean ± SEM, **P < 0.01, P values were calculated using the Student’s t-test. Red asterisks denote the absence of detectable transconjugant colonies.
Figure 3.

(a) E. coli WM 3064 carrying a pIS26-CRISPR/Cas9 plasmid targeting the mcr-1 gene conjugated with E. coli 001, E. coli 004 and E. coli 005 and the transposition efficiency was calculated. (b) Time course characterization of the Tn: IS26-CRISPR/Cas9 plasmid-curing system. Plasmid curing of pEc001, pUC19-mcr-1 and pUC19-IS26mcr-1 containing the mcr-1 gene. Plating on antibiotic LB agar plates was performed at 4, 8, 12 and 24 h after induction. The genotype of each plasmid-cured strain was confirmed by PCR using specific primers and the AR phenotype was confirmed using selective plates. The curing efficiency was calculated as PCR negative per 50 single colonies. Error bars represent the results from at least three biological replicates. After plasmid curing, Tn: IS26-CRISPR/Cas9 remained in the bacterial host as an immune system, and could block plasmid acquisition. (c) The Tn: IS26-CRISPR/Cas9 targeting the mcr-1 gene remained in E. coli 004 and E. coli 005 (resulting strains were ΔE. coli 004 and ΔE. coli 005). The resulting strain ΔE. coli 004 was used as a recipient and conjugated with E. coli WM3064: pEc001, E. coli WM3064: pEc002 and E. coli WM3064: pEc003, then the conjugation efficiency was calculated. (d) After plasmid curing, the resulting strains ΔE. coli 001, ΔE. coli 002 and ΔE. coli 003 with integrated Tn: IS26-CRISPR/Cas9 targeting replication genes IncX4, IncI2 and IncHI2, respectively, were also used as recipients conjugated with E. coli WM3064: pEc001, E. coli WM3064: pEc002, E. coli WM3064: pEc003, E. coli WM3064: pEc006 and E. coli WM3064: pEc007. The conjugation efficiency was calculated. (e) After plasmid curing, the resulting strains ΔE. coli 006 and ΔE. coli 007 with integrated Tn: IS26-CRISPR/Cas9 targeting blaKPC-2 and blaNDM-5 genes were also used as recipients conjugated with E. coli WM3064: pEc001 and E. coli WM3064: pEc002. Error bars represent conjugation results from at least three biological replicates and P < 0.01 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA, USA). Mean ± SEM, **P < 0.01, P values were calculated using the Student’s t-test. Red asterisks denote the absence of detectable transconjugant colonies.

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Besides targeting the mcr-1 gene, sgRNAs were designed to target the replication gene of IncI2, IncHI2 and IncX4 plasmids that harboured mcr-1, and sgRNAs targeting blaKPC-2 and blaNDM-5 genes were also developed. Since 24 h incubation time showed the highest mcr-1 gene-curing efficiency, 0.2 mg/L aTc treatment and 24 h induction time were used as the conditions to cure IncX4, IncI2 and IncHI2 plasmids, and blaKPC-2 and blaNDM-5 gene-carrying plasmids in clinical strains E. coli 001, E. coli 002 and E. coli 003, and E. coli 006 and E. coli 007, respectively. The results showed that pIS26-CRISPR/Cas9 targeting replication genes of IncX4, IncI2 and IncHI2 plasmids, and blaKPC-2 and blaNDM-5 genes could also re-sensitize antibiotic susceptibility of E. coli 001, E. coli 002 and E. coli 003, and E. coli 006 and E. coli 007, by removing the plasmids. The plasmid-curing efficiency reached 100% at 24 h. The plasmid-cured strains were named ΔE. coli 001, ΔE. coli 002, ΔE. coli 003, ΔE. coli 006 and ΔE. coli 007. The specific sgRNA and the plasmid-curing efficiency of each strain with 0.2 mg/L aTc treatment and 24 h induction time are shown in Table 2. The validation of the genotype and phenotype of each plasmid-cured strain is shown in Figures S4 and S5.

We also examined the stability of the Tn: IS26-CRISPR/Cas9 system after plasmid curing in all tested strains using PCR screening (Figures S3, S4 and S5). The results showed that the CRISPR/Cas9 system was stably maintained within the tested bacteria with appropriate supplementation of ST25 after eliminating the targeted plasmid. The transposition efficiency of pIS26-CRISPR/Cas9 into each recipient strain is shown in Table S3.

The integrated IS26-CRISPR-Cas9 system in bacteria-limited plasmid acquisition

We then examined whether the integrated IS26-CRISPR-Cas9 system in bacteria could provide immunization against exogenous plasmid acquisition. E. coli WM3064: pEc001, E. coli WM3064: pEc002, E. coli WM3064: pEc003, E. coli WM3064: pEc006 and E. coli WM3064: pEc007 were used as donor strains. ΔE. coli 001, ΔE. coli 002, ΔE. coli 003, ΔE. coli 004, ΔE. coli 006 and ΔE. coli 007, harbouring the IS26-CRISPR/Cas9 system targeting IncX4, IncI2 and IncHI2 replicon genes, mcr-1, blaKPC-2 and blaNDM-5 genes, respectively, were used as recipients in the conjugation assay. The efficiencies of Tn: IS26-CRISPR/Cas9 in preventing plasmid acquisition were examined using the sgRNAs targeting replication genes of IncX4, IncI2 and IncHI2 plasmids and mcr-1, blaKPC-2 and blaNDM-5 genes. Each pair of donors and recipients was as follows. To prevent the conjugation of mcr-1-harbouring plasmids, the donors + recipients were: E. coli WM3064: pEc001 + ΔE. coli 004; E. coli WM3064: pEc002 + ΔE. coli 004; and E. coli WM3064: pEc003 + ΔE. coli 004 (Figure 3c). To prevent the conjugation of IncX4, IncI2 and IncHI2 replicon type plasmids, the donors + recipients were E. coli WM3064: pEc001 + ΔE. coli 001; E. coli WM3064: pEc002 + ΔE. coli 002; and E. coli WM3064: pEc003 + ΔE. coli 003 (Figure 3d). To prevent the conjugation of the blaKPC-2 and blaNDM-5-harbouring plasmids, the donors + recipients were E. coli WM3064: pEc006 + ΔE. coli 006 and E. coli WM3064: pEc007 + ΔE. coli 007 (Figure 3e). The results showed that the recipients containing IS26-CRISPR/Cas9 systems demonstrated at least ∼2 log reduction of conjugation efficiency. In ΔE. coli 001 and ΔE. coli 003, the plasmid acquisition was limited entirely as no transconjugant colonies were detected compared with the control (without induction). The results suggested that the IS26-CRISPR/Cas9 system targeting replicon genes of IncX4, IncI2 and IncHI2, and mcr-1, blaKPC-2 and blaNDM-5 genes was capable of limiting the acquisition of plasmid.

Susceptibility testing

We then examined the MICs of colistin and meropenem for the parental strains and their plasmid-cured strains (Table S4). The results showed that the MICs of colistin in the plasmid-cured isolates were reduced from >4 to 1 mg/L. The MICs of meropenem in the cured isolates were reduced from 16 to <0.25 mg/L. The results indicated that targeting the IncX4, IncHI2 and IncHI2 replication genes and the mcr-1 gene effectively restored the colistin susceptibility among these strains. The blaKPC-2 and blaNDM-5 gene curing effectively restored the carbapenem susceptibility among these strains.

Discussion

HGT plays an essential role in acquiring new properties, such as pathogenicity and antibiotic resistance. Previous studies showed that ISs and composite transposons are frequently involved in the mobilization of ARGs.2 In this study, we constructed an IS26 composite transposon carrying the CRISPR/Cas9 system to eliminate antibiotic resistance plasmids. To test this system, a series of ‘Mobile’ Tn: CRISPR/Cas9-carrying plasmids were constructed. Conjugation experiments by co-cultivation of E. coli WM3064: pIS26-CRISPR/Cas9 with the E. coli recipient strains demonstrated that the plasmids carrying the Tn: CRISPR/Cas9-encoding transposon were successfully transferred to the recipient E. coli strains.

ARGs are often present in large or multicopy plasmids, which are capable of autonomously transferring among microbial populations, resulting in horizontal dissemination of drug resistance.19 Combating plasmid-mediated colistin resistance and carbapenem resistance is fundamental to restraining and preventing colistin resistance and carbapenem resistance dissemination in clinical isolates. In this study, plasmid-curing ability was observed in the high copy numbers of plasmids harbouring the mcr-1 gene (pUC19-mcr-1 and pUC19-IS26mcr-1) and the plasmids in clinical isolates carrying mcr-1 (pEc001, pEc002 and pEc003) and blaKPC-2 and blaNDM-5 genes (pEc006 and pEc007). Firstly, we compared the transposition efficiency of the Tn: IS26-CRISPR/Cas9 transposon between strains E. coli 001, E. coli 004 and E. coli 005. The result endorsed that the strains carrying an IS26 (E. coli 001 and E. coli 005) provided higher transposition efficiency than those that didn’t contain IS26 (E. coli 004). Our results were consistent with the previous reports6 in that the presence of an IS26 in a target plasmid significantly increased transposition efficiency. On the other hand, the results also showed the functionality of the system to mediate the transposition regardless of IS26 presence in the bacteria. This study aimed to use ISs to carry an engineered CRISPR/Cas9 system and make it move through the strain to fight against a multi-resistance plasmid. Once the suicide plasmid was transferred into the bacterium through conjugation, the Tn: IS26-CRISPR/Cas9 was transposed into the plasmid or chromosome. Unlike some previously described plasmid-borne CRISPR-Cas curing systems, the introduction of an exogenous plasmid may have caused a fitness cost and could not stably be maintained. In this study, the plasmid-cured strains containing the IS26-CRISPR/Cas9 were propagated in LB medium for 10 days by 1:100 dilution.33 The results showed that the IS26-CRISPR/Cas9 could stabilize within the host (data not shown).

To date, more than 4500 ISs belonging to 29 families have been identified.4 IS26 was chosen in this study because it is one of the relatively shorter types of IS, with great potential to simplify the process of experimental operation. More importantly, IS26 shows its presence frequently in multi-resistant regions (MRRs), often with more than 10 copies in some plasmids, and mediates the transfer of MDR genes by the form of TUs and Tns.6,34 The diversified modes of transfer mediated by IS26 may act in a pivotal role in the dissemination of resistance determinants in Gram-negative bacteria. The transposition mechanism and prevalence of IS26-related drug-resistant genes in clinical plasmids can facilitate the carriage of CRISPR/Cas9 per se as a tool to eliminate drug-resistant plasmids, as the type II CRISPR system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria, which can generate immune memory to foreign DNA.11,35,36 Most importantly, it was experimentally demonstrated that those short spacers can provide resistance against bacteriophage infection and plasmid transformation.37 The activity of Tn: IS26-CRISPR/Cas9 is therefore sufficient to eliminate even AR plasmids of high copy numbers from cells to re-sensitize AR bacteria to antibiotics, and effectively limit the spread of AR plasmids.

Although our system has achieved compelling results, some limitations still exist. One of the limitations of our study was that the IS26-CRISPR-Cas9 cassette was randomly integrated into the bacterial genome, making scarless plasmid curing impossible and may have caused rearrangement of the bacterial genome. In addition, our system relies on the inducer aTc to work and the concentrations of the inducing reagents are difficult to control in in vivo conditions. Thus, this may lead to a limitation for our system in in vivo applications as strictly controlled induction is almost impossible to be maintained in the animals. However, there are candidate promoters like the non-inducible constitutive BBa J23107 promoter, which fits well with the CRISPR/Cas9 system.38 In subsequent studies, using the BBa J23107 promoter or other non-inducible constitutive promoters to drive the expression of CAS9 will be ideal in practical applications. Taken together, in addition to plasmid curing, this system could further provide immunity in E. coli against the acquisition of plasmid-mediated ARGs. Our system also took advantage of an RP4 conjugation mechanism. The RP4 plasmid conjugation machinery has a broad host range, and it can conjugate into diverse bacterial species.27 Thus, there is great potential for the Tn: IS26-CRISPRCas9 plasmid curing system to serve as a promising tool to provide a new solution to the old dilemma of antibiotic resistance in diverse bacteria.

Funding

This work was supported by the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (grant 2019BT02N054), the Guangdong Major Project of Basic and Applied Basic Research (grant 2020B0301030007), the Program for Innovative Research Team in the University of Ministry of Education of China (grant IRT_17R39) and the 111 Project (grant 408 D20008).

Transparency declarations

The authors declare no competing interests.

Author contributions

J.S., L.C. and X.-P.L. designed the study. Y.-Z.H., T.-F.L., G.L., B.H. and X.K. performed the experiments. Y.-Z.H. and J.S. analysed the data. Y.-Z.H. made the figures. Y.-Z.H. wrote this manuscript. J.S., L.C., H.R. and X.-P.L. edited and revised the manuscript. Y.-H.L. coordinated the whole project. All authors contributed to the article and approved the submitted version.

Supplementary data

Tables S1 to S4, Figures S1 to S5 and Appendix 1 are available as Supplementary data at JAC Online.

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Author notes

†

Contributed equally to this work.

© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: [email protected].
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