CRISPR-AMRtracker: a novel toolkit to monitor the antimicrobial resistance gene transfer in fecal microbiota

doi:10.21203/rs.3.rs-2564758/v1

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CRISPR-AMRtracker: a novel toolkit to monitor the antimicrobial resistance gene transfer in fecal microbiota

https://doi.org/10.21203/rs.3.rs-2564758/v1

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The spread of antibiotic resistance genes (ARGs), especially those on plasmids, poses a major risk to global health. However, the extent and frequency of ARG transfer in microbial communities among human, animal, and environmental sectors is not well understood due to a lack of effective tracking tools. We have developed a new fluorescent tracing tool, CRISPR-AMRtracker, to study ARG transfer. It combines CRISPR/Cas9 fluorescence tagging, fluorescence-activated cell sorting, 16S rRNA gene sequencing, and microbial community analysis. The tool integrates a fluorescent tag immediately downstream ARGs, allowing for gene transfer tracking without affecting host cell antibiotic susceptibility, fitness, conjugation, or transposition. Our experiments show that sfGFP-tagged plasmid-borne mcr-1 can transfer across different bacteria species in feces, including Escherichia, Shigella, Lactobacillus, Staphylococcus, Enterococcus, and Bacillus. CRISPR-AMRtracker offers a powerful tool for monitoring ARG transfer in microbiota communities and can inform strategies to combat the threat of antibiotic resistance.

Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance

Biological sciences/Microbiology/CRISPR-Cas systems/CRISPR-Cas9 genome editing

Conjugation

Antibiotic resistance genes

horizontal gene transfer

mcr-1

CRISPR/Cas9

fluorescently labelling

Antimicrobial resistance (AMR) is a growing global health concern that poses a significant threat to the global health¹. To effectively combat the emergence and spread of AMR, it is crucial to understand the mechanisms, spectrum, and frequency by which antibiotic resistance genes (ARGs) are disseminated. Horizontal gene transfer (HGT) is one of the most significant routes for the spread of ARGs, particularly when mediated by conjugative plasmids.^{2, 3}. Conjugative plasmid transfer is a process by which bacteria transfer genes horizontally to other strain, species, genera, phyla and even domains. In addition to ARGs, these plasmids often carry other accessory genes, such as those metal resistances, catabolic pathways or virulence factors. HGT is a common event in the human, animal gut microbiome and environmental microbiome, including a collection of all microbes from various bacterial species as well as fungi and viruses^{4, 5}. Conjugative plasmids serve as the main vessels of gene flow in microbial communities, connecting distinct genetic pools among different biological sectors. Despite their importance, the dynamics of conjugative gene transfer in complex microbial communities, such as the gut microbiome, are still poorly understood.

Previous methods for monitoring plasmid-mediated HGT in natural environments have focused on identifying specific plasmid-encoded traits and phenotypes, such as antibiotic resistance or catabolic pathways that impose plasmid selection in the new hosts ⁶. However, these approaches are mostly cultivation-dependent and therefore have inherently limited the range of bacterial host profiling ⁶. Recent advancements in the field, such as using fluorescently-labelled plasmids, have expanded the range of host plasmids that can be studied, and these experiments have been conducted in various microcosms, including soil ⁷, water⁸, activated sludge ⁹ and gut ¹⁰. However, these studies have typically only focused on a few plasmid types such as RP4 or pKJK5, whereas important clinically relevant plasmids, such as those plasmids harbor carbapenemase genes or mobile colistin resistance genes (mcr), are rarely studied. Other techniques, such as transposition with Mu ¹¹, Tn5 ¹² and Tn7 ¹³, have been used to add fluorescent gene markers to plasmids, but these insertions are often random, which can lead to unintended consequences.

The CRISPR/Cas9 system is a highly efficient tool for creating a double-strand break (DSB) at a target site ¹⁴. By using a specific single guide RNA (sgRNA), the Cas9 endonuclease can make precise cuts, allowing for specific integration of a fluorescent gene into a plasmid. The system, composed of the Cas9 protein and the guide RNA (gRNA) ^{15 16}, has been used for various applications such as plasmid curing, gene deletions, point mutations, and gene insertions..

In this study, we utilized the CRISPR/Cas9 and lambda Red systems to integrate a sfGFP gene to the conserved regions of plasmids containing clinically significant antibiotic resistance genes (ARGs), including mcr-1, bla_NDM−1, bla_KPC−2, and bla_OXA−48, in bacteria such as Escherichia coli, Salmonella Typhimurium, and Klebsiella pneumoniae. We compared the antibiotic resistance, growth patterns, and transposition and conjugation abilities of the tagged strains with those of the wild-type strains. The fluorescently tagged strains were then used to monitor the dissemination of the mcr-1 gene in fecal microbiota. Our study provides a highly effective method for tracking ARG transfer in microbial populations.

2.1 Bacterial strains, plasmids, primers and growth conditions.

The strains used in this study are listed in Table S1, while the plasmids used are listed in Table S2. A list of primers used is provided in Table S3. Bacterial cultures including E. coli, S. typhimurium, K. pneumoniae, Acinetobacter baylyi, Bacillus subtilis, Enterobacter faecalis and Staphylococcus aureus were grown at 37°C in LB medium. Lactobacillus reuteri was grown in De Man, Rogosa and Sharpe (MRS) media. Diaminopimelic acid (DAP) was added at 50 mg/L for the growth of E. coli strain WM3064. Antibiotics were used at the following concentrations as needed: chloramphenicol (CHL) 25 mg/L, meropenem (MEM) 1 mg/L, Ampicillin (Amp) 100 mg/L, apramycin (APR) 30 mg/L, kanamycin (KAN) 30 mg/L, rifampin (RIF) 50 mg/L, streptomycin (STR) 2000 mg/L and colistin (CS) 2 mg/L.

2.2 Plasmid construction.

The plasmids pCasKP-oriT (cas9 plasmid with an oriT site), pSGKP-Tmcr-500GFP, pSGKP-Tarr-NDM500GFP, pSGKP-Tarr-KPC500GFP, pSGKP-Tarr-OXA500GFP (guide RNA plasmids harboring sfGFP gene and repair template targeting downstream ARGs), pJS05, pJS06 and pMQ (a plasmid with lacI^q used to inhibit the pTac promoter of sfGFP) were constructed using standard molecular cloning techniques (see Supplementary data, Appendix 1).

2.3 Labelling ARG plasmids with sfGFP gene.

The regions immediately downstream of ARGs, which are commonly conserved for the same resistance genes across different plasmid types, served as the target sites for insertion of the sfGFP gene. This was achieved using a two-plasmid CRISPR/Cas9 system^{17, 18}. The Cas9 plasmid pCasKP-oriT was transferred by conjugation to E. coli CSZ4, S. Typhimurium 14E1050, S. Typhimurium LS3479, E. coli HZE22, K. pneumoniae 50700 and K. pneumoniae 49210 and selected on LB agar plates containing APR at 30°C. The pCasKP-oriT recipient strains were incubated overnight at 30°C in LB-APR medium and then subcultured at 30°C in LB-APR, followed by 0.1 M L-arabinose induction, to make competent cells. The pSGKP plasmid vectors, including the gRNA and the repaired template, were transferred by electroporation as previously described [25]. Electroporants were selected on LB plates supplemented with RIF and APR, followed by PCR confirmation. Single colonies were transferred to LB agar plates containing 5% sucrose and incubated at 37°C to remove pSGKP-gfp and pCasKP-oriT vectors. Macroscopic green colonies were screened and confirmed by PCR and Sanger sequencing using primers MF and OR. The elimination of pCasKP-oriT was verified using primers CasKP-F and CasKP-R in PCR reactions, while primers SGKP-F and SGKP-R were used to verify the elimination of pSGKP-gfp.. The labelling efficiency (%) was calculated as the percent sfGFP-labelled clones out of the total of tested clones.

2.4 Donor strain construction.

Conjugation reporter donor strains were constructed from sfGFP gene-labelled strain.To do this, 100 ng of plasmid pMQ was electroporated into sfGFP gene-labelled strain and selected on LB-KAN plates. Red colonies were then selected and confirmed by PCR using primers Kan-F and Kan-R.

2.5 Conjugation assays.

Both the donor and recipient strains were grown to the log phase in their respective media, and the cell suspensions were adjusted to OD ₆₀₀ ~0.3 (~ 10⁸ CFU/ml cells). The cells were then washed in the media prior to mating. For broth mating, equal volumes of donor: recipient (500 µL each) cells were mixed and incubated at 37°C for 8 h. For agar mating, equal amounts of donor: recipient cells (10 µL) were transferred onto 0.45 µm nitrocellulose membrane filters on LB agar (MRS agar for L. reuteri). The mixtures were then incubated under aerobic conditions except for L. reuteri which was incubated anaerobically, at 37°C for 8 h. The cells were then suspended in sterile PBS and used for flow cytometry, fluorescence microscopy, or serially-diluted and spotted on LB plates containing appropriate antibiotics for bacterial enumeration. The conjugation frequency was calculated as the number of transconjugants per output recipients. The experiment was performed with 3 biological replicates.

2.6 Antimicrobial susceptibility testing.

The minimum inhibitory concentrations (MICs) of meropenem, campicillin and colistin were determined using the broth micro dilution method and the results were interpreted according to the guideline of Clinical and Laboratory Standards Institute (CLSI)¹⁹. The testing was performed with 3 biological replicates and repeated two times. E. coli ATCC 25922 was used as the quality control strain.

2.7 Bacterial growth curve assay.

The overnight cultures of bacterial strains were adjusted to an OD ₆₀₀ ~0.3 and then diluted 10 times with LB broth. 100 µL cells (~ 10⁶ CFU) were then transferred into 10 mL LB and cultured at 37°C at 180 rpm. The growth was monitored at OD₆₀₀ for 24 hours, and growth curves were constructed using Prism 8 (GraphPad, San Diego, Ca, USA).

2.8 Transposition assays.

Transposition assays were performed as previously described with minor modifications ²⁰. In brief, suicide plasmids pJS05 and pJS06, harboring the RK2 replicons and mcr-1 transposon, were electroporated into E. coli WM3064 to generate the donors WM3064/pJS05 and WM3064/pJS06. E. coli MG1655 was used as a recipient in the transposition assays. The donor strain was grown for 8 h at 37°C in LB supplemented with DAP until an OD₆₀₀ of ~ 0.3. The recipient strains were cultivated in LB at 37°C until an OD₆₀₀ of ~ 0.3 was reached. Both donor and recipient strains were centrifuged for 5 min at 6,000 rpm, and 10 µL donor and recipient cells were mixed by pipetting and placed on membrane filters on LB agar plates supplemented with DAP, followed by incubation at 37°C for 8 h. Membranes were then transferred to microcentrifuge tubes containing 900 µL PBS and vortexed to release the cells. Transposons were then identified as colonies growing on selective agar plates containing CS without DAP. The transposition frequency was calculated as transposons per recipient and conjugation assays were performed using 6 biological replicates.

2.9 Fluorescence microscopy of bacteria.

Bacteria were washed by PBS and centrifuged at 6,000 rpm, then suspended in PBS. A 1 µL suspension was dropped onto a microscope slide and covered with a glass coverslip. The bacteria were imaged at 50× magnification using a laser scanning confocal microscope (LSCM, LEICA, TCS SP8) using the TRITC and FITC channels.

2.10 Animal ethics statement.

Female C57BL/6 mice, 6 weeks old, were housed at the Laboratory Animal Center of Southern Medical University, Guangzhou, China. Animal care was in accordance with the guidelines of the American Association for Accreditation of Laboratory Animal Care criteria. All animal experiments were conducted in compliance with institution’s guidelines for animal use and the Guide for the Care and use of Laboratory Animals at laboratory animal center of South China Agricultural University (Guangzhou, China).

2.11 Isolation of live mouse gut bacteria.

Live gut bacteria from mice were isolated as previously described with minor modifications ¹⁰. Briefly, 250 µl of PBSC (pH 7.4 PBS containing 0.1% (w/v) L-cysteine) was added to pre-weighed stool samples in microcentrifuge tubes. The samples were mechanically disrupted using a motorized pestle and 750 µL PBSC was added, followed by vortex mixing and centrifugation at 1,000 rpm for 30 s. 750 µL of supernatant was then transferred into a new tube. The above step was repeated three more times to obtain a total ~ 3 ml supernatant. The resulting 3 mL of isolated cells were pelleted by centrifugation at 6,000 rpm for 5 min, and the supernatant was discarded. The cells were then suspended in 0.5–1.0 mL of PBSC.

2.12 Conjugation in natural recipient community.

The donor strain MQCSZ4GFP was streaked onto LB agar plates with CST and incubated at 37°C overnight. Single colonies were incubated into 4 mL LB broth media and grown for 4 h at 37°C. The recipient community was isolated anaerobically from fresh mouse feces immediately before conjugation as described above. The donor cells were washed twice in PBSC and adjusted to OD₆₀₀ 0.5. 10 µL aliquots were mixed 1:1 with recipient cells onto 0.45 µM membrane filters on BHI agar. The mating cultures were incubated at 37°C either aerobically or anaerobically for 8 h. An anaerobic condition were established using a commercial anaerobic pack to induce hypoxia (MGC, Shanghai, China). The cells were harvested from plates by scraping, suspended in 1 mL PBS, and used for downstream experiments.

2.13 Flow cytometry and FACS measurements.

An S3e FACS platform (Bio-Rad, Hercules, CA, USA) with green (488 nm) and mCherry (561 nm) lasers were used to quantify the numbers of donor and transconjugants. Bacteria were diluted 100-fold in PBS for optimal detection. The number of transconjugants was determined by counting the number of green fluorescence cells, while the number of recipient cells was calculated as the total number of bacteria minus the number of mCherry fluorescence cells. At least 50,000 cells were used for gated populations. The donor strain was used as the mCherry positive control, and recipient strains or gut bacteria were used as GFP negative controls.

2.14 Metagenomic 16S sequencing and analysis.

The microbial DNA from the FACS-sorted transconjugants and recipients was extracted using the E.Z.N.A. stool DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions. The V3-V4 hypervariable regions of the bacteria 16S rDNA genes were amplified using PCR, and the amplicons were sequenced using the Illumina MiSeq platform. The analysis was conducted using customized program scripts (https://docs.qiime2.org/2019.1/), and chimeras were removed. The sequences were then grouped into Operational Taxonomic Units (OTU)and the abundance and diversity of OTU for samples were analyzed. The representative OTUs were aligned using GeneBank (NCBI) databases.

2.15 Statistical analysis.

The statistical analysis was performed using GraphPad Prism 8 software. The results reported are the mean ± standard deviation obtained from at least three independent experiments.To determine significant differences between groups, unpaired t-tests or one-way analysis of variance (ANOVA) were used, and a p-value of less than 0.05 was considered significant..

2.16 Accession number(s).

The complete nucleotide sequence of pCasKp-oriT (ON988038), pSGKP-arr2-TKPC500GFP (ON988039), pSGKP-arr2-TNDM500GFP (ON988040), pSGKP-arr2-TOXA500GFP (ON988041), pSGKP-Tmcr-500GFP (OP009361) and pMQ (OP009360) were deposited in GenBank.

3.1 Integration of fluorescence tags at conserved ARG regions.

In this study, we developed a new tool and used the sfGFP fluorescence tag to track the spread of ARG plasmid in bacterial communities. Unlike previous studies, where a fluorescence tag was either inserted in a resistance gene or integrated randomly on a plasmid^{21, 22, 23}, our approach targets the conserved regions associated with different ARGs. These previous techniques were limited in their ability to quickly label various plasmids containing the same ARG, and they also posed a risk of causing unintended biological consequences due to the random nature of the insertions. To address these limitations, we developed a new fluorescence tagging system for plasmids that targets conserved regions associated with different ARGs. ARGs are often linked to different mobile genetic elements (MGEs), such as the, carbapenemase genes bla_KPC, bla_NDM and bla_OXA−48, which are associated with Tn4401/NTM_KPC²⁴, Tn125 ²⁵and Tn1999²⁶, respectively. The mcr-1 is often found in complete or partial Tn6330 with a core mcr-1-pap2 cassette ^{27, 28}. Our analysis of NCBI database genomes of bla_KPC, bla_NDM, bla_OXA−48 and mcr-1 genes showed that many of these gene cassettes have conserved regions across different plasmid types (as shown in Fig. 1a). For instance, the mcr-1-pap2 cassette (approx. 2000 bp) in Tn6330 is highly conserved in mcr-1-containing IncX4, IncI2 and IncHI2 plasmids.

Our analysis also indicated that the regions surrounding the AMR genes were consistently transferred along with the AMR genes during horizontal gene transfer. This made these regions ideal targets for fluorescence-based tracking. Our new system targets the conserved regions associated with specific ARGs, such as bla_KPC, bla_NDM, bla_OXA−48 and mcr-1. This allows us to label different clinically relevant plasmids containing the same ARGs, regardless of their plasmid background, using the same fluorescence tagging system. If the ARGs are located on the chromosome, our design can also insert the sfGFP gene into the chromosome.

Six clinical isolates containing different ARGs were selected for fluorescence tagging. These isolates were obtained from E. coli, S. Typhimurium and K. pneumoniae and carried mcr-1 (CSZ4, 14E1050 and LS3479), bla_KPC−2 (50700), bla_NDM−1 (HZE22), and bla_OXA−48 (49210). The mcr-1 gene was located on plasmids pCSZ4 (IncX4), p14E1050 (IncI2), and pLS3479 (IncHI2), while blaNDM-1, blaKPC-2 and blaOXA-48 were located on plasmids pHZE22 (IncHI2), p50700 (IncF) and p49210 (IncL) respectively (Table S2 and Fig. 1b).

3.2 Two plasmid CRISPR/Cas9 system was used for fluorescence tagging.

A two plasmid CRISPR/Cas9 system (consisting of plasmid pCas9-oriT and pSGKP-gfp) was utilized for fluorescence tagging (Table S2, Fig. 2a). Plasmid pCas9-oriT was engineered by adding an oriT region to the thermosensitive plasmid pCas9-apr, which provids Cas9 and lambda Red proteins [29]. This plasmid enabled the transfer of Cas9 and lambda Red genes to clinical isolates through conjugation. The plasmid pSGKP-gfp expressed the sgRNA under the control of the J23119 promoter²⁹, superfolder green fluorescent protein gene (sfGFP) under the control of the pTrc promoter, the sucrose-sensitive gene sacB ³⁰ as well as the homologous DNA fragment targeting the AMR gene. 500bp upstream and downstream elements of the sfGFP insertion site were PCR amplified and ligated to pSGKP-gfp at the XbaI site. The sfGFP gene on the plasmid pSGKP-gfp is regulated by a modified pTrc promoter, which can be repressed by the LacI^q protein (Fig. 2b). Four pSGKP-gfp plasmids (pSGKP-arr2-TKPC500GFP, pSGKP-arr2-TNDM500GFP, pSGKP-arr2-TOXA500GFP and pSGKP-Tmcr-500GFP), targeting bla_KPC, bla_NDM, bla_OXA−48 and mcr-1 gene cassettes were constructed.

To tag sfGFP gene in the conserved downstream region of the ARGs, the pCasKP-oriT plasmid was transferred into the clinical isolates E. coli CSZ4, S. Typhimurium 14E1050, S. Typhimurium LS3479, K. pneumoniae 50700, E. coli HZE22 and K. pneumoniae 49210, respectively, through conjugation. Then, the plasmid pSGKP-gfp with different homologous template (e.g. pSGKP-arr2-TKPC500GFP, pSGKP-arr2-TNDM500GFP, pSGKP-arr2-TOXA500GFP and pSGKP-Tmcr-500GFP) was electrotransformed into the aforementioned strains harboring pCasKP-oriT. After induction with L-arabinose, the sfGFP fluorescent tag was successfully inserted into the region downstream of mcr-1, bla_NDM−1, bla_KPC−2, bla_OXA−48 in the strains E. coli CSZ4, S. Typhimurium14E1050, S. Typhimurium LS3479, E. coli HZE22, K. pneumoniae 50700 and K. pneumoniae 49210, respectively (Fig. 2c). Further PCR screening and laser scanning confocal microscope (LSCM) showed that all these strains were successfully labelled with sfGFP at the targeted sites (Fig. 2d-e). The labeling efficiency ranged from 11.09% (pLS3479) to 100% (85.85% for p14E1050, and 100% for pCSZ4, pHZE22, p50700 and p49210), as shown in Fig. 2F.

3.3 Fluorescence labeling did not interfere normal functions of the bacterial strains.

To assess the effects of fluorescent labeling on host strains, we compared the minimum inhibitory concentration (MIC) values and growth curves of sfGFP-labeled strains to those of their parent strains. The results showed no differences in the MIC values of CST, MEM, or AMP in E. coli CSZ4, S. Typhimurium 14E1050, S. Typhimurium LS3479, E. coli HZE22, K. pneumoniae 50700, and K. pneumoniae 49210 between the sfGFP-labeled strains and their parent strains (Fig. 3a). Additionally, the growth kinetics of the sfGFP-labeled and parent strains were found to be the same (Fig. 3b and Fig S2). These findings suggested that the fluorescent labeling did not alter the antimicrobial susceptibility of the AMR genes or impose any significant fitness costs on the bacterial hosts.

We then examined whether the fluorescent tagging would impact the plasmid conjugation frequency. Our conjugation experiments using both broth and agar mating found no obvious differences in conjugation frequency between the sfGFP-labelled strains and their parent strains (Fig. 3c-d). We also investigated if the insertion with sfGFP segment would impact the transposition frequency. We cloned the Tn6330 harboring mcr-1 or mcr-1-sfGFP into the suicide plasmid pJS01 ^{28, 31}, producing the plasmid pJS05 (containing a 4673 bp Tn) and pJS06 (containing a 5582 bp Tn) (Fig. 3e-g), respectively. The transposition frequencies into E. coli MG1655 were 1.48 ± 0.66 × 10^− 6 and 1.98 ± 0.21 × 10^− 6 per transformed cell for pJS05 and pJS06, respectively. No significant transposition frequency differences were observed between pJS05 and pJS06 (Fig. 3h). These results indicated that the sfGFP gene labeling at the downstream conserved-region of ARGs did not impact antibiotic susceptibility, growth characteristics, conjugation frequency, or transposition frequency.

3.4 Monitoring ARGs transfer in complex microbiota.

To improve the detection of ARGs transfer from clinical isolated isolates to complex microbiota, we constructed a non-transferable ColE1 plasmid pMQ. The plasmid pMQ expresss mCherry fluorescence protein and lacI^q, which inhibits the sfGFP promoter pTrc if both plasmids co-existed. The design is to introduce the pMQ into sfGFP tagged clinical isolates, and the presence of pMQ will inhibit the sfGFP expression, resulting in only red fluorescence due to mCherry gene in pMQ. When the sfGFP labeled ARG plasmid transfers to other recipients, the sfGFP promoter inhibition disappear, leading to green fluorescence emission (Fig. 4a). This system allowed easy distinction and quantification of transconjugant cells using flow cytometry or rapid detection via a confocal microscope.

We then experimentally evaluated the design. The plasmid pMQ was transformed into E. coli CSZ4-sfGFP, K. pneumoniae 49210-sfGFP, E. coli HZE22-sfGFP to track the transfer of IncX4-mcr-1, IncL-bla_OXA−48 and IncHI2-bla_NDM−1 plasmids, respectively. As shown in Fig. 4B, E. coli C600 was fluorescent negative, while E. coli CSZ4-sfGFP, K. pneumoniae 49210-sfGFP, E. coli HZE22-sfGFP showed green fluorescence. Upon introduction of pMQ into E. coli CSZ4-sfGFP, K. pneumoniae 49210-sfGFP or E. coli HZE22-sfGFP, only red fluorescence was produced. In the conjugation system, both red and green fluorescence were observed, representing donor pMQ/AMR plasmids-harboring strains and E. coli C600 transconjugants, respectively. The ratio of green to red was higher in IncX4-mcr-1, IncL-bla_OXA−48 mating cultures (Fig. 4b), indicating a higher conjugation frequency of the two plasmids. To confirm the fluorescence detecting results, we randomly selected ~ 10 green and red colonies, conducted PCR and Sanger sequencing, and found that the green colonies were all from donors, while the green colonies were all from the E. coli EC600 transconjugants. These results indicated that our CRISPR-AMRtracker system successfully detected the AMR plasmid transfer in vitro.

The high conjugation frequency of the mcr-1-carrying IncX4 plasmid pCSZ4 (31229 bp, Fig S3) from E. coli CSZ4 to E. coli C600 strain prompted us to select it for further studies. To investigate the potential of IncX4-mcr-1 transfer in mixed bacterial populations, we firstly created a mixed recipient populations of four Gram-negative species: E. coli 25922, S. Typhimurium ATCC 14028, K. pneumoniae ATCC and A. baylyi ADP1. Using our above described pMQ/AMR-sfGFP system, we studied the IncX4-mcr-1 transfer in the mixed population using LSCM. Our results showed the fluorescence of both red and green cells in the conjugation cultures of E. coli 25922, S. Typhimurium ATCC 14028 and K. pneumoniae ATCC 700603 (Fig. 4c). To validate the fluorescence tracking results, we further conducted conjugation assay using single donor and recipient strains. The results showed that plasmid pCSZ4-sfGFP transferred successfully from donor to the recipient strains, including E. coli C600, S. Typhimurium ATCC 14028 and K. pneumoniae ATCC 700603, with frequencies of 1.12 ± 0.12 × 10^− 1, 7.03 ± 3.07 × 10^− 5 and 2.45 ± 0.01 × 10^− 5, respectively (Fig. 4d), but not to A. baylyi ADP1. This is consistent with the mixed population plasmid transfer results, supporting the potential application of our system in monitoring plasmid-mediated ARG transfer in natural mixed bacterial populations, such as the fecal microbiota.

3.5 Plasmid pCSZ4-sfGFP transfer in natural fecal microbiota community.

We then used the IncX4-mcr-1 CRISPR-AMRtracker system to examine plasmid-mediated transfer of ARGs in the fecal microbiota community under both aerobic and anaerobic conditions (Fig. 5a). The conjugation frequency of plasmid pCSZ4-sfGFP was 1.04×10^− 3 in the aerobic condition, which was about 5-fold lower under anaerobic conditions (2.00×10^− 4) (Fig. 5b). Transconjugants generated under aerobic conditions were isolated via cell sorting (FACS) and characterized by 16S rRNA gene amplicon sequencing. The recipient sample yielded 156 OTUs at the genus level, compared to 20 OTUs in the sort transconjugants sample. The sequenced transconjugants belonged to seven different genera, including Escherichia, Shigella, Brevibacillus, Lactobacillus, Staphylococcus, Enterococcus and Bacillus (Fig. 5c). To validate these results, we conducted the fluorescence-based mating approach on five pure strains of different species, including B. subtilis 163, B. subtilis ATCC 6633, E. faecalis ATCC 29212, S. aureus ATCC 29213 and L. reuteri 03. Green fluorescent cells were observed in all mating mixtures, except for B. subtilis ATCC 6633 (Fig. 5d).

HGT is a crucial process in the dissemination of ARGs among bacterial populations. However, the lack of effective tracking systems has hindered our comprehension of ARG transfer in complex bacterial communities. The use of a dual-fluorescence tracing system that distinguishes between donor and recipient strains offers a significant advantage in the study of HGT and the spread of ARGs.

Our study aimed to develop an efficient dual-fluorescence tracing system to facilitate the study of clinically significant plasmid-mediated transfer of AMR. Utilizing CRISPR/Cas9 and the lambda Red systems, the conserved regions of clinically relevant ARGs mcr-1, bla_NDM−1, bla_KPC−2, and bla_OXA−48 in clinical isolates were targeted and tagged with the sfGFP gene. We successfully labelled these important ARGs carried in IncX4, IncI2, IncHI2, IncF and IncL type plasmids, with 100% labelling success and relatively higher labelling efficiency, proving this method to be a useful tool for labeling different ARG-carrying plasmids in Enterobacteriaceae strains. The fluorescently labeled strains and their parent strains showed similar growth characteristics, antibiotic susceptibilities, and conjugation and transposition characteristics, indicating that the fluorescence tagging does not affect the normal function of the bacterial host. The sfGFP gene integrated at the downstream conserved region of the ARGs allows for monitoring of ARGs transfer via plasmids or mobile genetic elements (MGEs) transposition. This study provides a powerful tool for better understanding the spread of ARGs in complex bacterial systems, a crucial step towards developing effective strategies for controlling the spread of antibiotic resistance.

Our dual-fluorescence system presents a significant advantage over traditional methods, such as culture-dependent techniques or the use of helper strains with conjugative operons (e.g. RP4 IncP T4SS system) [3, 4, 30]. Our approach is highly effective in tagging clinically relevant AMR plasmids, and the use of pMQ enhances the detection of sfGFP-labeled AMR plasmid transfer in clinical bacterial hosts. Real-time monitoring of AMR plasmid transfer is made simple through the use of flow cytometry or fluorescent microscopy.

The transfer of IncX4-mcr-1 in this study can be observed under both anaerobic and aerobic conditions, with a conjugation frequency that was about 5 folds higher in aerobic condition. This result suggested an increase in oxygen levels can promote the spread of mcr-1 plasmid. Other studies have showed that an excess of ROS generation, due to disinfectant³², ultraviolet light³³ and carbamazepine³⁴, can further promote plasmid-mediated HGT. Additionally, inflammation has been shown to increase oxygen levels in the gastrointestinal tract, promoting plasmid transfer via conjugation³⁵. These findings suggest that changes in the anaerobic environment of the gut could affect the plasmid-mediated spread of ARGs and merit further investigation.

There are substantial evidences that fecal-derived bacteria act as reservoirs for the horizontal transfer of ARGs both between and within species ^{36, 37, 38}. Our results are in line with these findings, as we observed high conjugation frequencies and diverse transconjugants among fecal bacteria. Fecal bacteria also increased the likelihood of mcr-1 dissemination, as this gene has been commonly detected in Enterobacterales species such as E. coli, Shigella, K. pneumoniae, Salmonella enterica, E. aerogenes and E. cloacae ³⁹. Interestingly, we also detected transconjugants in a variety of bacteria including Brevibacillus, Lactobacillus, Staphylococcus, Enterococcus and Bacillus species. A recent study has also identified an mcr-1-carrying Bacillus cereus strain in tap water and importantly, the gene could be horizontally transferred to Enterococcus hirae in the gut of BALB/c J female mouse after inoculation of the B. cereus parent strain ⁴⁰. This finding, along with our results, highlights the potential for the transfer of mcr-1 inter-specifically in the gastrointestinal tract, and expands the bacterial host spectrum of mcr-1 beyond Enterobacterales.

In conclusion, our study presents a powerful tool, the CRISPR-AMRtracker system, for monitoring the spread of ARGs within complex bacterial communities. This system allows real-time monitoring of ARG transfer through plasmids or mobile genetic elements, and is a significant improvement over traditional methods. The integration of sfGFP gene in the ARG's neighboring region enables the easy tagging of different clinical important plasmids. Our results demonstrated the inter-specific transfer of mcr-1 in the fecal microbiota, expanding its bacterial host spectrum. The CRISPR-AMRtracker system has the potential to address the global AMR crisis by providing a deeper understanding of ARG spread and identifying key drivers of transfer in complex bacterial systems.

Funding

This work was supported by National Natural Science Foundation of China (31972735), Guangdong Major Project of Basic and Applied Basic Research (grant 2020B0301030007), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (32121004), Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2019BT02N054), Laboratory of Lingnan Modern Agriculture Project (NT2021006), Innovation Team Project of Guangdong University (2019KCXTD001), and the 111 Project (grant D20008).

Contributions

LC, JS conceived and designed the experiments. GL, TF-L, SY-Z，LJ-X，LW，XY-D，AG，YZ-H，RY-S performed the experiments and analyzed the data. JS, XP-L, YH-L supervised the project. GL wrote the manuscript. LC, HR, and LX-F revised manuscript. The authors have read and approved the manuscript.

Ethics declarations

Ethics approval.

Animal experimentation was approved by the by the ethics Committee of the Laboratory Animal Center of South China Agricultural University (Guangzhou, China), approval number 2020B041.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figure abstract. Summary diagram of development and application of CRISPR-AMRtracker.

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CRISPR-AMRtracker: a novel toolkit to monitor the antimicrobial resistance gene transfer in fecal microbiota

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Abstract

Figures

1. Introduction

2. Material And Methods

2.1 Bacterial strains, plasmids, primers and growth conditions.

2.2 Plasmid construction.

2.3 Labelling ARG plasmids with sfGFP gene.

2.4 Donor strain construction.

2.5 Conjugation assays.

2.6 Antimicrobial susceptibility testing.

2.7 Bacterial growth curve assay.

2.8 Transposition assays.

2.9 Fluorescence microscopy of bacteria.

2.10 Animal ethics statement.

2.11 Isolation of live mouse gut bacteria.

2.12 Conjugation in natural recipient community.

2.13 Flow cytometry and FACS measurements.

2.14 Metagenomic 16S sequencing and analysis.

2.15 Statistical analysis.

2.16 Accession number(s).

3. Results

3.1 Integration of fluorescence tags at conserved ARG regions.

3.2 Two plasmid CRISPR/Cas9 system was used for fluorescence tagging.

3.3 Fluorescence labeling did not interfere normal functions of the bacterial strains.

3.4 Monitoring ARGs transfer in complex microbiota.

3.5 Plasmid pCSZ4-sfGFP transfer in natural fecal microbiota community.

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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