Tool for capturing integron cassettes

Abstract

The invention relates to a tool for capturing integron cassettes, which includes a gene of a counterselective marker into which an integration site (attI) has been inserted to convert same into a blind reading marker for selecting recombination events. The tool also includes the integrase gene intI. The structure of the counterselective marker with the attI site can be integrated into the chromosome of a bacterium, especially of a strain of Vibrio cholerae without superintegron, while the intI gene is included in a plasmid in which the tfoX gene can also be inserted, the tfoX gene regulating the natural competence of V. cholerae. The invention also relates to methods and kits for detecting integron cassettes present in other bacteria by means of conjugation, and integron cassettes from DNA samples by means of natural transformation.

Description

[0001]

[0002] Tool for capturing integron cassettes

[0003] TECHNICAL SECTOR

[0004] The present invention falls within the field of biotechnology, more specifically, it relates to molecular biology tools for the detection of integron cassettes.

[0005] BACKGROUND OF THE INVENTION

[0006] Integrons are genetic platforms capable of capturing, storing, and rearranging genes encoded in small genetic elements called integron cassettes through site-specific recombination reactions. These structures are naturally found in the chromosomes of more than 17% of sequenced bacteria, and some, through their association with transposons and conjugative plasmids, have spread to clinical settings. Currently, they are present in approximately 50% of clinical isolates of Gram-negative bacteria and can carry a vast array of antibiotic resistance genes.In general, the stable part of integrons is composed of 3 key elements: the integrase gene (intí), which encodes a site-specific recombinase of the tyrosine recombinase family that carries out all recombination reactions within the integron; the integration site (attí) where integron cassettes are incorporated; and a constitutive promoter (P. c ) that allows the expression of the genes contained within the cassettes. The variable part of the integrons is made up of the collection of cassettes, generally composed of a single open reading frame (ORF) followed by a recombination site (atfC) that allows them to be recombined by the integrase, resulting in their integration and excision from the platform.

[0007] The successive integration of cassettes into the attl site leads to the formation of a collection of genes with adaptive functions, where those closest to the P cThey are expressed more intensely. Under stress conditions, integrase is expressed, which can cleave and reintegrate cassettes at the first position, where expression is highest. As mentioned, integrons are naturally present in the chromosomes of more than 17% of available sequenced genomes. The paradigm of these sedentary chromosomal integrons (SCIs) is the Vibrio cholerae superintegron (SI), a massive structure that houses approximately 200 cassettes, most of which encode genes with unknown functions. Some chromosomal integrons have been mobilized via transposons to conjugative plasmids that transport them by horizontal gene transfer; in this case, they are known as mobile integrons (MIs).In the 1950s, mobile integrons (MIs) played a key role in the emergence and rise of multidrug resistance to antibiotics, and today they carry more than 170 resistance cassettes against most antibiotics. Five classes of mobile integrons are known; class 1 is the most prevalent and clinically relevant, and also the best studied.

[0008] In recent years, applications of integrons or their elements as biotechnological tools have been described. Specifically, attC recombination sites have been used to clone DNA of interest, even large strands, and the possibility of determining the orientation of target genes when the DNA is inserted into the genome has been proposed, making it useful in gene therapy (CN 102517318A). The creation of attC sites de novo has also been described (EP2634256A1), by rewriting the primary sequence of attC sites to encode a second function, or to generate recombination between protein domains within multidomain proteins (W0201809991A1), generating synthetic recombination sites with custom sequences that can be inserted into a selected DNA region while preserving their functionality.

[0009] However, despite their importance, the routine detection of integron cassettes in pathogenic microorganisms remains complex. Classical methods, using PCR reactions (CN104894283A, CN101948909A, CN106222252A), are based on sequences supposedly conserved in the 3' region of the class 1 integron. Advances in next-generation sequencing have revealed that these sequences are not found in most class 1 integrons (the most common), nor are they present in integrons of classes 2 through 5. Therefore, the only effective way to identify and characterize integrons is by next-generation sequencing, a laborious and time-consuming method that requires specific and expensive equipment (sequencers) and qualified personnel for data analysis, thus limiting its routine implementation.

[0010] EXPLANATION OF THE INVENTION

[0011] Tool for capturing integron cassettes.

[0012] To solve the problem of the lack of tools for routinely detecting integron cassettes in bacteria and DNA samples, one aspect of this invention relates to a gene tool for capturing integron cassettes that includes a reporter system and the intl integrase gene.

[0013] The integron cassette capture tool is designed using a class 1 integron, which includes the most clinically relevant integrating factors (IMs), and whose integrase Intl 1 recognizes a broader spectrum of attC sites on integron cassettes than integrases of other integron classes. The invention includes cloning an integration site (attl) into a counterselective marker gene, with the aim of using it as a blind-reading marker to select recombination events such that the survival of the microorganism carrying the construct indicates the integration event. After inducing intl expression, the captured cassettes disrupt the gene of this counterselective marker, allowing bacterial survival. Before selecting these recombination events, growth under conditions non-toxic to this marker ensures bacterial viability.

[0014] To obtain this reporter gene, the attl site was cloned into the gene of a counterselective marker so that, upon expression, the resulting protein does not lose its normal function. The sacB gene was preferably selected as the specific reporter gene, and att / 1 as the integration site. A plastic region of sacB, specified later, was chosen where inserting 51 bp while respecting the reading frame does not affect the toxin's activity. Expression of sacB results in an enzyme that, in the presence of sucrose, metabolizes this disaccharide into glucose and levan, the latter being toxic to a large number of Gram-negative bacteria.One aspect of the invention, therefore, relates to a construct of a blind reporter that includes an attl site inserted into the gene of a counterselective marker, such that this insertion does not modify the functionality of the protein resulting from the gene, and the construct further includes the intl integrase gene. Preferably, the counterselective marker used to perform the construct is the sacB gene, so the attl site is inserted into the sacB gene (sacB:.attl) without modifying the functionality of the protein resulting from the translation of the sacB gene. The tool also includes the intl integrase gene. att / 1 and intl1 are preferably selected.

[0015] In this descriptive report, a counterselective marker is understood to be a marker that results in negative selection by eliminating and inhibiting the growth of the host organism.

[0016] This tool is included within a host bacterium in which the integron cassette capture mechanism will be activated with different objectives, such as capturing exogenous sample cassettes from other carrier bacteria as well as DNA present in the environment.

[0017] The counterselective marker gene, which includes the attl site, is inserted into the chromosome of the tool host bacterium, while the intl gene is inserted into a plasmid. The counterselective marker gene is cloned with its constitutive promoter. Intl, on the other hand, is under the control of an inducible promoter and is located on a high-copy-number plasmid. Preferably, this construct includes PBAD as the integrase promoter. This construct is preferentially inserted into V. cholerae.

[0018] A second option involves generating a plasmid version containing the integrase that also includes the tfoX gene, the regulator of natural competition in Vibrio cholerae, transcriptionally fused with the intl gene, such that PBAD induction leads to the expression of both genes. This construct also allows for better control of natural competition in Vibrio cholerae.

[0019] By high-copy-number plasmid, the expert in the field means those with "relaxed replication," the number of copies of which can vary between 15 and several hundred. However, the construct cannot be used directly on Vibrio cholerae because this species has a superintegron that would interfere with the objective of capturing integron cassettes from external samples.

[0020] One aspect of the invention relates to a Vibrio cholerae strain having the SI deleted, in whose chromosome a blind reporter construct with a counterselective marker and the attl integration site inserted into the counterselective marker gene has been included, without modifying the functionality of the protein resulting from the translation of said gene, and which, furthermore, includes a plasmid containing the integrase gene, intl. Preferably, the counterselective marker is the sacB gene (sacBr.atti).

[0021] Furthermore, the invention relates to a Vibrio cholerae ASI strain with the counterselective marker construct sacB'.'.attl as just described, which includes a plasmid with the intl gene and with the tfoX gene transcriptionally fused to the intl gene. Preferably, it relates to the V. cholerae N 16961 ASI strain with the construct and plasmid described in this paragraph, a strain that was deposited in the Spanish Type Culture Collection (University of Valencia Science Park, Calle del Catedrático Agustín Escardino Benlloch, 9, 46980 Paterna, Valencia) on 14 / 04 / 2024, where it received the reference number CECT31061.

[0022] The invention also relates to a method for detecting integron cassettes from a bacterial strain under study using a host bacterium containing the tool for capturing integron cassettes and placing both strains in conditions that allow the conjugation of plasmids from the strain under study to the host strain, as well as the recombination of integron cassettes present in the plasmids of the strain under study at the attl site of the host bacterium.

[0023] A second method for detecting integron cassettes allows for their detection from DNA samples. This method uses a host bacterium containing the tool for capturing integron cassettes and the DNA under study, through transformation. To perform this method, DNA samples of any origin are brought into contact with the host bacterium under conditions that allow the host bacterium to transform with the sample DNA and recombine the integron cassettes of the DNA under study at the attl site of the host bacterium.

[0024] Another aspect of the invention relates to a kit for detecting integron cassettes present in other bacteria under study or in DNA samples, which includes a V. cholerae ASI strain with the integron cassette capture tool described herein, the preferred strain being V. c / ?o / erae CECT31061. Optionally, the kit includes the V. cholerae ASI strain with the integron cassette capture tool as competent cells.

[0025] The DNA samples to be analyzed can be of human, animal, or environmental origin and can be clinical, food-related, or environmental, allowing for the analysis of the prevalence and spread of antibiotic resistance cassettes and, therefore, a better understanding of their ecology. Furthermore, by incorporating blind selection, these tools can be used to study the integron cassette content in saturated samples, analyzing their function, since chromosomal integrons are known to be a virtually infinite reservoir of integron cassettes with unknown functions.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] To complement the description being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is included as an integral part of said description, in which, for illustrative and non-limiting purposes, the following has been represented:

[0028] Figure 1. Base of the tool for capturing integron cassettes. To capture a cassette, integrase (intí) expression is induced and the bacteria are incubated in the presence of sucrose; the bacteria die if the sacB gene is intact or survive if an integron cassette is inserted into the attl site, interrupting the transcription of the sacB gene that metabolizes sucrose, resulting in a toxic compound.

[0029] Figure 2. Schematic of the tool for capturing integron cassettes and their background noise. A. Platform details. The sacB::attl construct is cloned onto the chromosome. intH and tfoX are cloned under the control of the PBAD promoter onto a plasmid. B. Frequency of escape mutants (FME) of the system, comparing the sacB::attl1 construct with the wild-type gene. Dots represent results from independent experiments.

[0030] Figure 3. Cassette capture using the tool for integron cassette capture by conjugation in V. cholerae ASI. Recombination frequencies (RF) were obtained by calculating the number of recombinants (selected by phenotype, purple, or by blind selection using the tool of the invention, blue) out of the total number of bacteria. Background noise, corresponding to the number of bacteria growing when no cassette is conjugated, is shown in gray. sacB::attl1 represents the tool, and attl represents a similar control without the sacB gene. Dots represent results from independent experiments.

[0031] Figure 4. Cassette capture using the natural transformation integron cassette capture tool in V. cholerae ASI. Recombination frequencies (RF) were obtained by calculating the number of recombinants divided by the total number of bacteria. pppC167, gDNA A096, and gDNA A096 + GTT* represent the integron cassette capture frequencies from these samples, respectively. Background noise, corresponding to the number of bacteria growing when no sample is supplied for analysis, is shown in gray. Dots represent results from independent experiments.

[0032] PREFERRED EMBODIMENT OF THE INVENTION

[0033] The present invention is illustrated by the following examples, which are not intended to limit its scope. Tables describing the primers (Table 1), plasmids (Table 3), and strains (Table 4) used or generated in these examples are also included.

[0034] Figure 1 shows the cloning of an integration site (attí) within the SacB gene, with the aim of using it as a blind readout marker to select recombination events so that the survival of the microorganism carrying the construct indicates the integration event. After inducing intl expression, the captured cassettes disrupt the SacB gene, allowing bacterial survival in the presence of sucrose. Before selecting these recombination events, growth in sucrose-free medium ensures bacterial viability.

[0035] Example 1. Construction of the blind reporter.

[0036] An att / 1 site (SEQ ID NO: 17) from E. coli was inserted into the sacB gene of Bacillus subtilis, generating sacB::attl1 (SEQ ID NO: 18) by SOE-PCR (Splicing by Overlap Extension). This was done by first amplifying the att / 1 site from the pA121 plasmid with primers SEQ ID 1 and 2, and also two fragments of the sacB gene, from the pC525 plasmid, with primer pairs SEQ ID 3 and 4 and SEQ ID 5 and 6, and then joining them by another PCR with primers SEQ ID 3 and 6, obtaining sacB::attl1 (SEQ ID NO: 18).

[0037] Example 2. Development of a tool to capture integron cassettes.

[0038] In this example we describe the construction of the tool that includes sacB::attl (SEQ ID NO: 18).

[0039] To clone into the final host, in this example the V. cholerae strain C536, which is a laboratory strain derived from the B522 V. cholerae strain N16961 ASI, registered in the Spanish Type Culture Collection as CECT30916), the regions adjacent to the Tn7 region of chromosome 1 of strain C536 were amplified to use as homology regions in the insertion of our constructs, using primers SEQ ID NO 7 and 8 for the left homology region, and 9 and 10 for the right homology region. A zeocin resistance marker was also amplified, using primers SEQ ID 11 and 12 from plasmid pA249, to join it to the 3' end of the region of interest, sacB::attl (SEQ ID NO: 18), amplified with primers SEQ ID 3 and 6 as described in Example 1. The two homology regions, the region of interest and the zeocin resistance selection marker, were joined by SOE-PCR using primers SEQ ID 7 and 10.

[0040] The construct generated with SEQ ID 7 and 10 primers by SOE-PCR of sacB::attl1 was then introduced into the host strain, C536, by natural transformation, using the Instant Ocean protocol. Briefly, 150 µL of autoclaved chitin (Apollo Scientific) was inoculated into artificial seawater (7 g / L Instant Ocean [Aquahum Systems]) with 100 pL of bacteria at an optical density of 1 in 750 µL of artificial seawater (7 g / L Instant Ocean [Aquahum Systems]) and incubated statically at 30°C for 24 h. Then, 550 µL of supernatant was removed, and 2 pg of the PCR product to be transformed was added. The mixture was then incubated statically again at 30°C for 24 h. Finally, the cells were plated with the construct resistance marker (zeocin), generating strain C869.

[0041] Next, by electroporation, the pC274 plasmid, which contains the integrase gene intl1 (SEQ ID NO: 19), and the Vibrio cholerae natural competition regulator tfoX (SEQ ID NO: 20), was introduced into strain C869, thus generating strain C874, deposited in the Spanish Collection of Type Cultures with reference number CECT31061 (Figure 2A).

[0042] This construct was tested with a kill assay to ensure the toxin functioned correctly and to evaluate the tool's background noise. Briefly, the CECT31061 strain was incubated overnight at 30°C in LB medium with 100 pg / mL spectinomycin and 1% glucose. The following day, serial dilutions of the culture were plated under survival conditions (LB medium + 100 pg / mL spectinomycin + 1% glucose, incubated at 30°C) and under kill conditions (LB medium without NaCl + 10% sucrose, incubated at 20°C). We obtained an escape mutant frequency (EMF) of 10⁴ 6(Figure 2B). By escape mutants we mean the “background noise”, that is, the bacteria that survive but have not taken up DNA at the integration site.

[0043] Example 3. Method for detecting integron cassettes by conjugation using the V. cholerae strain that includes a tool for capturing integron cassettes.

[0044] One of the methods for detecting integron cassettes using the tool whose construction is indicated in the previous examples is based on the conjugation of plasmids containing integron cassettes.

[0045] In this conjugation example, the suicide plasmid pA123 was used. This plasmid is based on the pSW23T plasmid with an attC site, thus ensuring the delivery of one of the recombination substrates in its single-stranded form, which is its recombinogenic form (this plasmid with the attC site emulates integron cassettes). The pA123 donor strain (A123) conjugates the plasmid, which enters the recipient cell—in this example, strain CECT31061—in single-stranded form. Because it is a suicide plasmid, meaning it requires a specific protein (P1, TT) for its replication, the recipient strain, CECT31061, which contains the tool described in Example 2, cannot sustain its replication. The only way for the pA123 vector to remain in the recipient cells is for it to recombine with the attH site contained in the CECT31061 tool. The efficiency of cassette uptake is measured by calculating the recombination frequency (FR).

[0046] For cassette detection using this method, the donor strain A123 was cultured overnight in LB medium supplemented with 25 pg / mL chloramphenicol and 0.3 mM diaminopimelic acid (DAP). The recipient strain CECT31061 (internally designated C874) was cultured overnight in LB medium supplemented with 50 pg / mL zeocin, 100 pg / mL spectinomycin, and 1% glucose (to repress intH expression) at 30°C in LB medium. The following day, the donor strain A123 culture was diluted 1 / 100 in LB with chloramphenicol and DAP, as described above, and the recipient strain CECT31061 was diluted 1 / 100 in LB with the same antibiotics mentioned above and 0.2% arabinose (to induce intl1) at 30°C. These cultures were incubated to an ODeoo = 0.7. Then, after washing the cultures, the recipient and donor cultures were mixed in a 4:1 ratio in 1 mL of total volume, centrifuged for 2 min at 7000 rpm, and resuspended in 100 pl of LB.This volume was spread onto a conjugation membrane (mixed cellulose ester membrane of M i 11 i pore, 47 mm in diameter and 0.45 µm pore size) on a Peth plate containing LB + 0.3 mM DAP + 0.2% arabinose and incubated overnight (18 hours) at 30°C to allow conjugation and recombination to occur. The following day, the membrane-contained cells were resuspended in 5 mL of LB, after which serial 1:10 dilutions were performed up to 10 minutes. 7Five pL of each dilution were plated onto LB medium supplemented with various antibiotics and reagents. To count total recipient bacteria, plates were plated onto LB medium containing 50 pg / mL zeocin and 1% glucose, incubated at 30°C. To count recombinant bacteria based on the phenotype conferred by the integrated suicide plasmid, plates were plated onto LB medium containing 50 pg / mL zeocin, 2.5 pg / mL chloramphenicol, and 1% glucose, incubated at 30°C. To count recombinant bacteria based on survival by insertion into sacB, plates were plated onto LB medium without NaCl containing 10% sucrose, incubated at 20°C. The recombination frequency was calculated as the proportion of recombinant colony-forming units (CFU) relative to the total number of recipient CFUs, i.e., CFUs of CECT31061. In this case, we obtained a recombination frequency (FR) of 10' 3 in the case of phenotype detection, and 10' 3In the case of detection by our tool, frequencies very similar to the control that only contains one attH (C650 strain, table 3) (Figure 3).

[0047] These results show, on the one hand, that the tool described here is able to detect integron cassettes in the classic recombination assay, without needing to know what phenotype is conferred by the captured cassette and, on the other hand, that conjugation is useful as a way of detecting integron cassettes.

[0048] Example 4. Method for detecting integron cassettes by natural transformation using the V. cholerae strain that includes a tool for capturing integron cassettes.

[0049] Another method for detecting integron cassettes with the tool described in Example 2 focuses on directly testing DNA samples. For this, we used the host bacterium CECT31061, which contains the tool for capturing integron cassettes and whose natural transformation capacity is enhanced by the expression of the tfoX gene.

[0050] For the detection of cassettes with this method, several DNA samples were used: i) pppC167: a POR product amplified with the SEQ ID primers 13 and 14 of the pC167 plasmid containing a mobile class 1 integron array composed of two resistance cassettes, the aacA61 gene cassette in the first position and the catB3 gene cassette in the second position (both described in Hipólito, A. et al. (2023) Profile and resistance levels of 136 integron resistance genes, npj Antimicrob Resist 1, 13), in the 5'>3' direction; ii) gDNA A096: a genomic extraction from strain A096 corresponding to Vibrio cholerae N 16961 with its superintegron (179 cassettes); and finally iii) gDNA A096 + GTT*: the same genomic DNA sample amplified with the Equ¡Ph¡29 polymerase (ThermoFisher) with the GGT* R primer that enriches the GTTRRY sequence-rich regions characteristic of the attC sites of integron cassettes.

[0051] For the cassette capture assay, the host strain of the integron cassette capture tool CECT31061 was used as the cassette recipient strain. This strain was cultured overnight in LB medium supplemented with 50 pg / mL zeocin, 100 pg / mL spectinomycin, and 1% glucose (to repress intl1 and tfoX expression) at 30°C. The following day, it was diluted 1 / 100 in LB with the same antibiotics and 0.2% arabinose (to induce intH and tfoX) at 30°C for 4 h 30 min. Afterward, 300 pL of the culture were inoculated into 400 pL of artificial seawater (7 g / L Instant Ocean [Aquarium Systems]) and 300 pL of autoclaved chitin (Apollo Scientific). Next, approximately 2 pg (with the exception of the gDNA A096 + GTT* sample where it is difficult to purify and measure the DNA concentration and from which the total volume obtained, i.e. 15 pL, was added) of the DNA samples in different tubes and incubated statically at 30°C for 24 h.

[0052] The following day, the cells were resuspended by vortexing the mixture for 30s, after which 1:10 to 10 dilutions were performed. -7 Five milliliters (pL) of each dilution were plated onto LB agar plates supplemented with the various antibiotics and reagents listed below. To count the total recipient bacteria, plates were plated onto LB agar containing 50 pg / mL zeocin and 1% glucose, incubated at 30°C. To count recombinant bacteria by survival after insertion of one of the DNA samples used as cassettes into sacB, plates were plated onto LB agar without NaCl containing 10% sucrose, incubated at 20°C. The recombination frequency was calculated as the proportion of recombinant colony-forming units (CFU) relative to the total number of recipient CFUs, i.e., CFUs of CECT31061. For sample pppC167, we obtained a recombination frequency (RF) of 2.1 x 10⁻⁶ -5In the case of the gDNA A096 sample, we obtained a frequency of 3.5x10 -6 , and in the case of the gDNA sample A096 + GTT*, we obtained a recombination frequency of 8.9x10 -6 Finally, in the case of the control tube without a sample, the detection frequency (or, in this case, of escape mutants) was 4.3x10 -6(Figure 4). This allowed us to deduce potential cassette uptake events. Sample pppC167 appeared clearly positive, as did sample gDNA A096 + GTT*. Sample gDNA A096, being below the escape mutant frequency, was likely negative. To confirm this result, the contents of the sacB::attl1 platform were amplified with primers SEQ ID NO 15 and 16. A 340 bp band indicates no cassette is inserted, while a larger band indicates cassette uptake. We were able to confirm the capture of the catB3 cassette in over 60% of the colonies obtained for sample pppC167 and detected 11 cassettes in 21 colonies tested for sample A096 + GTT*. Sample gDNA A096 was indeed negative. Table 1. Primers and sequences used

[0053]

[0054]

[0055] In the GGT*R primer, the asterisks represent phosphorothioate bonds, the R bases purines, and the Y bases pyrimidines. Table 2. Plasmids used and generated

[0056] Table 3. Strains used and generated

Claims

CLAIMS 1. A tool for capturing integron cassettes that includes an attl site inserted into the gene of a counterselective marker and the intl integrase gene, wherein the attl integration site is inserted into the gene of the counterselective marker without altering the functionality of the protein encoded by said gene.

2. Tool for capturing integron cassettes according to claim 1 wherein the integration site is attl1.

3. Tool for capturing integron cassettes according to any of the preceding claims wherein the counterselective marker gene is sacB.

4. Tool for capturing integron cassettes according to claim 3 wherein the sacB counterselective marker gene is cloned with its constitutive promoter.

5. Tool for capturing integron cassettes according to any of claims 3-4 wherein the sacB gene with the attl integration site inserted corresponds to the sequence SEQ ID NO:

18.

6. Tool for capturing integron cassettes according to any of the preceding claims, wherein the gene encoding the counterselective marker and having the attl integration site inserted is included in the chromosome of a host bacterium.

7. Tool for capturing integron cassettes according to claim 6 wherein the host bacterium is a strain of Vibrio cholerae lacking a superintegron.

8. Tool for capturing integron cassettes according to any of the preceding claims wherein the intl gene is included in a plasmid.

9. Tool for capturing integron cassettes according to claim 8 which, in addition to the intl gene, includes the tfoX gene in the same plasmid.

10. Tool for capturing integron cassettes according to claim 9 wherein The tfoX gene corresponds to the sequence SE ID NO:

20.

11. Tool for capturing integron cassettes according to any of claims 9-10 wherein the tfoX gene is transcriptionally fused to the intl gene.

12. Tool for capturing integron cassettes according to any of claims 8-11 wherein the plasmid containing the intl gene is of high copy number.

13. Tool for capturing integron cassettes according to any of claims 8-12 wherein intl is under the control of an inducible promoter.

14. Tool for capturing integron cassettes according to claim 13 wherein the inducible promoter is PBAD.

15. Host bacterium that includes a tool as described in any of the preceding claims.

16. Host bacteria of the Vibrio cholerae species deposited with the reference CECT31061.

17. A method for detecting integron cassettes of a bacterial strain under study comprising contacting the strain under study with the host bacterium defined in any of claims 15-16 under conditions that allow conjugation of plasmids of the strain under study and recombination of integron cassettes of the strain under study at the attl site of the host bacterium.

18. Method for detecting integron cassettes in DNA samples comprising contacting the DNA sample under study with the host bacterium defined in any of claims 15-16 under conditions that allow transformation of the host bacterium with the DNA from the sample and recombination of the integron cassettes of the DNA under study at the attl site of the host bacterium.

19. Kit for the detection of integron cassettes in bacteria under study that includes any of the bacteria defined in claims 15-16.

20. Kit for the detection of integron cassettes in bacteria under study according to claim 19 wherein the host bacteria including the integron cassette collection tool are competent cells.

21. Kit for the detection of integron cassettes in DNA samples including any of the bacteria defined in claims 15-16.

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