Vibrio cholerae strains for capturing integron cassettes

Abstract

The present invention relates to a V. cholerae strain in which a platform has been built to detect integron cassettes. The platform includes a blind reporter system built by inserting an attl integration site into the gene of the toxin of a toxin / antitoxin system, which is inserted in the chromosome of the bacterium; the gene of the corresponding antitoxin and the gene of the intl integrase, both included in plasmids. The invention also relates to V. cholerae strains in which the superintegron (ASI) has been deleted, to the method for obtaining said strains, and to V. cholerae ASI strains in which the platform for capturing integron cassettes has been included. The invention includes methods and kits for detecting integron cassettes by using the strains and platforms built.

Description

[0001]

[0002] Vibrio cholerae strains for capturing integron cassettes

[0003] TECHNICAL SECTOR

[0004] The present invention falls within the biotechnology sector, 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 reorganizing 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 three key elements: the integrase gene (intl), which encodes a site-specific recombinase of the tyrosine recombinase family that carries out all recombination reactions within the integron; the integration site (attl) 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 integrons consists of the collection of cassettes, generally composed of a single open reading frame (ORF) followed by a recombination site (attC) that allows them to be recombined by integrase, resulting in their integration and excision from the platform.

[0007] 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 care expressed more intensely. Under stress conditions, integrase is expressed, which can cleave and reintegrate cassettes at the first position, where expression is highest. As indicated, integrons are naturally present in the chromosomes of more than 17% of available sequenced genomes. The paradigm of these sedentary chromosomal integrons (SCI) is the superintegron (SI) of Vibrio cholerae, a massive structure that houses approximately 200 cassettes, most of which encode genes with unknown functions. Some chromosomal integrons have been mobilized through transposons to conjugative plasmids that carry them by horizontal transfer; in this case, they are known as mobile integrons (MI).In the 1950s, MIs played a pivotal 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, as well as 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 ones, and the possibility of determining the orientation of target genes when inserting genomic DNA has been proposed, making it useful in gene therapy (CN 102517318A). The creation of attC sites de novo has also been described (EP2634256A1), 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 its functionality.

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

[0010] EXPLANATION OF THE INVENTION

[0011] Vibrio cholerae strains for capturing integron cassettes.

[0012] To address the problem of the lack of tools for routinely detecting integron cassettes in DNA samples, one aspect of this invention relates to an integron cassette capture gene platform that includes a reporter system and the integrase intl gene.

[0013] The integron cassette capture platform is designed from a class 1 integron, to which the most clinically relevant MIs belong, and whose integrase Intl 1 recognizes a broader spectrum of attC sites on integron cassettes than integrases from other integron classes. The invention involves cloning an integration site (attl) within the toxin gene of a toxin / antitoxin (TA) system, with the goal of using it as a blind readout marker to select for recombination events such that the survival of the microorganism carrying the construct is evidence of the integration event. After inducing intl expression, the captured cassettes disrupt the toxin gene, allowing bacterial survival in the absence of antitoxin. Before selecting for these recombination events, controlled expression of the antitoxin ensures bacterial viability.

[0014] To obtain this reporter, the attl site was cloned within the gene of a toxin from a TA system so that, upon expression, the resulting protein maintains its normal function. Preferably, the ccdB gene is selected as the specific reporter, accompanied by the gene for its antitoxin, ccdA, and attH is preferably selected as the integration site. A plastic region of ccdB, specified below, was chosen where introducing 51 bp while respecting the reading frame does not influence the toxin's activity. Expression of ccdB produces a toxin that binds to the GyrA subunit of bacterial DNA gyrase, preventing its correct function. CcdB is part of a type II toxin / antitoxin system, with CcdA as the antitoxin that inhibits the binding of CcdB to GyrA.One aspect of the invention, therefore, relates to a blind reporter construct that includes an attl site inserted into the toxin gene of a toxin / antitoxin system, such that said insertion does not modify the functionality of the protein resulting from the toxin gene, and the construct further includes the antitoxin gene of the toxin / antitoxin system and the intl integrase gene. Preferably, the toxin / antitoxin system used to carry out the construct is the CcdB / CcdA system whereby the attl site is inserted into the ccdB gene (ccdB..attl) without modifying the functionality of the protein resulting from the translation of the ccdB gene, and the construct also includes the ccdA gene and the intl integrase gene. AttH and intl1 are preferably selected.

[0015] This construct is embedded within a host bacterium, in which the integron cassette uptake mechanism will be activated for different purposes, such as uptake of exogenous sample cassettes.

[0016] The toxin gene, which includes the attl site, is introduced into the chromosome of the host bacterium of the construct, while the antitoxin gene and intl are each on a separate plasmid. On the one hand, the toxin gene is cloned under an inducible promoter, while the antitoxin gene is cloned under the control of a constitutive promoter and, preferably, on a thermosensitive replication origin plasmid. On the other hand, intl is under the control of an inducible promoter and on a high-copy-number plasmid. Preferably, this construct includes PTetM as the toxin promoter, P cs as the antitoxin promoter and PBAD as the integrase promoter. This construct is preferentially introduced into . cholerae.

[0017] Another option is to generate a version of the reporter containing an intein, to have a version that allows for greater control over the toxin, in case it is necessary to limit its effects on the host cell.

[0018] By high copy number plasmids, those skilled in the art understand those that have “relaxed replication” whose number can vary between 15 and several hundred copies.

[0019] However, the construct cannot be used directly in Vibrio cholerae because this species has a superintegron that would interfere with the objective of capturing integron cassettes from external samples. Therefore, starting from the V. cholerae N 16961 strain, the V. cholerae N 16961 ASI strain has been developed, in which the superintegron (SI) contained on chromosome 2 has been deleted, to be used as a host bacterium in which to insert the blind reporter construct. The V. cholerae N16961 ASI strain has been deposited in the Spanish Type Culture Collection (Science Park of the University of Valencia, Calle del Catedrático Agustín Escardino Benlloch, 9, 46980 Paterna, Valencia) on 25 / 07 / 2023, where it has received the reference number CECT30916.

[0020] The V. cholerae IS is a large chromosomal integron, containing more than 100 kb and 170 cassettes. In order to use V. cholerae as a host bacterium, it was necessary to delete the IS because it interfered with attempts to generate a cassette capture tool. However, this deletion process was hampered by the presence of 19 toxin-antitoxin (TA) systems in the IS, which are involved in its stabilization. Indeed, TA systems, due to their additive effect, are known stabilizers of DNA segments, including the superintegron (Fraikin N, et al. 2020. Type II toxin-antitoxin systems: evolution and revolutions. J Bacteriol 202:e00763-19). To delete the SI, a sequential deletion method of the TA systems was devised and developed, which we called SeqDelTA, using the natural competence of V. cholerae and homologous recombination to produce allelic exchanges.The successive exchanges were designed to eliminate the toxin but leave the antitoxin intact, ensuring that the event was not lethal to the bacteria.

[0021] Another aspect of the invention therefore relates to a method for deleting the SI of V. cholerae which includes the sequential deletion of the TA systems and which includes the following steps:

[0022] 1 - prepare a construction with:

[0023] - a left homology region (LHR) that is homologous to a V. cholerae DNA fragment between intIA and the TA system antitoxin gene to be eliminated and is approximately 500 bp in size;

[0024] - a right homology region (RHD) that is homologous to a V. cholerae DNA fragment comprising the complete gene of the TA system antitoxin to be eliminated and, where necessary, the promoter region of the corresponding toxin gene, and has an approximate size of 500 bp;

[0025] - the gene for a selective marker; and joining these three elements into a single fragment that has the gene for the selective marker located between the RHI and RHD;

[0026] 2 - introduce the construct obtained in the previous step into the bacteria in which the TA systems are to be eliminated and induce allelic exchange with the bacteria's chromosome;

[0027] 3 - repeat steps 1-2 for all TA systems that you want to eliminate with the gene of a selective marker different from the one used in the previous construction;

[0028] 4 - Perform a clean deletion of the remaining SI residues on the chromosome and of the last resistance marker, after step 3. This is done by cloning, into a counterselectable suicide plasmid, two DNA fragments whose sequence is homologous to the two fragments adjacent to the SI; the subsequent introduction of the cloned suicide plasmid into the strain whose SI is to be deleted; and the induction of allelic exchange between the suicide plasmid construct and the bacterial chromosome.

[0029] Preferably, in step 3 the same RHI is chosen as in step 1. Also preferably, 3 genes from 3 different selective markers are selected, which alternate in the different successive constructions.

[0030] Optionally, if the organization of the TA system requires it, it may be necessary to design alternative RHIs and maintain two selective markers in consecutive allelic replacements and, in a subsequent step, eliminate both selective markers.

[0031] One aspect of the invention, therefore, relates to a strain of V. cholerae in which the SI has been deleted. Preferably, this strain is V. cholerae CECT30916.

[0032] Another aspect of the invention relates to a strain in which the construction of the blind reporter has been included. Preferably, it refers to the V. cholerae N 16961 ASI strain with the platform for capturing integron cassettes, which has been deposited in the Spanish Type Culture Collection (Science Park of the University of Valencia, Calle del Catedrático Agustín Escardino Benlloch, 9, 46980 Paterna, Valencia) on July 25, 2023, where it has received the reference number CECT30917.

[0033] The invention also relates to a method for detecting integron cassettes from DNA samples using the host bacteria containing the platform for capturing integron cassettes and the DNA under study by conjugation.

[0034] Another aspect of the invention relates to a kit for the detection of integron cassettes in DNA samples that includes a V. cholerae ASI strain with the platform for capturing integron cassettes described herein, preferably as competent cells, with the preferred strain being V. cholerae CECT30917.

[0035] The samples to be analyzed can be of human, animal, or environmental origin, and can be clinical, food, or environmental, allowing for the analysis of the prevalence and spread of antibiotic resistance cassettes and, therefore, for understanding the ecology of these elements. Furthermore, by including blind selection, these tools can be used to study the content of integron cassettes in tested samples, analyzing their function, since chromosomal integrons are known to be a virtually infinite reservoir of integron cassettes of unknown function.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] To complement the description being made and in order to help better understand the characteristics of the invention, a set of drawings is attached as an integral part of said description, in which the following has been represented for illustrative and non-limiting purposes:

[0038] Figure 1. Base of a platform for integron cassette capture. A. In a resting state, we induce ccdA expression, which allows the bacteria to survive. B. To capture a cassette, we induce integrase (intl) expression and block ccdA' expression. The bacteria die if the ccdB gene is intact, or survive if a cassette is inserted into the attl site, interrupting ccdB gene transcription.

[0039] Figure 2. Schematic of the platform for capturing integron cassettes and their background noise. A. Platform details. The ccdB::attl1::NPU construct is cloned into the chromosome under the control of the PyetM promoter. ccdA is cloned under the control of the P promoter. C S is cloned into a plasmid, and intl1 is also cloned into a plasmid under the control of the PvanM promoter. B. FME of the system, compared with the intein-less ccdB::att!1 construct and the wild-type toxin.

[0040] Figure 3. Schematic of the SeqDelTA technique. To delete the SI we used fragments constructed by SOE-PCR that included a resistance marker (zeo R , carb R or cm R ), an RHI and an RHD that leaves the antitoxin gene (Antitox.) intact but deletes the toxin gene (Tox.). Successive deletions eliminated the TA system and the cassettes contained between the previous TA system and that of the current deletion.

[0041] Figure 4. Cassette capture using the conjugative integron capture platform in V. cholerae ASI. Recombination frequencies (RF) were obtained by calculating the number of recombinants (either by phenotype (violet) or by blind selection using our platform (pink) relative to the total number of bacteria. ccdB::attl1::NPU represents the platform, and attl represents a similar control without the ccdB gene.

[0042] PREFERRED EMBODIMENT OF THE INVENTION

[0043] The present invention is illustrated by the following examples, which are not intended to be limiting in scope. Also included are tables describing the primers (Table 1), plasmids (Table 3), and strains (Table 4) used or generated in these examples.

[0044] Figure 1A depicts the cloning of an integration site (attH) within the CcdB toxin gene of the CcdB / A toxin / antitoxin system, with the aim of using it as a blind reading marker to select recombination events so that the survival of the microorganism carrying the construct demonstrates the integration event. After inducing intl expression, as shown in Figure 1B, the captured cassettes disrupt the CcdB gene, allowing bacterial survival in the absence of CcdA. Before selecting these recombination events, controlled expression of the antitoxin ensures bacterial viability.

[0045] Example 1. Construction of the blind reporter.

[0046] An E. coli attH site (SEQ ID NO: 37) was inserted into the Vibrio fischeri ccdB gene, generating ccdBKo::attl1 (SEQ ID NO: 38). This construct was synthesized at IDT (Integrated DNA Technologies, USA) with a STOP codon, and its functionality was regenerated by SOE-PCR (Splicing by Overlap Extension) (POR). This was done by first amplifying two fragments, from SEQ ID NO: 38, with the primer pairs SEQ ID 1 and 2 and SEQ ID 3 and 4, and then joining them by another POR with primers SEQ ID 1 and 4, obtaining ccdB::attl1 (SEQ ID NO: 39).

[0047] Likewise, we also generated a version of the reporter containing an intein, ccdB::attl1::NPU (SEQ ID NO: 41) (from the IDT-synthesized fragment ccdBKo::att!1::NPU (SEQ ID NO: 40), using primer pairs SEQ ID 5 and 2, and SEQ ID 3 and 6. The final fragment was amplified with primers SEQ ID 5 and 6. The objective of introducing NPU is to have a version where we have more control over the toxin, in case it is necessary to limit its effects on the host cell (López-lgual, R. et al. “Engineered toxin-intein antimicrobials can selectively target and kill antibiotic-resistant bacteria in mixed populations.” Nature Biotechnology, 2019, 37 (7), pp.755-760). The blind reporter was tested in its different constructions as described in the following examples.

[0048] Example 2. Development of a platform for capturing integron cassettes.

[0049] To generate the platform for capturing integron cassettes, a thermosensitive origin of replication plasmid (pA884) containing ccdA under the control of the P promoter was first generated. C S. The plasmid was constructed using the Gibson Assembly technique (Gibson DG. “Enzymatic assembly of overlapping DNA fragments.” Methods Enzymol. 2011;498:349-61) by assembling the ccdA gene (SEQ ID NO 46) amplified with primers SEQ ID NO 9 and 10 from plasmid pA202, the P C S amplified with primers SEQ ID NO 11 and 12 from plasmid pA249 and the backbone fragment amplified from plasmid pA128 with primers SEQ ID NO 13 and 14. Then, with plasmid pA884, the V. cholerae N 16961 ASI strain, which is described below and has the reference CECT30916, was transformed.

[0050] In this example we describe the construction of the platform including PTetM ccdB::attl::NPU (SEQ ID NO: 42). A plasmid (pB629) was first constructed by amplifying the ccdB::attl::NPU fragment (SEQ ID NO: 41 ), synthesized in IDT as described in example 1 , with primers SEQ ID NO: 15 and 16 and the backbone of plasmid pA839 with primers SEQ ID NO: 7 and 8. The generated products were then ligated by Gibson Assembly and electroporated into the CcdB-resistant E. coli strain TT3813 (A118). To clone into the final host, in this example the V. cholerae strain CECT30916 described below, the regions adjacent to the superintegron deletion zone of chromosome 2 of V. cholerae CECT30916 were amplified to use as regions of homology in the insertion of our constructs.They were amplified using primers SEQ ID NO 17 and 18, and 19 and 20 and, by SOE-PCR, each of the homology regions was joined to one of the ends of the region of interest, PTGIM ccdB::attl::NPU (SEQ ID NO: 42), amplified with primers SEQ ID 21 and 22 from plasmid pB629.

[0051] After electroporation of pA884, the PyetM ccdB::attl::NPU fragment was introduced into the host strain, V. cholerae N16961 ASI, by natural transformation using the Instant Ocean protocol. Briefly, 80 mg of autoclaved chitin (Sigma-Aldrich) was inoculated with 1 ml of bacteria at an optical density of 1 in artificial seawater (7 g / liter Instant Ocean [Aquahum Systems]) and incubated statically at 30°C for 24 h. The supernatant was then changed to fresh medium, and 500 ng of the POR product to be transformed was added. The cells were then incubated statically at 30°C for 24 h. Finally, the cells were plated with the construct's resistance marker.

[0052] Next, by electroporation, the plasmid pB907, which contains the intH integrase gene (SEQ ID NO 45), was introduced into the V. cholerae CECT30916 strain, thus generating the B916 strain, deposited in the Spanish Type Culture Collection with reference number CECT30917 (Figure 2A).

[0053] This construct was tested with a killing assay to ensure proper toxin function and assess the background noise of the tool. Briefly, strain CECT30917 was incubated at 30°C in carbenicillin broth overnight, ensuring antitoxin production and thus bacterial survival. The following day, serial dilutions of the culture were performed under survival conditions (100 pg / mL carbenicillin, incubated at 30°C to maintain the thermosensitive plasmid) and death conditions (LB with 100 ng / mL anhydrotetracycline, which induces P expression). Tet, and incubating at 42°C to lose the thermosensitive plasmid). We obtained an escape mutant frequency (EMF) of 10' 6 (Figure 2B) By escape mutants we mean “background noise,” that is, bacteria that survive but have not taken up DNA at the integration site.

[0054] Example 3. Construction of the . cholerae ASI strain.

[0055] To generate the ASI mutant of . cholerae N16961, we developed a method called SeqDelTA, which is represented in Figure 3. After identifying the 19 toxin-antitoxin (TA) systems contained in the superintegron of . cholerae N16961, we prepared successive constructs designed with regions of homology and a selective marker. A left region of homology (LHR) was designed and maintained throughout the procedure. This LHR was located in the catB9 gene and was called LHR because it was closest to the intIA gene (the integrase gene of . cholerae). For successive constructs, a right region of homology (RHR) was designed to hybridize with the complete gene for the antitoxin of the TA system to be eliminated and with part of the gene for the corresponding toxin. It was called LHR because it was furthest from the intIA gene.In this way, the toxin was eliminated, but the antitoxin was left intact, ensuring that the event was not lethal to the bacteria (Figure 3). Three resistance genes for three different antibiotics were used as selective markers (Zeo. R , Cm R and Carb R), using a resistance gene different from the previous construct in each successive construct. Each of these three fragments from each successive construct was obtained by PCR and subsequently joined by SOE-PCR, placing the resistance marker between the RHI and the RHD. Each successive construct was introduced by natural transformation into the Vibrio cholerae strain resulting from the previous TA system deletions. This strategy allowed the resistance marker introduced in the previous step to be eliminated while TA system deletion was progressed through changes in the RHD. When necessary due to the orientation and / or order of the genes in the TA system, alternative RHIs were designed and two resistance markers were maintained in consecutive allelic replacements, before the new step allowed both to be eliminated.

[0056] As an example of the procedure followed in successive delatios, to delete the first TA system (VGA 311-312), using as a template the DNA of . cholerae N 16961 , we amplified the zeocin resistance gene with primers SEQ ID NO: 23 and 24, common to the 3 markers used (zeocin, carbenicillin and chloramphenicol), and which hybridize with the left homology region amplified with primers VGA 299 F (SEQ ID NO: 25) and LRHI VGA 300 R (SEQ ID NO: 26), and on the other hand with the right homology region amplified with primers LRHD VGA 311 F (SEQ ID NO: 27) and VGA 312 R (SEQ ID NO: 28). These 3 fragments were joined using the SOE-PCR technique, with primers SEQ ID NO: 25 and 28 and the fragment SEQ ID NO: 43 was obtained. The fragment SEQ ID NO: 43 was introduced into V.cholerae N16961 by natural transformation and allelic exchange occurred by homologous recombination giving rise to strain A400 which lacks a TA system due to interruption of the toxin gene, but maintains the antitoxin gene and has Zeo as a resistance marker. R .

[0057] As indicated, each toxin / antitoxin system was deleted following these same steps, advancing from TA system to TA system away from the intH gene. In addition, after each step, the deletion of the corresponding toxin was verified by PCR from several colonies, and the loss of the deleted marker, i.e., the one used in the deletion of the previous TA system, was checked phenotypically.

[0058] As already mentioned, in some cases, the arrangement of the toxin and antitoxin genes required us to take an intermediate step. For example, obtaining strain A024 is included here.

[0059] To do this, we amplified the zeocin resistance gene with primers SEQ ID NO: 23 and 24, which hybridize with the left homology region amplified with primers VGA 421 F (SEQ ID NO: 29) and LRHI VGA 422 R (SEQ ID NO: 30), and on the other side with the right homology region amplified with primers LRHD VGA 443 F (SEQ ID NO: 31) and VGA 447 R (SEQ ID NO: 32). These 3 fragments were joined using the SOE-PCR technique, with primers SEQ ID NO: 29 and 32 and the fragment SEQ ID NO: 44 was obtained. The fragment SEQ ID NO: 44 was introduced into V. cholerae N 16961 by natural transformation and the allelic exchange occurred as mentioned above giving rise to the strain A024 that lacks the TA system that was intended to be eliminated, has as a resistance marker Zeo R but maintains the previous Carb resistance marker R Both resistance markers will be removed with the next build.

[0060] Once all the TA systems were deleted, we obtained the V. cholerae A066 strain that preserves the gene of the last selective marker used (in this example Zeo R ), the catB9 gene on which the RHI was designed, the first TA system and the start of the superintegron (i.e., intlA, attIA, and the first 8 cassettes up to the catB9 gene).

[0061] To achieve a clean deletion of the superintegron, a suicide counterselectable plasmid (pMP7) was used. Replication of pMP7 depends on the cellular TT protein and encodes the ccdB toxin gene under the control of the inducible PBAD promoter. The region of interest was cloned into plasmid pMP7, in this case a fragment that allows the clean deletion of possible superintegron remnants, including the zeocin resistance marker. Since plasmid pMP7 does not replicate in the host, selection for its resistance marker recovers colonies in which pMP7 has recombined with the region of interest, thus introducing a duplication of this same region. By inducing ccdB expression, bacteria that have lost it are selected by a second allelic exchange between the two duplicated regions (excising plasmid pMP7 from the chromosome), either resulting in a clean deletion or returning to the original genotype.

[0062] In this example, the region of interest was the two regions adjacent to the superintegron, one on each side, and was started from the V. cholerae A066 strain generated after the deletion of the last TA system as described in this same example. To provide homology for allelic substitution, two fragments were amplified from V. cholerae N16961 strain DNA, A with primers RHI link pMP7 F (SEQ ID NO: 33) and LRHI+DR (SEQ ID NO: 34), and B with primers RHD F (SEQ ID NO: 35) and RHD link pMP7 R (SEQ ID NO: 36), joining them by SOE-PCR with primers SEQ ID NO: 33 and 36, giving rise to a 1000 bp fragment without superintegron residues and without any resistance marker. Both the generated fragment and plasmid pMP7 were digested with Nael and Hindlll enzymes and ligated together to generate pA097, which was electroporated into CcdB-resistant E. coli P3914 (A116), giving rise to strain A097. pA097 was then introduced into V.cholerae A066 by conjugation. The conjugation assay was performed by growing the donor strain (A097) in LB containing 25 pg / mL chloramphenicol, 0.3 mM DAP, and 1% glucose, and the recipient strain (V. cholerae A066) in LB containing 50 pg / mL zeocin to an OD of 0.6. After washing the cultures, the donor and recipient strains were mixed in 1 mL of total volume in 4:1 portions, centrifuged for 2 min at 7000 rpm, and resuspended in 100 pl of LB. This volume was spread onto a conjugation membrane (Millipore mixed cellulose ester membrane, 47 mm diameter, 0.45 µm pore size) on a Peth LB + 0.3 mM DAP + 1% glucose plate and incubated overnight (18 hours) for pA097 conjugation. Integration of pA097 into the recipient A066 genome was selected by washing the filter in 5 ml of LB and plating senated dilutions onto Cm (2.5 µg / ml) and 1% glucose plates.Chloramphenicol-resistant colonies were subsequently grown in liquid LB medium and plated on LB agar plates supplemented with 0.2% arabinose to express ccdB and select for the second allelic exchange involving excision of pA097. Following this process, we Sanger sequenced the superintegron region and selected a colony that presented a clean deletion of the superintegron, generating strain B522, which was deposited in the Spanish Type Culture Collection and received the reference number CECT30916.

[0063] Example 4. Method for detecting integron cassettes using the V. cholerae strain that includes a platform for capturing integron cassettes.

[0064] The method for detecting integron cassettes using the platform whose construction is indicated in the previous examples is based on a conjugation / recombination assay.

[0065] In this example, the suicide plasmid pA123 was used, which is based on the plasmid pSW23T with an attC site and which, therefore, ensures the delivery of one of the recombination substrates in its single-stranded form, which is its recombinogenic form (this plasmid with an attC site emulates integron cassettes). The donor strain of pA123 (A123) conjugates the plasmid which enters the recipient cell, in this example the CECT30917 strain, in single-stranded form. Being a suicide plasmid, that is, a plasmid that requires a specific protein (P¡, TT) for its replication, the recipient strain, CECT30917, which contains the platform described in example 2, cannot sustain its replication. The only way for the pA123 vector to be maintained in recipient cells is for it to recombine with the att / 1 site contained in the CECT30917 platform. The efficiency of cassette uptake is measured by calculating the recombination frequency (RF).

[0066] For cassette detection by this method, the donor strain A123 was grown overnight in LB medium supplemented with 25 pg / mL chloramphenicol and 0.3 mM diaminopimelic acid (DAP). The recipient strain CECT30917 (internally designated B916) was grown overnight in LB medium supplemented with 50 pg / mL zeocin, 100 pg / mL carbenicillin, 50 pg / mL spectinomycin, and 1% glucose (to repress intl expression) at 30°C (to ensure maintenance of the thermosensitive plasmid pA884, which includes the ccdA antitoxin gene, and thus bacterial survival). The following day, the donor strain culture was diluted 1 / 100 in LB containing chloramphenicol and DAP as described above, and the recipient strain CECT30917 was diluted 1 / 100 in LB containing the same antibiotics mentioned above and 0.2% arabinose (to induce intH) at 30°C (to ensure maintenance of plasmid pA884). These cultures were incubated until an OD = 0.6.Next, after washing the cultures, 1 mL of recipient and donor cells were mixed in a 4:1 ratio, followed by centrifugation for 2 min at 7000 rpm and resuspension in 100 pl of LB. This volume was spread onto a conjugation membrane (Millipore mixed cellulose ester membrane, 47 mm diameter and 0.45 pm pore size) on a Petri dish containing LB + 0.3 mM DAP + 0.2% arabinose and incubated overnight (18 hours) at 30°C for conjugation and recombination to occur. The following day, the cells contained in the membrane were resuspended in 5 mL of LB, after which serial dilutions of 1:10 were made up to 10'. 7and 5 pL of each dilution were plated on LB medium supplemented with different antibiotics and reagents. To count the total recipient bacteria, they were plated in 50 pg / mL zeocin, 100 pg / mL carbenicillin, 50 pg / mL spectinomycin and 1% glucose, incubating at 30°C. To count the recombinant bacteria by the phenotype conferred by the integrated suicide plasmid, they were plated in 50 pg / mL zeocin, 100 pg / mL carbenicillin, 50 pg / mL spectinomycin, 2.5 pg / mL chloramphenicol and 1% glucose, incubating at 30°C. To count recombinant bacteria by survival per insertion within ccdB, cultures were incubated in 50 pg / mL zeocin, 50 pg / mL spectinomycin, 1% glucose, and 100 ng / mL anhydrotetracycline at 42°C. The recombination frequency was calculated as the proportion of recombinant colony-forming units (CFU) with respect to the total number of recipient CFU. In this case, we obtained a recombination frequency (RF) of 10'. 2in the case of detection by phenotype, and 10' 3 In the case of detection by our platform, frequencies similar to the control that only contains one attH (strain A980, table 3) (Figure 4).

[0067] Table 1. Primers used

[0068] Table 2. Plasmids used and generated Table 3. Strains used and generated

Claims

CLAIMS 1. Method for deleting the superintegron (SI) from chromosome 2 of Vibrio cholerae by sequential deletion of the toxin / antitoxin (TA) systems, which includes the following steps: 1 - prepare a construction with: - a left homology region (LHR) that is homologous to a DNA fragment of . cholerae between intl and the TA system antitoxin gene to be eliminated and is approximately 500 bp in size; - a right homology region (RHD) that is homologous to a DNA fragment of . cholerae comprising the complete gene of the TA system antitoxin to be eliminated and, optionally, the promoter region of the corresponding toxin, and has an approximate size of 500 bp; - the gene for a selective marker; and joining these three elements into a single fragment that has the gene for the selective marker located between the RHI and RHD; 2 - introduce the obtained construct into the bacteria in which the TA systems are to be eliminated and induce allelic exchange with the bacteria's chromosome; 3 - repeat steps 1-2 for all TA systems that you want to eliminate with the gene of a selective marker different from the one used in the previous construction; 4 - perform a clean deletion of the remaining SI residues on the chromosome and of the last resistance marker, after step 3.

2. Method for deleting the SI of chromosome 2 of . cholerae according to claim 1 wherein, in step 3, the same RHI as in step 1 is selected.

3. Method for deleting the SI of . cholerae according to any of the preceding claims, in which 3 genes from 3 different selective markers are selected and alternated in successive constructions.

4. Method for deleting the SI of . cholerae according to any of the preceding claims, which includes the use of a different RHI than that defined in step 1 and the maintenance of two selective markers in consecutive allelic replacements.

5. Strain of . cholerae characterized by the deletion of the SI from its chromosome 2 (V. cholerae ASI strain) by the method defined in any of claims 1-4.

6. V. cholerae ASI strain deposited in the Spanish Type Culture Collection with reference CECT30916.

7. V. cholerae strain according to any of claims 5-6 including a platform for capturing integron cassettes including: - an attl integration site inserted into the gene encoding a toxin of a toxin / antitoxin system, - the antitoxin gene of the toxin of said toxin / antitoxin system, - the intl integrase gene, where the attl integration site is inserted into the toxin gene without modifying the protein's functionality and where the gene encoding the toxin and having the attl integration site inserted is included in the chromosome of the V. cholerae strain or host bacterium, the gene encoding the antitoxin is included in a plasmid, and the intl gene is included in a plasmid.

8. V. cholerae strain according to claim 7 wherein the integration site is attH.

9. V. cholerae strain according to any of claims 7-8, wherein the toxin / antitoxin system included in the platform for capturing integron cassettes is the ccdB / ccdA system.

10. V. cholerae strain according to claim 9, wherein the ccdB gene with the attl integration site included in the platform for capturing integron cassettes corresponds to the sequence SEQ ID NO:

39.

11. V. cholerae strain according to any of claims 7-9, wherein the toxin gene included in the platform for capturing integron cassettes includes the NPU gene of an intein following the attl integration site.

12. V. cholerae strain according to claim 11, wherein the ccdB gene with the attl integration site and the NPU intein inserted, included in the platform for capturing integron cassettes, corresponds to the sequence SEQ ID NO:

41.

13. V. cholerae strain according to any of claims 7-12 wherein the platform for capturing integron cassettes includes the antitoxin gene in a plasmid.

14. V. cholerae strain according to any of claims 7-13 wherein the platform for capturing integron cassettes includes the intl integrase gene on a plasmid.

15. V. cholerae strain according to claim 14, wherein the plasmid containing the intl gene is of high copy number.

16. V. cholerae strain according to any of claims 7-15, wherein the promoters of the toxin gene and the intl gene included in the platform for capturing integron cassettes are inducible promoters and the promoter of the antitoxin gene included in the platform for capturing integron cassettes is a constitutive promoter.

17. V. cholerae strain according to claim 16, wherein the promoter of the toxin gene is PyetM, the promoter of the intl gene is PBAD and the promoter of the antitoxin gene is PcS.

18. V. cholerae strain according to any of claims 7-17 wherein the integrase gene is intH.

19. V. cholerae strain deposited in the Spanish Type Culture Collection with reference CECT30917.

20. Method for detecting integron cassettes of a bacterial strain under study that includes contacting the strain under study with the V. cholerae host bacterium defined in any of claims 7-19 under conditions that allow the conjugation of plasmids of the strain under study, and the recombination of the integron cassettes of the strain under study at the attl site of the platform to capture integron cassettes included in the host bacterium.

21. Kit for the detection of integron cassettes in DNA samples that includes any of the bacteria defined in claims 7-19.

22. Kit for the detection of integron cassettes in DNA samples according to claim 21, wherein the bacteria are competent cells.