Probiotic plasmids and uses thereof

Abstract

The present disclosure relates generally to conjugated plasmids comprising a multiple entry exclusion system derived from a plasmid carrying a gene conferring undesired traits of interest, such as pathogenic traits, such as antimicrobial drug resistance (AMR), virulence determinants, metal resistance, or other undesired traits, such as, for example, multiple entry exclusion systems derived from plasmids carrying a gene conferring undesired traits of interest. And the use of those probiotic plasmids for protecting bacterial populations (e.g., present in the gut, present in the environment, etc.) from obtaining plasmids imparting the undesirable traits and thereby preventing the propagation of the undesirable traits (e.g., AMR, virulence, metal resistance, etc.) within microbiota.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Australian Provisional Patent Application No. 2021903809 filed on November 25, 2021, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure generally relates to conjugated plasmids, including multiple entry-exclusion systems derived from plasmids carrying genes that confer undesirable traits of interest, such as pathogenic traits, such as antimicrobial resistance (AMR), virulence determinants, metal resistance or other undesirable traits, and the use of those probiotic plasmids to protect bacterial populations (e.g., present in the intestine, present in the environment, etc.) from acquiring plasmids that confer the undesirable trait and thereby preventing the spread of the undesirable trait (e.g., AMR, virulence or metal resistance, etc.) within the microbial population. Background Art

[0004] Antimicrobials are fundamental to many aspects of modern medicine, including treating infections, preventing infections during surgical procedures, and protecting immunocompromised patients from infections. However, over the past few decades, the use of antibiotics and other antimicrobials in human medicine, animal husbandry, veterinary practice, and aquaculture has increased dramatically. Although there is a growing trend towards improved antimicrobial stewardship in many countries, historical overuse of antimicrobials has selected for many mutant strains that are resistant to commonly used antimicrobials. Therefore, one of the major threats facing society is the increase in the number of antimicrobial resistant (AMR) bacteria such as Escherichia coli, Klebsiella pneumoniae, and Salmonella. The ability of these bacteria to resist the effects of antibiotics is largely mediated by invasive plasmids carrying antimicrobial resistance genes (ARGs), which, once acquired, cannot be removed due to the use of an 'addiction system'. Similarly, virulence factors are bacteria-associated molecules that are required for bacteria to cause disease when infecting eukaryotic hosts such as humans. A large number of virulence factors encoded by bacterial pathogens are also plasmid-borne. For example, plasmids in species of the genera Salmonella, Shigella, Escherichia coli, and Yersinia carry virulence genes (Pilla and Tang 2018). In short, virulence factors important for microbial pathogenesis and the virulence plasmids that encode these factors play an important role in disease. Virulence plasmids help bacteria infect humans, animals, and plants through a variety of mechanisms. Some virulence factors are toxins that damage or kill animal cells, others help bacteria attach to and invade animal cells, and still others protect bacteria from the immune system.

[0005] Many pathogenic bacteria carry AMR, virulence or metal resistance genes on extrachromosomal mobile genetic elements (such as plasmids). Large plasmids are effectively transferred between bacteria through cell-cell contact (conjugation), and once acquired, they ensure their own survival in the bacterial population through the addiction system. Due to their self-propagating properties, bacteria can quickly acquire these plasmids by being exposed to environments where AMR or virulence or metal resistance is endemic or prevalent (such as hospitals, clinics, contaminated sites, environments with metal toxicity, etc.). Once acquired, these plasmids are unlikely to be lost or removed due to their addiction properties. Therefore, it is highly desirable to first protect bacteria from the effects of AMR or virulence plasmid acquisition to reduce the burden of pathogenic plasmids and control transmission.

[0006] Currently, strategies for combating virulence / AMR transmission are limited. Plasmid cures (in which the AMR plasmid is removed from the bacterial population) and anti-plasmid approaches are being explored to reduce the prevalence of ARGs and sensitize the bacteria to antibiotics. However, no plasmid-cure treatment options are currently approved and available. In fact, few cure mechanisms have been tested in vivo, or even in experimental models. Although work on plasmid-cure approaches has made great strides, each of the above strategies has its own limitations and requires further investigation before a safe and effective treatment can be achieved. Therefore, there remains a need to address AMR burden and transmission.

[0007] Any discussion of documents, acts, materials, devices, articles or the like contained in this specification is not to be taken as an admission that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present disclosure as they existed before the priority date of each appended claim. Summary of the invention

[0008] The present disclosure is based, among other things, on the recognition of the inventors that novel strategies are needed to combat certain traits in bacterial populations, such as the prevalence of AMR, bacterial virulence, and metal resistance as examples, particularly in clinical or bioremediation settings. To this end, the inventors have developed a novel strategy to prevent bacterial populations from acquiring and / or spreading the plasmids using recombinant conjugated plasmids comprising a multiple entry exclusion system (EES). Plasmid replication incompatibility (Inc) and EES are two plasmid-carrying properties that can inhibit plasmid transfer to recipient bacteria carrying the same or closely related plasmids. EES is specifically present in most known conjugated plasmid types, and can inhibit entry of closely related plasmids into recipient bacteria by 10 to 10,0000 times, depending on the plasmid type. The inventors have exploited these plasmid-carrying properties to develop probiotic plasmids that can protect microbial populations from acquiring AMR or virulence plasmids. Specifically, the inventors have developed probiotic plasmids comprising multiple variant EES from the AMR plasmid of interest. Plasmids of incompatibility group F are the largest group of AMR plasmids and typically house multiple replicons. Using their novel approach, the inventors constructed protective probiotic plasmids carrying three genetic variants of the IncF EES gene. The inventors tested the novel probiotic plasmids in in vitro conjugation experiments and found that bacteria carrying these probiotic plasmids could protect >99.99% of their populations from acquiring different IncF plasmid variants carrying AMR. The inventors also demonstrated that increased expression of the EES gene (e.g., using an inducible promoter) can significantly increase protection against invasive AMR plasmids.

[0009] In one example, the present disclosure provides a recombinant plasmid comprising an entry exclusion system (EES) from a plurality of plasmid variants, wherein the plasmid variants comprise genes conferring a trait of interest. In one embodiment, the recombinant plasmid is a conjugated plasmid.

[0010] In one example, the trait of interest is an undesirable trait (e.g., a pathogenic trait) in a bacterial population. For example, the trait of interest may be antimicrobial resistance (AMR), bacterial virulence, or heavy metal resistance. In one example, the trait is AMR. In one example, the trait is bacterial virulence.

[0011] In one example, the recombinant plasmid (eg, a recombinant conjugated plasmid) includes EES from two plasmid variants that include genes conferring the trait of interest (eg, from a pathogenicity plasmid).

[0012] In one example, the recombinant plasmid (eg, a recombinant conjugated plasmid) includes EES from at least three plasmid variants that include genes conferring the trait of interest (eg, from a pathogenicity plasmid).

[0013] The EES in the recombinant plasmid (eg, recombinant conjugative plasmid) may be from different incompatibility groups. Alternatively, the EES in the recombinant conjugative plasmid may be from the same incompatibility group.

[0014] In one example, one or more of the EES are from a plasmid variant selected from the following incompatibility groups (Inc), or plasmids with any other known and unknown Inc groups: IncF, IncI, IncA, IncC, IncL, IncM, IncN, IncX, and IncH. For example, one or more of the EES are from a plasmid variant of incompatibility group F (IncF). In some examples, a plurality of EES in the recombinant conjugated plasmid are from a plasmid variant of IncF (e.g., a traS gene variant). In another example, all of the EES in the recombinant conjugated plasmid are from a plasmid variant of IncF (e.g., all of the EES are traS gene variants).

[0015] In one example, the recombinant plasmid (e.g., a recombinant conjugated plasmid) includes one or more of the following:

[0016] an EES comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75% identical, or at least 80% identical, or at least 85% identical) to the sequence shown in SEQ ID NO: 1 (traS_F variant);

[0017] EES comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75% identical, or at least 80% identical, or at least 85% identical) to the sequence shown in SEQ ID NO: 2 (traS_R100 variant); and / or

[0018] An EES comprising a polynucleotide sequence that is at least 70% identical (eg, at least 75% identical, or at least 80% identical, or at least 85% identical) to the sequence shown in SEQ ID NO:3 (traS_SLT variant).

[0019] In another example, the recombinant conjugated plasmid includes one or more of the following:

[0020] EES comprising a polynucleotide sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 1 (traS_F variant);

[0021] EES comprising a polynucleotide sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 2 (traS_R100 variant); and / or

[0022] An EES comprising a polynucleotide sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 3 (traS_SLT variant).

[0023] In one example, the recombinant conjugated plasmid includes one or more of the following:

[0024] EES comprising a polynucleotide sequence that is at least 95% identical (e.g., at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) to the sequence shown in SEQ ID NO: 1 (traS_F variant);

[0025] EES comprising a polynucleotide sequence that is at least 95% identical (e.g., at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) to the sequence shown in SEQ ID NO: 2 (traS_R100 variant); and / or

[0026] EES, comprising a polynucleotide sequence that is at least 95% identical (e.g., at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) to the sequence shown in SEQ ID NO:3 (traS_SLT variant).

[0027] In yet another example, the recombinant plasmid (e.g., a recombinant conjugated plasmid) includes one or more of the following:

[0028] EES, comprising the polynucleotide sequence shown in SEQ ID NO: 1 (traS_F variant);

[0029] EES comprising the polynucleotide sequence shown in SEQ ID NO: 2 (traS_R100 variant); and / or

[0030] EES, said EES comprising the polynucleotide sequence shown in SEQ ID NO: 3 (traS_SLT variant).

[0031] In some instances, the recombinant plasmid (e.g., recombinant conjugated plasmid) includes the EES variants named as the traS_F variant herein. In some instances, the recombinant plasmid (e.g., recombinant conjugated plasmid) includes the EES variants named as the traS_R100 variant herein. In some instances, the recombinant plasmid (e.g., recombinant conjugated plasmid) is named as the EES variants of the traS_SLT variant herein. For example, the recombinant plasmid (e.g., recombinant conjugated plasmid) may include the EES variants named as the traS_F variant herein and the EES variants named as the traS_R100 variant herein. For example, the recombinant plasmid (e.g., recombinant conjugated plasmid) may include the EES variants named as the traS_F variant herein and the EES variants named as the traS_SLT variant herein. For example, the recombinant plasmid (e.g., recombinant conjugated plasmid) may include the EES variants named as the traS_100 variant herein and the EES variants named as the traS_SLT variant herein. For example, the recombinant plasmid (eg, a recombinant conjugated plasmid) may include an EES variant designated herein as the traS_F variant, an EES variant designated herein as the traS_100 variant, and an EES variant designated herein as the traS_SLT variant.

[0032] Alternatively or in addition, the recombinant plasmid (e.g., a recombinant conjugated plasmid) includes one or more EES from a plasmid variant of the following: incompatibility group L (IncL), incompatibility group C (IncC), incompatibility group M (IncM) and / or incompatibility group A (IncA). In one example, the plasmid includes an EES from a plasmid variant of incompatibility group L (IncL). In one example, the plasmid includes an EES from a plasmid variant of incompatibility group C (IncC). In one example, the plasmid includes an EES from a plasmid variant of incompatibility group M (IncM). In one example, the plasmid includes an EES from a plasmid variant of incompatibility group A (IncA). In another example, one or more of the EES are from a plasmid variant of incompatibility group L (IncL), and one or more of the EES are from a plasmid variant of incompatibility group C (IncC). In another example, one or more of the EES is from a plasmid variant of incompatibility group L (IncL), and one or more of the EES is from a plasmid variant of incompatibility group M (IncM). In another example, one or more of the EES is from a plasmid variant of incompatibility group C (IncC), and one or more of the EES is from a plasmid variant of incompatibility group M (IncM). In another example, one or more of the EES is from a plasmid variant of incompatibility group L (IncL), one or more of the EES is from a plasmid variant of incompatibility group C (IncC), and one or more of the EES is from a plasmid variant of incompatibility group M (IncM).

[0033] For example, the recombinant plasmid (e.g., a recombinant conjugated plasmid) may include one or more (or each) of the following:

[0034] an EES comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75% identical, or at least 80% identical, or at least 85% identical) to the sequence shown in SEQ ID NO:9 (excL variant);

[0035] an EES comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75% identical, or at least 80% identical, or at least 85% identical) to the sequence shown in SEQ ID NO: 10 (excC variant);

[0036] EES comprising a polynucleotide sequence (excM variant) that is at least 70% identical (e.g., at least 75% identical, or at least 80% identical, or at least 85% identical) to the sequence shown in SEQ ID NO: 11; and / or

[0037] An EES comprising a polynucleotide sequence (excA variant) that is at least 70% identical (eg, at least 75% identical, or at least 80% identical, or at least 85% identical) to the sequence shown in SEQ ID NO: 12.

[0038] For example, the recombinant conjugated plasmid may include one or more (or each) of the following:

[0039] EES comprising a polynucleotide sequence that is at least 90% identical to the sequence shown in SEQ ID NO:9 (excL variant);

[0040] EES comprising a polynucleotide sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10 (excC variant);

[0041] EES comprising a polynucleotide sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 11 (excM variant); and / or

[0042] An EES comprising a polynucleotide sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 12 (excA variant).

[0043] For example, the recombinant plasmid (e.g., a recombinant conjugated plasmid) may include one or more (or each) of the following:

[0044] an EES comprising a polynucleotide sequence that is at least 95% identical (e.g., at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) to the sequence shown in SEQ ID NO:9 (excL variant);

[0045] an EES comprising a polynucleotide sequence that is at least 95% identical (e.g., at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) to the sequence shown in SEQ ID NO: 10 (excC variant);

[0046] EES comprising a polynucleotide sequence that is at least 95% identical (e.g., at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) to the sequence shown in SEQ ID NO: 11 (excM variant); and / or

[0047] An EES comprising a polynucleotide sequence (excA variant) that is at least 95% identical (e.g., at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) to the sequence shown in SEQ ID NO: 12.

[0048] For example, the recombinant plasmid (e.g., a recombinant conjugated plasmid) may include one or more (or each) of the following:

[0049] EES, comprising the polynucleotide sequence shown in SEQ ID NO: 9 (excL variant);

[0050] EES, comprising the polynucleotide sequence shown in SEQ ID NO: 10 (excC variant);

[0051] EES comprising the polynucleotide sequence shown in SEQ ID NO: 11 (excM variant); and / or

[0052] EES comprising the polynucleotide sequence shown in SEQ ID NO: 12 (excA variant).

[0053] In one example, one or more of the EES is operably linked to a constitutive promoter. For example, the constitutive promoter can be selected from a promoter native to the EES or a strong integrin promoter (Pc) or another suitable (including inducible, such as ara) promoter.

[0054] Alternatively or additionally, one or more of the EES is operably linked to an inducible promoter. For example, the inducible promoter may be an L-arabinose inducible promoter.

[0055] In some examples, each EES is operably linked to a separate promoter.

[0056] In some examples, one or more EES are operably linked to the same promoter.

[0057] In one example, the recombinant plasmid (e.g., a recombinant conjugated plasmid) includes a plurality of plasmid replication incompatibility (Inc) control regions (inc-control). For example, the Inc control region is from a plasmid variant selected from the following Inc group, and a plasmid having any other known or unknown Inc group: IncF, IncI, IncA, IncC, IncL, IncM, IncX, IncN, IncH.

[0058] In some examples, the Inc control region and the EES are from the same Inc group.

[0059] In one example, one or more of the Inc control regions are from a plasmid variant of IncF.

[0060] In one example, the recombinant plasmid (e.g., a recombinant conjugated plasmid) further comprises a selectable marker or gene. For example, the selectable marker or gene can be an antibiotic resistance gene. For example, the selectable marker confers selectable enzyme activity.

[0061] The present disclosure also provides a bacterial cell, which includes the recombinant conjugated plasmid of the present disclosure. Preferably, the bacterium is a probiotic. For example, the probiotic can be selected from the group consisting of the following members: Enterobacteriaceae, Lactobacillus, Bifidobacteria, Streptococcus, Lactococcus, Enterococcus, Propionibacterium, Faecalibacterium, Pediococcus, Ruminococcus, Megasphaera and Bacillus.

[0062] The present disclosure also provides a composition comprising one or more living bacterial cells as described herein.

[0063] In one example, the one or more live bacterial cells are lyophilized. The lyophilized bacterial cells may be microencapsulated and / or provided in a capsule.

[0064] In one example, the composition is a food or beverage product. For example, the food or beverage product can be a dairy product (e.g., a yogurt drink).

[0065] The present disclosure also provides a method for preventing or reducing the acquisition and / or spread of a plasmid conferring a trait of interest within a bacterial population, the method comprising introducing into the population one or more bacterial cells as described herein or a composition as described herein comprising the one or more bacterial cells.

[0066] In one example, the trait of interest is an undesirable trait, such as a pathogenicity trait conferred by a pathogenicity plasmid.

[0067] In one example, the trait of interest may be antimicrobial resistance (AMR). According to this example, one or more of the bacterial cells introduced into the bacterial population may include a recombinant conjugated plasmid comprising EES from a plurality of AMR-conferring plasmid variants.

[0068] In one example, the trait of interest may be bacterial virulence. According to this example, one or more of the bacterial cells introduced into the bacterial population may include a recombinant conjugated plasmid comprising EES from a plurality of plasmid variants that confer bacterial virulence.

[0069] In one example, the bacterial population is an intestinal bacterial population, and the method includes administering the one or more bacterial cells or a composition comprising the one or more bacterial cells to a subject in need thereof. For example, the method may include orally administering the one or more bacterial cells or a composition comprising the one or more bacterial cells to a subject.

[0070] In one example, the subject is a human.

[0071] In one example, the subject is a non-human animal. For example, the non-human animal can be a livestock species or a companion animal.

[0072] In another example, the bacterial population into which the probiotic is introduced is a population of bacteria that colonizes plants, and the method comprises contacting the plant or the soil in which the plant grows with the one or more bacterial cells or a composition comprising the one or more bacterial cells.

[0073] In another example, the bacterial population into which the probiotics are introduced is present in an environment selected from the group consisting of a healthcare environment, a soil environment, an environment including a water source, an environment including wastewater, an environment including industrial waste, an environment including agricultural waste, an environment including sewage, and / or an environment including biosolids, and the method comprises introducing the one or more bacterial cells or a composition comprising the one or more bacterial cells into the environment. For example, the method may be a method of bioremediation.

[0074] The present disclosure also provides a food product or beverage comprising one or more bacterial cells as described herein or a composition comprising the one or more bacterial cells.

[0075] In one example, the food product or beverage is a dairy product. For example, the food product or beverage can be yogurt (e.g., drinkable yogurt). BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 The traS gene sequences synthesized for cloning into a plasmid backbone are provided for (A) traS_R100 (SEQ ID NO: 4), (B) traS_F (SEQ ID NO: 5), and (C) traS_SLT (SEQ ID NO: 6). The -35 and -10 regions of the promoter are shaded green with underlining, the RBS sequence is shown in bold and underlined, and the coding region is shaded grey.

[0077] Figure 2 Vector maps of probiotic plasmids constructed with individual IncF entry exclusion gene variants are provided: (A) traS_R100, (B) traS_F, and (C) traS_SLT.

[0078] Figure 3 A synthetic sequence of TraS_3 is provided, encoding three IncF entry exclusion gene variants traS_R100, traS_F and traS_SLT, which are combined with the native promoters of traS_R100, traS_SLT and the integrator promoter Pc of tras_F and restriction sites (HindIII+XbaI). The -35 and -10 regions of the promoter are shaded and underlined in green, the PBS sequence is shown in bold and underlined, and the coding region is shaded in grey.

[0079] Figure 4 Provides carrying Figure 3 Vector map of the probiotic plasmids of the three IncF traS variants (traS_3).

[0080] Figure 5 A vector map of the pBAD33_Gm plasmid backbone including the IncF variant traS_R100 under the control of an arabinose-inducible promoter is provided.

[0081] Figure 6 A synthetic sequence of TraS_3 is provided, encoding three IncF entry exclusion gene variants, traS_R100, traS_F and traS_SLT, flanked by XbaI+HindIII restriction sites for cloning in the pBAD33-Gm backbone. The -35 and -10 regions of the promoter are shaded green and underlined, the RBS sequence is shown in bold and underlined, and the coding region is shaded grey.

[0082] Figure 7A vector map of a probiotic plasmid with a pBAD33_Gm backbone is provided, in which the coding sequences of the three IncF entry exclusion gene variants traS_R100, traS_F and traS_SLT have been cloned. IncF EES variants traS_R100 and traS_SLT are each under the control of an arabinose inducible promoter, while IncF EES variant traS_F is under the control of its native promoter.

[0083] Figure 8 A summary of the results of conjugation experiments performed with IncF plasmid from donor J53 to recipient BW25113Rf with or without different probiotic plasmid constructs of the present disclosure is provided.

[0084] Fig. 9 An alignment of different variants of the exclusion protein present in the IncA, C, IncM and IncL plasmid sequences found in GenBank is provided. (A) All IncC plasmids in GenBank have only one Exc protein type. The Exc proteins of IncC and IncA belong to the same exclusion group and block each other's entry. (B) and (C) show the exclusion protein variants from IncL and IncM plasmids.

[0085] Fig.10 The nucleotide sequence of the exc(L)_exc(C)_fosA3 gBlock is provided. The underlined stretches of nucleotides from 5'-3' are the coding regions of the IncL_exc, IncC_exc and fosA3 genes, respectively.

[0086] Fig.11 A physical map of the conjugative probiotic plasmid PB1.1 (size: 62702 bp) is provided. The two orange areas are for two independent insertion locations.

[0087] Fig.12 The nucleotide sequence of the probiotic plasmid PB.1.1 is provided.

[0088] Fig.13 A summary of the results of conjugation experiments with IncM, IncC or IncL plasmids with or without the probiotic plasmid construct PB1.1 of the present disclosure is provided. The blue bars are used to transfer different AMR plasmids into empty J53Az bacteria during the liquid mating experiment. The orange bars are used to transfer the corresponding plasmids into J53Az carrying the conjugated probiotic plasmid PB1.1. When the recipient bacteria have the probiotic plasmid, the transfer of the AMR plasmid is significantly reduced. The conjugation frequency is the ratio of the transconjugants per recipient. The data shown are the averages of three independent liquid mating experiments with standard errors.

[0089] Fig.14 A summary of the results of mouse experiments is provided, demonstrating the acquisition of different AMR plasmids with or without the probiotic plasmid construct PB1.1 disclosed herein. AMR-colonized mice were co-housed (8 hours per day) with control mice that did not receive probiotic plasmid treatment (none) or (probiotic) mice treated with probiotic plasmids. At different time points, AMR plasmids were detected in exposed mice from the non-treated group (none). AMR plasmids were not detected in mice that received probiotic plasmids (probiotics). Circles of different colors represent different mice in each group.

[0090] Fig.15 A summary of the results of antibiotic treatment after 120 hours of exposure to mice colonized with AMR is provided. After 120 hours of exposure to mice colonized with AMR plasmids, CTX in sucrose water was provided to probiotic plasmid-colonized and non-colonized control mice for 2 days. In mice whose intestinal flora were not colonized with probiotic plasmids (none), CTX-resistant bacterial colonization was greatly enhanced, but was only detected in 2 mice out of 9 mice, and the level in mice colonized with probiotic plasmids (probiotics) was very low. Circles of different colors represent different mice in the group.

[0091] Explanation of Sequence Listing Symbols

[0092] SEQ ID NO: 1 provides the polynucleotide sequence of the F plasmid variant entry exclusion system designated traS_R100.

[0093] SEQ ID NO: 2 provides the polynucleotide sequence of the F plasmid variant entry exclusion system designated traS_F.

[0094] SEQ ID NO: 3 provides the polynucleotide sequence of the F plasmid variant entry exclusion system designated traS_SLT.

[0095] SEQ ID NO: 4 provides the polynucleotide sequence of the F plasmid variant entry exclusion system designated traS_R100 with the native promoter, RBS and restriction sites.

[0096] SEQ ID NO: 5 provides the polynucleotide sequence of the F plasmid variant entry exclusion system designated traS_F with the native promoter, RBS and restriction sites.

[0097] SEQ ID NO: 6 provides the polynucleotide sequence of the F plasmid variant entry exclusion system designated traS_SLT with the native promoter, RBS and restriction sites.

[0098] SEQ ID NO: 7 provides the polynucleotide sequence of a construct comprising three F plasmid variants designated traS_R100, traS_SLT with the native promoter, and traS_F with the integron promoter Pc, RBS and flanking HindIII and XbaI restriction sites entering the exclusion system.

[0099] SEQ ID NO: 8 provides a polynucleotide sequence of a construct comprising (i) a common RBS having a traS_R100 variant polynucleotide sequence, (ii) a traS_F variant polynucleotide sequence having its natural promoter and RBS, and (iii) a traS_SLT variant polynucleotide sequence having an araBAD promoter and a common RBS, wherein HindIII and XbaI restriction sites are located on the sides of the three traS variant sequences.

[0100] SEQ ID NO:9 provides the polynucleotide sequence of the C plasmid variant entry exclusion system designated ExcC.

[0101] SEQ ID NO: 10 provides the polynucleotide sequence of the L plasmid variant entry exclusion system designated ExcL.

[0102] SEQ ID NO: 11 provides the polynucleotide sequence of the M plasmid variant entry exclusion system designated ExcM.

[0103] SEQ ID NO: 12 provides the polynucleotide sequence of the A plasmid variant entry exclusion system designated ExcA.

[0104] SEQ ID NO: 13 provides the nucleotide sequence of exc(L)_exc(C)_fosA3 gBlock.

[0105] SEQ ID NO: 14 provides the polynucleotide sequence of the probiotic plasmid PB1.1. DETAILED DESCRIPTION

[0106] General Technology

[0107] Throughout this specification, references to individual steps, features, compositions of matter, groups of steps or features, or groups of compositions of matter shall be considered to cover both one and more (i.e., one or more) of such steps, features, compositions of matter, groups of steps or features, or groups of compositions of matter, unless expressly stated otherwise or the context requires otherwise.

[0108] Those skilled in the art will appreciate that, in addition to those specifically described, the present disclosure is susceptible to changes and modifications. It should be understood that the present disclosure includes all such changes and modifications. The present disclosure also includes all steps, features, compositions and compounds mentioned or indicated in this specification individually or collectively, and any and all combinations of any two or more of the steps or features.

[0109] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0110] Unless expressly stated otherwise, any example of the present disclosure herein should be considered applicable mutatis mutandis to any other example of the present disclosure.

[0111] Unless specifically defined otherwise, all technical and scientific terms used herein should be interpreted as having the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0112] Unless otherwise indicated, the recombinant DNA, recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in the literature in sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Vols. 1 and 2, IRL Press (1991); DM Glover and BD Hames (eds.), DNA Cloning: A Practical Approach, Vols. 1-4, IRL Press (1995 and 1996); and FM Ausubel et al., (eds.), Current Protocols in Molecular Biology, Greene Publishing Associates (1996). Pub. Associates) and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988); and JE Coligan et al., (eds.), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0113] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer, or group of multiple steps or elements or integers, but not the exclusion of any other step or element or integer, or group of multiple elements or integers.

[0114] The term "and / or", for example, "X and / or Y" should be understood to mean "X and Y" or "X or Y", and should be considered to provide clear support for both meanings or either meaning.

[0115]

[0026] As used in this disclosure, including the claims, the words "a" and "an" mean "one or more."

[0116] As used herein, the terms "about" and "approximately" are interchangeable and should generally be understood to refer to a numerical range around a given number, as well as all the numbers in the numerical range. In addition, all numerical ranges herein should be understood to include each integer within the range.

[0117] As used herein, the terms "such as" and "ie" are used merely as examples and are not intended to be limiting, and should not be construed to refer only to those items expressly enumerated in the present disclosure.

[0118] Plasmids

[0119] The present disclosure provides a recombinant conjugated plasmid comprising multiple entry exclusion systems (EES) from a variety of plasmid variants, wherein the plasmid variant from which the EES is derived includes one or more genes that confer a trait of interest (e.g., a trait typically undesirable in a bacterial population). Exemplary traits undesirable in a bacterial population include, but are not limited to, antimicrobial resistance (AMR) and bacterial virulence. In some instances, the EES is derived from a pathogenic plasmid variant. As used herein, the term "pathogenicity" is understood in the art to be, for example, causing pathogenicity, such as the morbidity and / or mortality of an organism or a population of an organism. In general, pathogenic plasmids typically carry genes for resistance to antibiotics or heavy metals, genes for the metabolism of atypical substrates, or genes for many species-specific properties, such as metabolic properties or virulence factors. Therefore, the recombinant conjugated plasmid of the present disclosure can include multiple EES from pathogenic plasmid variants carrying genes that confer any one or more undesirable traits in these undesirable traits.

[0120] Plasmids disclosed herein utilize the properties of EES to inhibit plasmid transfer to recipient bacteria carrying the same or closely related plasmids. EES are specifically present in most known conjugated plasmid types and can inhibit the acquisition of closely related plasmids in recipient bacteria by 10 to 10,0000 times, depending on the plasmid type. The inventors have found that recombinant plasmids including EES from a variety of plasmid variants can be used to prevent or reduce the acquisition and / or spread of a range of closely related plasmid variants (e.g., such as pathogenic plasmid variants) with the same or related EES within a bacterial population.

[0121] As used herein, "plasmid" will be understood to mean a circular double-stranded DNA molecule, which forms an extrachromosomal self-replicating gene element that can be used as a medium for introducing nucleic acids into bacteria and eukaryotic cells. Bacterial plasmids are usually circular covalently closed and supercoiled. In some instances, plasmids can be spread from one bacterium to another bacterium (including bacteria of other species) by a process known as "conjugation". This host-to-host transfer of genetic material is a mechanism of horizontal gene transfer. Therefore, as used herein, "conjugated plasmid" will be understood to be a plasmid that can carry out host-to-host or horizontal transfer.

[0122] The term "recombinant" used in the context of the plasmid of the present disclosure is understood to mean an artificial or synthetic plasmid made using a plasmid backbone and components from one or more plasmids capable of expressing one or more DNA expression cassettes or operons. It will be appreciated by those skilled in the art that any number of plasmid backbones may be used to construct a recombinant plasmid of the present disclosure. However, exemplary plasmid backbones include pBCSK+, pBAD33_Gm, pJIBE401 and pJIMKCore_M as described herein. Such backbones may be modified to carry genes encoding replication and partitioning systems and multiple entry exclusion systems, in particular.

[0123] However, it will also be appreciated by those skilled in the art that constructs including multiple entry exclusion systems (EES) from multiple plasmid variants according to the present disclosure may also be delivered to host bacteria by other recombinant means (e.g., free phage (pseudolysogenic) or transposable gene elements or other integration and conjugation elements). In this regard, although it is a particularly preferred embodiment to introduce multiple EES into host bacteria using plasmids as described herein, it will be appreciated by those skilled in the art that concepts may be extended to other delivery means, and these alternative embodiments are contemplated herein. Therefore, in other examples, the present disclosure also provides a free phage, transposable gene element or other integration and conjugation element, comprising multiple EES from multiple plasmid variants, wherein the plasmid variant from which the EES is derived comprises one or more genes conferring traits of interest (e.g., traits typically not desired in bacterial populations). Exemplary traits not desired in bacterial populations include, but are not limited to, AMR and bacterial virulence.

[0124] As used herein, "entry exclusion system" or "EES" is understood to mean a genetic element or system that allows entry exclusion. "Entry exclusion" refers to a property of a plasmid by which a cell containing the plasmid becomes a poor recipient of similar plasmids during additional conjugation rounds. The entry exclusion system of a plasmid operates by inhibiting the physical entry of an incoming plasmid into a cell in which the incoming plasmid exhibits an exclusion phenotype. During bacterial division, inclusion of an entry exclusion system protects the plasmid from competition with related plasmids when it is separated. Therefore, entry exclusion (i) prevents incompatible incoming plasmids from eliminating pre-existing (e.g., less desirable but closely related) plasmids within the host cell, (ii) avoids uneconomical excess DNA transfer, and (iii) avoids death of the recipient cell due to lethal conjugation (zygosis).

[0125] Entry exclusion proteins from different sources are not equal in effectiveness, but exhibit variable exclusion indexes, which can be increased by overexpressing exclusion proteins. As used herein, the term "exclusion index" refers to the frequency of a given plasmid transferred to a plasmid-free receptor divided by the frequency of a receptor transferred to the same plasmid or a plasmid with a similar entry exclusion system. For example, plasmid F shows an EI of 100-300 in mating between E. coli because it is transferred to a plasmid-free receptor 100-300 times better than to F+ receptors (Achtman et al., 1977; Achtman and Skurray, 1977). In a preferred embodiment, the exclusion index or protection index is the ratio of the conjugation frequency to an empty receptor (without an exclusion system) and to a receptor bacterium with a probiotic plasmid (with a cloned exclusion gene). The exclusion index indicates the multiple of plasmid transfer inhibition. Therefore, the higher the exclusion index value, the stronger the protection against obtaining the tested plasmid.

[0126] As described herein, recombinant conjugated plasmid (or other medium for delivering DNA construct described herein) includes multiple EES from multiple plasmid variants.In an example, recombinant conjugated plasmid includes EES from two plasmid variants, and the two plasmid variants include the gene for giving the proterties paid attention to.In an example, recombinant conjugated plasmid includes EES from at least three (for example, 3 kinds or 4 kinds or 5 kinds or 6 kinds or 7 kinds or 8 kinds or 9 kinds or 10 kinds or more) plasmid variants, and the plasmid variant includes the gene for giving the proterties paid attention to.In some instances, two or more EES in EES in recombinant conjugated plasmid are from the same incompatibility group.In some instances, all EES in recombinant conjugated plasmid are from the same incompatibility group.In other examples, EES in recombinant conjugated plasmid are from different incompatibility groups (for example, EES can be derived from 2,3,4 or more different incompatibility groups).

[0127] An incompatibility group indicates that two plasmids cannot stably coexist for several generations in the same bacterial cell line. Plasmids that are incompatible with each other are assigned to the same "incompatibility group" or "Inc". In contrast, plasmids classified into different incompatibility groups may be able to coexist in the same bacterial cell. Therefore, "plasmid incompatibility" refers to the inability of plasmids to stably coexist in the same cell when the plasmids have similar or identical plasmid replication and / or plasmid partitioning systems, that is, each plasmid is separated into daughter cells with entry exclusion genes during cell division. In the absence of selective pressure on the two plasmids, two incompatible plasmids occupying the same cell tend to separate or partition to different cells during cell division. The stable intracellular coexistence of one plasmid with another requires that each plasmid can control its own replication / partitioning independently of the other plasmid, so that a stable copy number can be established and maintained. However, the inability of a given plasmid to maintain a stable copy number in the presence of another plasmid is a characteristic feature of incompatibility. When two plasmids of the same incompatibility group are found in the same cell, competition for cellular resources may result. Whichever plasmid replicates faster, or offers some other advantage, will usually be represented to a disproportionate degree in the copies allowed by the incompatibility system.Surprisingly, two plasmids can also be incompatible when they both have the same function of partitioning themselves into daughter cells.

[0128] Plasmids usually belong to only one of many existing incompatibility groups. Known incompatibility groups have more than 30. Examples include but are not limited to: IncN, IncW, IncL, IncM, IncT, IncU, IncW, IncY, IncB / O, IncI1, IncK, IncCom9, IncFI, IncFII, IncFIII, IncHI1, IncHI2, IncX, IncA, IncC, IncD, IncFIV, IncFV / FO, IncFVI, IncH1 3, IncI2, IncI, IncI, IncV, IncP, IncQ, etc., including variants thereof. Therefore, in some instances, one or more EES in the EES in the recombinant plasmid are derived from a plasmid variant selected from the incompatibility group (Inc) described above. In some examples, one or more (or all) of the EES in the recombinant plasmid are from a plasmid variant selected from the following incompatibility group (Inc): IncF, IncI, IncA, IncC, IncM, IncL, IncN, IncX, IncP, and IncH.

[0129] In a specific example, one or more of the EES are derived from a plasmid variant of IncF. In another example, all of the EES are derived from a plasmid variant of IncF. For example, a recombinant conjugated plasmid may include EES from at least 2 (e.g., at least 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 or more) plasmid variants of IncF.

[0130] As described herein, in some instances, entry into the exclusion system can be derived from plasmid variants of incompatibility group F, which is the largest group among AMR plasmids. Due to the considerable variation in its replication system / genes, it has been a challenge to date to cure and / or protect all plasmids within IncF using existing plasmid-based methods (see, e.g., Kamruzzaman M et al., 2017 and Bikard et al., 2014). F plasmids use two different EES genes, traT and traS, for exclusion. Most F plasmid exclusion activity is thought to be attributable to TraS (EI of about 200, compared to 20 for TraT). Therefore, the EI of F plasmid EES is also thought to be gene dose-dependent. In this regard, Skurray et al. and Achtman et al. (Skurray, Willetts et al., 1976, Achtman, Kennedy et al., 1977) showed that when traS and traT were cloned in multicopy plasmids, the EI increased to 10,000. In one example, one or more of the EES in the recombinant conjugated plasmid is a traS gene variant.

[0131] The present disclosure provides three gene variants of the entry exclusion gene traS from the F plasmid, named traS-R100, traS_F and traS_SLT, which together represent most of the IncF plasmid variations.

[0132] In one example, the recombinant conjugated plasmid of the present disclosure includes an EES, the EES including a polynucleotide sequence that is at least 70% (e.g., at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical to the sequence shown in SEQ ID NO: 1 ("traS_F variant EES"). For example, the recombinant conjugated plasmid of the present disclosure may include an EES, the EES including the polynucleotide sequence shown in SEQ ID NO: 1.

[0133] In one example, a recombinant conjugated plasmid of the present disclosure includes an EES comprising a polynucleotide sequence that is at least 70% (e.g., at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical to the sequence shown in SEQ ID NO: 2 ("traS_R100 variant EES"). For example, a recombinant conjugated plasmid of the present disclosure may include an EES comprising the polynucleotide sequence shown in SEQ ID NO: 2.

[0134] In one example, the recombinant conjugated plasmid of the present disclosure includes an EES, which includes a polynucleotide sequence that is at least 70% (e.g., at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical to the sequence shown in SEQ ID NO: 3 ("traS_SLT variant EES"). For example, the recombinant conjugated plasmid of the present disclosure may include an EES, which includes the polynucleotide sequence shown in SEQ ID NO: 3.

[0135] In some examples, traS_F variant EES, traS_R100 variant EES, and traS_SLT variant EES are combined in the recombinant conjugated plasmid of the present disclosure.

[0136] Those skilled in the art will appreciate that the recombinant conjugated plasmids comprising the F plasmid variant EES genes described herein may further comprise EES from other incompatibility groups of bacteria (e.g., bacteria known to be pathogenic). Exemplary additional EES genes from other incompatibility groups of bacteria include, but are not limited to, trbK (IncPα plasmid), eex (IncN plasmid pKM101 and IncW plasmid R388), exc (IncI plasmid R144), eexA and eexB (IncHI1 plasmid R27), eexC (IncC plasmid), mbeD (ColE1-like plasmid-non-self-propagating), sec10 / prgA (pAD1 and other sex information plasmids), pif (pSAM2 plasmid), and homologs, orthologs, and variants thereof.

[0137] Alternatively or in addition, the recombinant conjugated plasmid of the present disclosure may include one or more (e.g., 2, 3 or 4 or more) EES from the following plasmid variants: incompatibility group L (IncL), incompatibility group C (IncC), incompatibility group M (IncM) and / or incompatibility group A (IncA). This may be an alternative or supplement to the EES from the plasmid variant of incompatibility group F as described in the previous examples.

[0138] In a specific example, a recombinant conjugated plasmid of the present disclosure includes an EES from a plasmid variant of incompatibility group L (IncL), the EES including a polynucleotide sequence that is at least 70% (e.g., at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical to the sequence shown in SEQ ID NO: 9. For example, a recombinant conjugated plasmid of the present disclosure may include an EES, the EES including the polynucleotide sequence shown in SEQ ID NO: 9.

[0139] In a specific example, the recombinant conjugated plasmid of the present disclosure includes an EES from a plasmid variant of incompatibility group C (IncC), the EES including a polynucleotide sequence that is at least 70% (e.g., at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical to the sequence shown in SEQ ID NO: 10. For example, the recombinant conjugated plasmid of the present disclosure may include an EES, the EES including the polynucleotide sequence shown in SEQ ID NO: 10.

[0140] In a specific example, a recombinant conjugated plasmid of the present disclosure includes an EES from a plasmid variant of incompatibility group M (IncM), the EES including a polynucleotide sequence that is at least 70% (e.g., at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical to the sequence shown in SEQ ID NO: 11. For example, a recombinant conjugated plasmid of the present disclosure may include an EES, the EES including the polynucleotide sequence shown in SEQ ID NO: 11.

[0141] In a specific example, a recombinant conjugated plasmid of the present disclosure includes an EES from a plasmid variant of incompatibility group A (IncA), the EES including a polynucleotide sequence that is at least 70% (e.g., at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical to the sequence shown in SEQ ID NO: 12. For example, a recombinant conjugated plasmid of the present disclosure may include an EES, the EES including the polynucleotide sequence shown in SEQ ID NO: 12.

[0142] In some examples, the recombinant conjugated plasmid of the present disclosure includes an EES from a plasmid variant of IncL as described herein and an EES from a plasmid variant of IncC as described herein. The recombinant conjugated plasmid may further include an EES from a plasmid variant of IncF as described herein.

[0143] In some examples, the recombinant conjugated plasmid of the present disclosure includes an EES from a plasmid variant of IncL as described herein and an EES from a plasmid variant of IncM as described herein. The recombinant conjugated plasmid may further include an EES from a plasmid variant of IncF as described herein.

[0144] In some examples, the recombinant conjugated plasmid of the present disclosure includes an EES from a plasmid variant of IncL as described herein and an EES from a plasmid variant of IncA as described herein. The recombinant conjugated plasmid may further include an EES from a plasmid variant of IncF as described herein.

[0145] In some examples, the recombinant conjugated plasmids of the present disclosure include EES from a plasmid variant of IncC as described herein and EES from a plasmid variant of IncM as described herein. The recombinant conjugated plasmids may further include EES from a plasmid variant of IncF as described herein.

[0146] In some examples, the recombinant conjugated plasmids of the present disclosure include EES from a plasmid variant of IncA as described herein and EES from a plasmid variant of IncM as described herein. The recombinant conjugated plasmids may further include EES from a plasmid variant of IncF as described herein.

[0147] In some examples, the recombinant conjugated plasmid of the present disclosure includes an EES from a plasmid variant of IncA as described herein and an EES from a plasmid variant of IncC as described herein. The recombinant conjugated plasmid may further include an EES from a plasmid variant of IncF as described herein.

[0148] In some examples, the recombinant conjugated plasmids of the present disclosure include EES from a plasmid variant of IncL as described herein, EES from a plasmid variant of IncC as described herein, and EES from a plasmid variant of IncA as described herein. The recombinant conjugated plasmid may further include EES from a plasmid variant of IncF as described herein.

[0149] In some examples, the recombinant conjugated plasmids of the present disclosure include EES from a plasmid variant of IncL as described herein, EES from a plasmid variant of IncM as described herein, and EES from a plasmid variant of IncA as described herein. The recombinant conjugated plasmid may further include EES from a plasmid variant of IncF as described herein.

[0150] In some examples, the recombinant conjugated plasmids of the present disclosure include EES from a plasmid variant of IncL as described herein, EES from a plasmid variant of IncC as described herein, and EES from a plasmid variant of IncM as described herein. The recombinant conjugated plasmid may further include EES from a plasmid variant of IncF as described herein.

[0151] In some examples, the recombinant conjugated plasmids of the present disclosure include EES from a plasmid variant of IncL as described herein, EES from a plasmid variant of IncA as described herein, and EES from a plasmid variant of IncC as described herein. The recombinant conjugated plasmid may further include EES from a plasmid variant of IncF as described herein.

[0152] In some examples, the recombinant conjugated plasmids of the present disclosure include EES from a plasmid variant of IncC as described herein, EES from a plasmid variant of IncA as described herein, and EES from a plasmid variant of IncM as described herein. The recombinant conjugated plasmid may further include EES from a plasmid variant of IncF as described herein.

[0153] In some examples, the recombinant conjugated plasmid of the present disclosure includes EES from a plasmid variant of IncL as described herein, EES from a plasmid variant of IncC as described herein, EES from a plasmid variant of IncM as described herein, and EES from a plasmid variant of IncA as described herein. The recombinant conjugated plasmid may further include EES from a plasmid variant of IncF as described herein.

[0154] In some examples, the recombinant conjugated plasmids of the present disclosure include EES from a plasmid variant of IncF as described herein, EES from a plasmid variant of IncL as described herein, EES from a plasmid variant of IncC as described herein, EES from a plasmid variant of IncM as described herein, and EES from a plasmid variant of IncA as described herein.

[0155] Alternatively or in addition, one or more of the EESs contained in the recombinant conjugated plasmid of the present disclosure may be derived from a multidrug resistant organism or pathogen, including but not limited to organisms belonging to the genera Acinetobacter, Citrobacter, Enterobacter, Enterococcus, Escherichia, Klebsiella, Serratia or Staphyloccus. Exemplary multidrug resistant organisms include Acinetobacter baumannii, such as ATCC isolate No. 2894233-696-101-1, ATCC isolate No. 2894257-696-101-1, ATCC isolate No. 2894255-696-101-1, ATCC isolate No. 2894253-696-101-1, or ATCC isolate No. 2894254-696-101-1; Citrobacter freundii, such as freundii), such as ATCC isolate No. 33128, ATCC isolate No. 2894218-696-101-1, ATCC isolate No. 2894219-696-101-1, ATCC isolate No. 2894224-696-101-1, ATCC isolate No. 2894218-632-101-1, or ATCC isolate No. 2894218-659-101-1; Enterobacter cloacae cloacae), such as ATCC isolate No. 22894251-659-101-1, ATCC isolate No. 22894264-659-101-1, ATCC isolate No. 22894246-659-101-1, ATCC isolate No. 22894243-659-101-1, or ATCC isolate No. 22894245-659-101-1; Enterococcus faecalis rococcusfaecalis), such as ATCC isolate No. 22894228-659-101-1, ATCC isolate No. 22894222-659-101-1, ATCC isolate No. 22894221-659-101-1, ATCC isolate No. 22894225-659-101-1, or ATCC isolate No. 22894245-659-101-1;Enterococcus faecium, such as ATCC isolate No. 51858, ATCC isolate No. 35667, ATCC isolate No. 2954833_2694008, ATCC isolate No. 2954833_2692765, or ATCC isolate No. 2954836_2694361; Escherichia coli, such as ATCC isolate CGUC 11332, CGUC 11350, CGUC 11371, CGUC 11378, or CGUC 11393; Klebsiella pneumoniae, such as ATTC isolate No. 27736, ATTC isolate No. 29011, ATTC isolate No. 20013, ATTC isolate No. 33495, or ATTC isolate No. 35657; Serratia marcescens marcescens, such as ATCC isolate No. 43862, ATCC isolate No. 2338870, ATCC isolate No. 2426026, ATCC isolate No. SIID 2895511, or ATCC isolate No. SIID 2895538; or Staphylococcus aureus, such as ATCC isolate No. JHH 02, ATCC isolate No. JHH 02, ATCC isolate No. JHH 03, ATCC isolate No. JHH 04, ATCC isolate No. JHH 05, or ATCC isolate No. JHH 06. ;

[0156] Alternatively or in addition, one or more of the EES contained in the recombinant plasmid of the present disclosure can be derived from a virulence plasmid carrying a virulence gene encoding a virulence factor / virulence determinant. For example, pathogenic Escherichia coli, Shigella genus and other enteric bacteria that cause dysentery, such as Salmonella typhi (typhoid) and Yersinia pestis (Y. pestis, plague), usually rely on plasmids to carry virulence factors. Therefore, one or more of the EES contained in the recombinant conjugated plasmid of the present disclosure can be derived from a virulence plasmid.

[0157] The EES gene / variant can each be linked to a promoter to drive its expression in a host cell. As used herein, the term "promoter" is understood to define a regulatory DNA sequence, usually located upstream of a gene or sequence to be expressed, that mediates the initiation of transcription by directing RNA polymerase to bind to DNA and initiate RNA synthesis.

[0158] The promoter for inclusion in the recombinant plasmid of the present disclosure may be an endogenous promoter, a heterologous promoter, or a combination thereof.

[0159] In some examples, the promoter is a constitutive promoter (eg, T7, SP6, T3, Integron (Pc), or other suitable constitutive promoter).

[0160] In other examples, one or more EES genes in the EES gene can be under the control of an inducible promoter. An inducible promoter can be a nucleotide sequence or an operator system that guides the conditional expression of the EES gene in the presence of a certain compound, nutrient, amino acid, sugar, peptide, protein or condition (e.g., light, oxygen, heat, cold). Alternatively, an inducible promoter can include one or more repressor elements so that the transcription of the induced EES gene does not need to be present in the presence of a certain compound, nutrient, amino acid, sugar, peptide, protein or condition. Any suitable inducible promoter, system or operator known in the art can be used. Non-limiting examples of inducible promoters contemplated include a lactose regulatory system (e.g., a lactose operon system), a sugar regulatory system, a metal regulatory system, a steroid regulatory system, an alcohol regulatory system, an IPTG inducible system, an arabinose regulatory system (e.g., an arabinose operon system, e.g., an ARA operon promoter, pBAD, pARA, PARAE, ARAE, ARAR-ParaE, portions thereof, combinations thereof, etc.), a synthetic amino acid regulatory system (e.g., see Rovner AJ et al., (2015) Nature 518(7537): 89-93), a fructose repressor, a tac promoter / operator (pTac), a tryptophan promoter, a PhoA promoter, a recA promoter, a proU promoter, a cst-1 promoter, a tetA promoter, a cadA promoter, a nar promoter, a PL promoter, a cspA promoter, etc., or combinations thereof. In a specific example, the inducible promoter is an L-arabinose inducible promoter.

[0161] In some examples, two or more or all of the EES genes within the recombinant conjugated plasmid are operably linked to the same promoter in the plasmid, ie, a single promoter drives expression of multiple EES gene variants.

[0162] In other examples, each EES gene variant is operably linked to a separate promoter.

[0163] In some examples, the entry exclusion gene is linked to its native promoter and ribosome binding site (RBS). For example, the recombinant conjugated plasmid may include traS_F variant EES, traS_R100 variant EES, and traS_SLT variant EES, each of which is operably linked to its native promoter and RBS. For example, the recombinant conjugated plasmid of the present disclosure may include the polynucleotide sequence shown in SEQ ID NO: 4-6 or a sequence that is at least 70% (e.g., at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%) identical to the sequence shown in SEQ ID NO: 4-6.

[0164] In some instances, the recombinant plasmid of the present disclosure may include a toxin-antitoxin system or an additive system to prevent survival of plasmid-free segregants. As used herein, the terms "toxin-antitoxin system," "addiction system," and similar terms (e.g., killing system, killing-anti-killing, post-separation killing, poison-antidote, plasmid addiction system, or programmed cell death) are used to describe a genetic system that has evolved in a plasmid to ensure that host cells are selectively killed without receiving any copies of the plasmid. The molecular basis of the "toxin-antitoxin system" requires the presence of at least two genes: one specifies a stable toxic agent, i.e., a "toxin," and the other encodes an unstable factor that prevents the lethal effect of the gene encoding the toxic agent, i.e., an "antitoxin." The toxin and antitoxin genes may each be operably connected to a promoter within the plasmid to drive the expression of the corresponding gene. The toxin-antitoxin system for inclusion in a bacterial plasmid is known in the art and is contemplated herein.

[0165] Most plasmids are capable of independent replication. In order for a plasmid to replicate independently in a cell (e.g., a bacterial cell), it must have a stretch of DNA that can serve as a "replication origin." Plasmids that are capable of autonomous replication in a host cell are also referred to as replicons. In the case of a recombinant conjugated plasmid capable of autonomous replication according to the present disclosure, the plasmid will include a replication origin.

[0166] Other plasmids may stably integrate into the host cell's genome, and thereby replicate along with the host genome.

[0167] The recombinant conjugated plasmid may further include one or more plasmid replication incompatibility (Inc) control regions (inc-control). In a specific example, one or more inc-control regions are from one or more of the described plasmid incompatibility groups. For example, one or more inc-control regions are from a plasmid incompatibility group selected from the following: IncF, IncI, IncA, IncC, IncL, IncM, IncX, IncP, IncN and IncH. However, those skilled in the art will appreciate that the inc-control region may be from any known or yet to be identified plasmid incompatibility group.

[0168] Plasmids of the present disclosure may also include a number of other genetic elements, such as genes for plasmid-specific replication initiation proteins (Rep), repeating units called repeats, DnaA boxes, and adjacent AT-rich regions. In addition, plasmids may contain one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments obtained or derived from one or more natural and / or artificial sources.

[0169] The recombinant conjugated plasmid of the present disclosure may further include one or more selectable markers or genes to enable selection of cells (e.g., bacterial cells) comprising the plasmid. Selectable marker genes suitable for inclusion in plasmids will be known to those skilled in the art. In one example, selectable markers may be antibiotic resistance genes. Antibiotic resistance genes confer resistance to antibiotic compounds, such as ampicillin, kanamycin, chloramphenicol, tetracycline, rifampicin, neomycin, hygromycin, erythromycin, methotrexate and gentamycin. In another example, selectable markers are genes that confer selectable enzyme activity (e.g., beta-galactosidase or lactose operon).

[0170] In one example, the recombinant plasmid of the present disclosure may include the entry exclusion gene variants designated as traS_F, traS_R100, and traS_SLT as described herein (by reference to SEQ ID NOs: 1-3), operably linked to a promoter and RBS sequence having flanking HindIII and XbaI restriction sites within the pBCSK+ backbone (i.e., pJIMK_traS_3, Figure 4 ). For example, the recombinant plasmid of the present disclosure may include the sequence shown in SEQ ID NO:7.

[0171] In one example, the recombinant plasmid of the present disclosure may include entry excluder gene variants designated as traS_F, traS_R100, and traS_SLT as described herein (by reference to SEQ ID NOs: 1-3), wherein at least one of the traS variants is operably linked to the pBAD arabinose operon promoter shown in SEQ ID NO: 8 ( Figure 6 For example, traS variants can be combined within the pBAD33_Gm backbone (e.g., pJIMK_ara_traS_R100, Figure 5 For example, constructs including traS variants can be cloned into the HindIII+XbaI sites of the pBAB33_Gm backbone to generate pJIMK_ara_traS_3, wherein IncF EES variants traS_R100 and traS_SLT are each under the control of an arabinose-inducible promoter, and IncF EES variant traS_F is under the control of its native promoter ( Figure 7 ).

[0172] In one example, the recombinant plasmids of the present disclosure may include entry exclusion gene variants named IncC, IncL, and IncM as described herein (respectively by reference to SEQ ID NOs: 9-11), which are operably linked to promoter and RBS sequences, and the promoter and RBS sequences have any suitable restriction sites within the cloning vector, for example, pBCSK+ backbone. For example, the recombinant plasmids of the present disclosure may include the sequences shown in SEQ ID NOs: 9-11. Alternatively, the recombinant plasmids of the present disclosure may include the sequences shown in SEQ ID NOs: 11 and 13.

[0173] In one example, the recombinant plasmid of the present disclosure may include entry exclusion gene variants designated as IncC, IncL, IncM and IncA as described herein (respectively by reference to SEQ ID NOs: 9-12), operably linked to a promoter and a PBS sequence, wherein the promoter and RBS sequence have any suitable restriction sites within a cloning vector, e.g., pBCSK+ backbone. For example, the recombinant plasmid of the present disclosure may include the sequences shown in SEQ ID NOs: 9-12. Alternatively, the recombinant plasmid of the present disclosure may include the sequences shown in SEQ ID NOs: 11 and 13.

[0174] Probiotics

[0175] The present disclosure also provides a bacterial cell comprising a recombinant conjugated plasmid as described herein. That is, a bacterial cell or a progeny of such a bacterial cell into which a recombinant conjugated plasmid of the present disclosure has been introduced. Such bacterial cells can be used as probiotics to prevent or reduce the acquisition and / or spread of plasmids (such as pathogenic plasmids) expressing incompatible entry exclusion system genes in bacterial populations. Therefore, the bacteria as described herein can be probiotics.

[0176] The terms "probiotic," "probiotics," or the like used in connection with the bacteria of the present disclosure should be understood to mean bacteria that enhance the growth and / or health of beneficial bacteria in a particular environment (e.g., the gastrointestinal tract, soil, or water system of a subject). Alternatively or in addition, "probiotics," "probiotics," or the like may help reduce the growth and / or prevalence of pathogenic bacteria in a particular environment (e.g., the gastrointestinal tract, soil, or healthcare environment). For example, administering a sufficient amount of probiotics to a subject should confer health benefits to the subject (e.g., a human or animal host), such as by improving the balance of gastrointestinal microorganisms. In another example, a probiotic administered in sufficient amounts or otherwise introduced into soil, wastewater, biosolids, or other environments can improve the balance of microorganisms in the environment and cause one or more beneficial results.

[0177] The probiotics of the present disclosure are generally non-pathogenic and may exhibit (i) one or more beneficial functions within the gastrointestinal tract of humans and non-human animals, or (ii) one or more beneficial qualities that make them suitable for bioremediation applications.

[0178] Exemplary probiotic, non-pathogenic bacteria include Escherichia coli, non-pathogenic members of the Enterobacteriaceae family, and other Bacillus species, including but not limited to: Bacillus coagulans; Bacillus coagulans Hammer; and Bacillus brevis subsp., coagulans, Bacillus laevolacticus, Bacillus subtilis, Bacillus uniflagellatus, Bacillus laterosporus, Bacillus laterosporus BOD, Bacillus megaterium, Bacillus polymyxa, Bacillus licheniformis, Bacillus brevis subsp. pumilus) and Bacillus stearothermophilus. Exemplary probiotic Lactobacillus species include, but are not limited to, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus DDS-1, Lactobacillus GG, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus delbruekii, Lactobacillus bulgaricus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus salivari ... salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus salivarius, L helveticus and Lactobacillus sporogenes.Exemplary probiotic Sporolactobacillus species include all Sporolactobacillus species, for example, Sporolactobacillus P44. Exemplary probiotic Bifidobacterium species include, but are not limited to, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium bifidus, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium infantis, Bifidobacterium longum, and any genetic variants thereof. Other strains that can be used include members of the genus Lactococcus due to probiotic activity, such as Lactococcus lactis, Lactococcus diacetylactis, Lactococcus cremoris, and Streptococcus (Enterococcus). For example, Enterococcus faecium is commonly used as a livestock probiotic and can therefore be used as a co-administered agent. Similarly, Ruminococcus and Megasphaera can be used in applications where the bacteria are administered as probiotics to livestock species.

[0179] For example, the probiotic may be selected from the group consisting of Escherichia, Klebsiella, Citrobacter, Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Enterococcus, Propionibacterium, Faecalibacterium, Pediococcus, Ruminococcus, Megasphaera, and Bacillus.

[0180] Non-pathogenic Escherichia coli is an exemplary bacterium that can be used as a probiotic in the present disclosure because of its ability to colonize the highly acidic environment of the gastrointestinal tract, particularly in the human gastrointestinal tract.

[0181] For example, transformation of bacterial cells with the recombinant conjugated plasmids of the present disclosure can be performed by any means known in the art, including but not limited to protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829 or Dubnar and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

[0182] The growth of these various probiotic species to form cell cultures, cell pastes and spore preparations is generally well known in the art. The skilled artisan will be able to determine appropriate growth / culture conditions depending on whether the bacteria are aerobic, facultative anaerobic or obligate anaerobic. Such culture methods are known in the art.

[0183] In some examples, probiotics are encapsulated, for example, to protect the bacteria and maintain viability. Protection of the bacteria can be achieved if the majority of cells are still alive or still metabolically active, or more encapsulated cells are still alive when compared to unencapsulated cells treated under the same conditions. Methods of encapsulation (e.g., microencapsulation) of bacteria are known in the art.

[0184] Alternatively or in addition, probiotics can be freeze-dried. The conventional meaning of the term "freeze drying" (also referred to as freeze drying (lyophilisation), freeze drying (lyophilization) or freeze drying (cryodesiccation)) is to cool a liquid sample, convert a freezable solution into ice, crystallize a crystallizable solute, and form an amorphous matrix including a non-crystalline solute associated with an unfrozen mixture, and then evaporate (sublimate) water from the amorphous matrix. In this process, the evaporation (sublimation) of the frozen water in the material is usually carried out under reduced ambient pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase. Freeze drying generally includes the steps of pretreatment, freezing, primary drying and secondary drying. Freeze drying methods are known in the art. An exemplary method is described in WO 2015000972, the entire contents of which are incorporated herein by reference.

[0185] In other examples, the probiotics may be spray dried or extruded.

[0186] Composition

[0187] Also disclosed herein are compositions (e.g., probiotic compositions) comprising the recombinant conjugated plasmids of the present disclosure and probiotics. Such compositions can be used to prevent or reduce the spread and / or acquisition of plasmids that impart undesirable traits (e.g., pathogenic plasmids as described herein) in bacterial populations. For example, the compositions can be used to prevent or reduce the spread and / or acquisition of plasmids that impart multidrug resistance (also referred to as AMR), virulence, and / or metal resistance in pathogenic bacterial populations, and contribute to colonization (i.e., recolonization) of the gastrointestinal tract or other environments (e.g., as in the case of bioremediation).

[0188] In some embodiments, the composition can be formulated for administration to a subject. For example, the probiotics of the present disclosure can be formulated in a composition suitable for administration to a human or animal subject. In one example, the composition is used for administration to a human. In another example, the composition is used for administration to an animal. Exemplary animals that may be particularly useful for the compositions of the present disclosure include livestock species (e.g., cattle, sheep, horses, pigs, donkeys, poultry), companion animals (e.g., dogs, cats), performance animals (e.g., racehorses, camels, greyhounds), and captive wild animals. In one example, the animal is a ruminant. Exemplary ruminants include cattle, sheep, goats, buffaloes, deer, or camels. In another example, the animal can be a hindgut fermenter. An exemplary hindgut fermenter is a horse. In another example, the animal can be a bird species, such as poultry.

[0189] In other examples, the composition can be formulated for application to an environment selected from the group consisting of a healthcare environment, a soil environment, an environment including a water source, an environment including wastewater, an environment including industrial waste, an environment including agricultural waste, an environment including sewage, and / or an environment including biosolids.

[0190] Probiotics and compositions comprising the probiotics can be formulated for administration or application by any route determined by those skilled in the art to be suitable. For example, according to examples in which probiotics and compositions comprising the probiotics are used for administration to subjects, compositions can be formulated for oral administration (e.g., as ingestible liquids or solids, oral drenches, feed additives, foods (e.g., dairy products, such as drinkable yogurt) or capsules), topical administration (e.g., as emulsions or creams), intranasal administration or parenteral administration. In one example, the compositions of the present disclosure are formulated for oral administration, such as food, beverages, boluses, drenches or capsules. Therefore, the compositions may further include one or more physiologically acceptable excipients, carriers or additives suitable for human or non-human animal intake. Physiologically acceptable excipients, carriers or additives suitable for human or non-human animal intake are known in the art and are described herein. For example, such carriers may allow the probiotics of the present disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. The choice of carrier will depend on the form of the composition, the expected method of administration, the expected shelf life and storage considerations. In some instances, the composition can be a food or beverage product (e.g., a dairy product, such as drinkable yogurt). In some instances, the composition can be a tablet, a pill, a caplet or a capsule. Suitable excipients include but are not limited to fillers, such as sugars, including but not limited to lactose, sucrose, mannitol and sorbitol; cellulose preparations, such as but not limited to corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone (PVP). If desired, disintegrants can be added, such as but not limited to cross-linked polyvinylpyrrolidone, agar or alginic acid or its salt, such as sodium alginate. Compositions that can be used orally include but are not limited to capsules made of gelatin, and soft sealed capsules made of gelatin and plasticizers (such as glycerol or sorbitol). In some instances, the composition can be formulated in a buffer. Those skilled in the art will appreciate that a suitable buffer can be used. Examples of suitable buffers include, but are not limited to, phosphate, calcium carbonate, bicarbonate, phosphocitrate, and histidine. In other examples, the composition can be formulated with a carrier having a low oxygen diffusion rate (e.g., an ingestible oil). The composition can further include an antioxidant.

[0191] In some examples, the composition may include a preservative or stabilizer. In addition, depending on the method of manufacture, the composition may include one or more cryoprotectants known in the art.

[0192] Depending on the example of whether the composition is a food product (such as a dairy product), a feed / nutritional supplement or a beverage product, the probiotic or the composition comprising the probiotic can be prepared by or shipped to the manufacturer. The probiotic or the composition comprising the probiotic can then be formulated as a food product, a feed / nutritional supplement or a beverage product by adding additional ingredients suitable for the product.

[0193] In some examples, the compositions of the present disclosure are stable when stored at ambient room temperature (e.g., 20° C. and 25° C.), for example, stable when stored at ambient room temperature for at least one month or longer. In other examples, such as when the composition is formulated into a dairy or beverage product, it may be necessary to refrigerate the composition to maintain the viability of the probiotics.

[0194] In some examples, the compositions of the present disclosure are packaged in a container. The container may contain a single dose or multiple doses of a composition as described herein.

[0195] Preferably, the composition includes a probiotic of the present disclosure in an amount sufficient to provide, at least in part, a benefit to the health of the subject or the environment to which it is administered.

[0196] Depending on the application in which the probiotic composition is formulated for a human or non-human animal, the composition will include an amount of probiotics sufficient to provide a health benefit to a human or non-human animal, at least in part. An amount sufficient to achieve this effect is defined as a "therapeutically effective amount". The effective amount for this purpose may vary according to many factors known to those skilled in the art, including but not limited to the species of the subject, the anatomical structure of the digestive system (e.g., a four-chamber or single-chamber stomach), the size / weight of the subject, the composition of the diet of the subject (existing and future), whether the subject is breastfeeding, whether the subject is pregnant, and the results to be achieved. Therefore, the appropriate dose (e.g., CFU / strain) of the probiotics formulated in the composition of the present disclosure can be determined by those skilled in the art taking into account one or more of the above factors.

[0197] In one example, a unit or dose of a composition of the present disclosure may include about 10 2 CFU is about 10 14 CFU, or about 10 3 CFU is about 10 13 CFU, or about 10 4 CFU is about 10 13 CFU, or about 10 5 CFU is about 10 13 CFU, or about 106 CFU is about 10 13 CFU, or about 10 6 CFU is about 10 12 CFU, or about 10 7 CFU is about 10 11 CFU, or about 10 8 CFU is about 10 10 CFU, or about 10 9 CFU is about 10 10 CFU of the probiotic bacteria of the present disclosure. For example, each unit or dose of the composition of the present disclosure may include about 5×10 7 CFU, or about 6×10 8 CFU, or about 10 9 CFU, or about 10 10 CFU of probiotics.

[0198] method

[0199] Also provided herein are methods for preventing or reducing the spread and / or acquisition of plasmids that confer undesirable traits (e.g., pathogenicity plasmids as described herein) in a bacterial population, the method comprising introducing a probiotic as described herein or a composition comprising the probiotic into the population.

[0200] In one example, the method is used to prevent or reduce the spread and / or acquisition of antimicrobial resistance-conferring plasmids in a bacterial population. For example, the method may include introducing a probiotic as described herein or a composition comprising the probiotic into a bacterial population, wherein one or more of the probiotic cells introduced into the population comprises a recombinant conjugated plasmid comprising an entry exclusion system (EES) from a plurality of AMR-conferring plasmid variants, as described herein.

[0201] In one example, the method is used to prevent or reduce the spread and / or acquisition of a plasmid that confers virulence to a bacterial population. For example, the method may include introducing a probiotic as described herein or a composition comprising the probiotic into a population, wherein one or more of the probiotic cells introduced into the population comprises a recombinant conjugated plasmid comprising an EES from a plurality of plasmid variants that confer virulence to a bacterial population, as described herein.

[0202] In another example, the method is used to prevent or reduce the spread and / or acquisition of a plasmid that confers heavy metal resistance in a bacterial population. For example, the method may include introducing a probiotic as described herein or a composition comprising the probiotic into a bacterial population, wherein one or more of the probiotic cells introduced into the population comprises a recombinant conjugated plasmid comprising an EES from a plurality of plasmid variants that confer heavy metal resistance, as described herein.

[0203] For example, the method can prevent or reduce the spread and / or acquisition of plasmids that confer pathogenicity traits (such as AMR and / or bacterial virulence) in bacterial populations of the gastrointestinal tract during conjugation rounds. In one example, the method can prevent or reduce the spread and / or acquisition of plasmids that confer pathogenicity traits in bacteria of the Enterobacteriaceae family. "Enterobacteriaceae" contains 14 major genera and 6 additional genera, and is known to have different characteristics. Typical examples are Escherichia, Salmonella and Klebsiella. Virulence genes are usually found in enteric bacteria on large plasmids (about 60kb or larger). Enteric bacteria are usually pathogens that infect the gastrointestinal tract of birds and / or mammals. The method of the present disclosure can be used to target a population of enteric bacteria in the intestine, and in subsequent conjugation rounds, a recombinant conjugated plasmid is introduced into the progeny of the bacterial population. In the process of doing so, the method can prevent or reduce the spread and / or acquisition of plasmids that confer pathogenicity traits (such as AMR and / or bacterial virulence) to the bacterial population of the intestine.

[0204] In other examples, the method is used to prevent or reduce the spread and / or acquisition of pathogenic plasmids as described herein in bacterial populations within an environment. The environment into which the probiotics or compositions comprising the probiotics are introduced can be selected from a healthcare environment, a soil environment, an environment comprising a water source, an environment comprising wastewater, an environment comprising industrial waste, an environment comprising agricultural waste, an environment comprising sewage, and / or an environment comprising biosolids. Thus, in one example, the method can be used for bioremediation.

[0205] The reduction in the spread or acquisition of pathogenic plasmids in a bacterial population can be any reduction in the prevalence of pathogenic plasmids relative to a bacterial population that has not been introduced into the probiotic or the present disclosure, such as a reduction of at least 10% (e.g., a reduction of at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40% or more). The reduction in the spread or acquisition of pathogenic plasmids in a bacterial population may produce one or more therapeutic results. As used herein, the terms "treating," "treat," or "treatment," and variants thereof, refer to clinical interventions using non-pathogenic bacteria that are designed to alter the natural course of the individual or cell being treated during a clinical pathology process, and may also refer to environmental remediation efforts. When used in conjunction with a biological organism or infection, the desired therapeutic effect includes ameliorating, eliminating, reducing, preventing or otherwise alleviating or managing the deleterious effects of a biological organism by improving and / or controlling colonization of the gastrointestinal tract or other environment of both humans and animals by pathogenic bacteria carrying such traits, by curing the pathogenic population in the intestinal tract or other environment of a subject to lose multidrug resistance or metal resistance or virulence (e.g., Enterobacteriaceae).

[0206] In one example, the probiotic of the present disclosure or a composition comprising the probiotic is administered once or more daily, weekly, biweekly, monthly or bimonthly, wherein the daily, weekly, biweekly, monthly or bimonthly dosage includes the amount of the probiotic as described above.

[0207] In some embodiments, the probiotics or compositions comprising the probiotics are administered to a human or animal subject. In one example, the probiotics or compositions are administered to a human. In another example, the probiotics or compositions are administered to an animal. Exemplary animals for which the probiotics and compositions of the present disclosure may be particularly useful are described herein.

[0208] As described herein, the probiotics and compositions can be administered by any route determined to be suitable by one skilled in the art. For example, depending on the example of administering the probiotics and compositions to a human or animal subject, the composition can be formulated for oral administration (e.g., as an ingestible liquid or solid, oral drenching, feed additive, food (e.g., dairy products) or capsule), topical administration (e.g., as an emulsion, cream or gel), intranasal administration or parenteral administration. In one example, the composition of the present disclosure is administered orally, for example, as food, beverage, bolus, drenching or capsule.

[0209] In one example, the probiotic or a composition comprising the probiotic can be applied as a topical cream, lotion or gel. For example, a cream, lotion or gel comprising the probiotic can effectively reduce or prevent the acquisition or spread of pathogenic plasmids within a bacterial population on the skin, thereby controlling antibiotic resistant pathogens on the skin. In this regard, pathogenic Pseudomonas, Staphylococcus and / or Enterococci are often associated with infection in severe burns.

[0210] Preferably, the probiotics are administered to the subject in a therapeutically effective amount. As described herein, this can vary according to many factors known to those skilled in the art, including but not limited to the species of the subject, the anatomical structure of the digestive system (e.g., four-chamber or single-chamber stomach), the size / weight of the subject, the composition of the subject's diet (existing and future), whether the subject is breastfeeding, whether the subject is pregnant, and the outcome to be achieved. Therefore, the appropriate dose (e.g., CFU / strain) of the probiotics formulated in the compositions of the present disclosure can be determined by those skilled in the art taking into account one or more of the above factors.

[0211] In one example, a unit or dose of a composition administered to a subject may include about 10 2 CFU is about 10 14 CFU, or about 10 3 CFU is about 10 13 CFU, or about 10 4 CFU is about 10 13 CFU, or about 10 5 CFU is about 10 13 CFU, or about 10 6 CFU is about 10 13 CFU, or about 10 6 CFU is about 10 12 CFU, or about 10 7 CFU is about 10 11 CFU, or about 10 8 CFU is about 10 10 CFU, or about 10 9 CFU is about 10 10 CFU of the probiotic bacteria of the present disclosure. For example, the method may include administering a probiotic comprising about 5×10 7 CFU, or about 6×10 8 CFU, or about 10 9 CFU, or about 10 10 Composition of probiotics in CFU.

[0212] Examples

[0213] Example 1: Probiotic plasmids with a single exclusion gene inhibit IncF plasmid transfer

[0214] Probiotic plasmids pJIMK_traS_F, pJIMK_traS_R100, pJIMK_traS_SLT were constructed by cloning different traS variants from IncF plasmid into pBCSK+ backbone.

[0215] Bacterial strains and plasmids used:

[0216] Table 1 provides details of the individual bacterial strains and plasmids used to generate the probiotic plasmids designated pJIMK_traS_F, pJIMK_traS_R100, pJIMK_traS_SLT.

[0217] Table 1. Bacterial strains and plasmids used

[0218]

[0219] Analysis of the entry-exclusion system in IncF plasmids:

[0220] The inventor has obtained and checked the traS sequence of the entry exclusion in the responsible IncF plasmid of those IncF plasmids available in GenBank, and then used MEGAX software to carry out multiple sequence alignment.Then carry out BlastN and BlastP search on traS nucleotide and amino acid sequence.Three major variants of traS sequence are identified from available IncF plasmid sequence in GenBank.These three traS variants are named as traS_F, traS_R100 and traS_SLT.Variant traS_F obtains (GenBank accession number AP001918.1) from Escherichia coli K-12 plasmid F, traS_R100 obtains (GenBank accession number AP000342.1) from Shigella flexneri plasmid R100, and traS_SLT obtains (GenBank accession number AE006471.2) from Salmonella typhimurium plasmid pSLT. The complete nucleotide sequences of the three trsS variants, designated traS_R100, traS_F and traS_SLT, are shown in SEQ ID NOs: 1-3, respectively.

[0221] Construction of probiotic plasmid:

[0222] The gBloek elements of traS_R100, traS_F and traS_SLT genes (IDT, USA) and their natural promoters and ribosome binding sites (RBS, respectively) were artificially synthesized using appropriate restriction sites. Figure 1AC and SEQ ID NOs: 4-6).

[0223] Then the synthesized traS_R100, traS_SLT and traS_F genes were digested at 37°C for 1 hour with HindIII+BamHI, BamHI+EcoRI and BamHI+XbaI restriction enzyme combinations, respectively. Then the digested DNA samples were purified using a PCR purification kit (Thermo Fisher, USA). At the same time, the cloning vector pBCSK+ (chloramphenicol resistance) was digested for 2 hours with the corresponding restriction enzyme combination, and then purified. Then the purified traS gene variants were cloned individually into the cloning vector pBCSK+ to construct probiotic plasmids pJIMK_traS_R100, pJIMK_traS_F and pJIMK_traS_SLT ( Figure 2 ). These probiotic plasmids were transformed into rifampicin-resistant E. coli BW25113Rf to produce BW25113Rf (pJIMK_traS_R100), BW25113Rf (pJIMK_traS_F), BW25113Rf (pJIMK_traS_SLT). BW25113Rf with probiotic plasmids and empty BW25113Rf were then used as recipients for transfer of the corresponding IncF plasmids from the donor J53 strain (Table 2).

[0224] Table 2. IncF plasmid-specific entry-exclusion system in recipient bacteria prevents acquisition of the corresponding IncF plasmid

[0225]

[0226] In vitro conjugation:

[0227] In vitro plasmid conjugation was measured using a standard filter mating assay (Rodriguez-Grande and Femandez-Lopez 2020). Donors with the corresponding plasmids and recipient bacteria (Table 2) were grown overnight in LB medium with appropriate antibiotics. The culture (1 mL) was collected by centrifugation at 10,000g for 5 minutes, washed twice with 1 mL of saline, and suspended in 50 μL of saline. The donor and recipient suspensions were mixed. The mixture was placed on a nitrocellulose filter (Amersham Hybond-C extra, 82 mm) on an LB agar plate and then incubated overnight at 37 ° C. The mixture of cultures was collected in 5 mL of saline, and 100 μL of the mating mixture with different dilutions was spread onto a nutrient agar plate containing rifampicin (90 μg / mL) and plasmid-specific antibiotics that conferred resistance to the incoming plasmid and incubated overnight at 37 ° C. The transconjugant colonies were then counted and the conjugation frequency was measured by dividing the number of transconjugants by the number of donor bacteria. In addition, the colonies were spliced ​​onto CHROMagar directional plates containing appropriate antibiotics to confirm that they were not spontaneous rifampicin-resistant mutants of the donor strain. The presence of the incoming plasmid gene in the transconjugant was confirmed by colony PCR using a single colony as a template.

[0228] Exclusion Index / Protection Index Measurement:

[0229] The exclusion index or protection index is the ratio of the conjugation frequency of empty recipients (without the exclusion system) relative to recipient bacteria with the probiotic plasmid (with the cloned exclusion gene). The exclusion index indicates the fold difference in the inhibition of plasmid transfer. Therefore, higher exclusion index values ​​indicate greater protection against the acquisition of the tested plasmid.

[0230] Probiotic plasmids with a single exclusion gene inhibited the conjugative transfer of different IncF plasmids in recipient bacteria by nearly 200-fold (Table 2, Figure 8 ). This transfer inhibition is specific to different plasmid types. For example, tras_R100 in recipient bacteria strongly inhibits the transfer of R100 plasmid.

[0231] Example 2: Probiotic plasmids with a combination of various exclusion gene variants inhibit conjugative transfer of several IncF plasmids

[0232] Since the three variants of traS identified by the inventors (i.e., traS_R100, traS_SLT, and tras_F) represent the majority of IncF plasmids present in the GenBank database, the inventors combined all three traS gene variants of IncF plasmids and their native promoters for traS_R100, traS_SLT, and integron promoter Pc for traS_F with their native RBS into a single vector system, and constructed a probiotic plasmid to protect host bacteria from acquiring most IncF plasmids. The new probiotic plasmid, named pJIMK_traS_3, was constructed by combining the three traS variants with their native promoters two-in-one with integron promoter Pc and its own RBS with flanking HindIII and XbaI restriction sites into a single construct (SEQ ID NO: 7). The restriction-digested and purified trsS-3 gene was then cloned into the HindIII+XbaI site of pBCSK+ to construct pJIMK_traS_3 ( Figure 3 and 4 ). The probiotic plasmid pJIMK_traS_3 was then transferred into BW25113Rf to construct BW25113Rf (pJIMK_traS_3) and used for conjugative transfer of different IncF plasmid variants from the J53 strain to measure the level of transfer inhibition. The inventors were able to demonstrate that bacteria carrying the probiotic plasmid pJIMK_traS_3 can successfully protect recipient bacteria from acquiring all IncF plasmid variants, similar to the protection of the traS gene against specific F plasmids tested (Table 3, Figure 8 ). This result shows that probiotic plasmids with different combinations of exclusion gene variants can be constructed to prevent the acquisition of various plasmid types. It also shows that a probiotic plasmid comprising a combination of three traS exclusion gene variants can prevent the acquisition of most IncF plasmid types.

[0233] Table 3. Probiotic plasmids with multiple entry-exclusion systems from IncF plasmid variants can protect bacteria from acquiring several different IncF plasmid variants

[0234]

[0235] Example 3: Increased expression of one or more exclusion genes in a probiotic plasmid significantly increases protection against a target plasmid.

[0236] In this example, the inventors evaluated whether the increase of the expression of the traS variant was excluded increased the transfer inhibition of the plasmid in the recipient bacteria. The expression of the gene concerned can be conveniently controlled by cloning the gene into an expression vector under an arabinose-inducible promoter with araB and araC genes. In this example, the pBAD33_Gm expression vector (gentamicin resistance, plasmid No. 65098, Addgene company) is used to generate an expression vector including the traS variant of the gene exclusion, to control the expression of the traS variant gene by adding different concentrations of arabinose.

[0237] The effect of traS gene expression on the transfer inhibition of plasmids in recipient bacteria was specifically evaluated for variant traS_R100. Briefly, the traS_R100 sequence with a consensus RBS region was synthesized and cloned into the HindIII+XbaI sites of pBAD33_Gm to construct pJIMK_ara_traS_R100 ( Figure 5 ). It was found that increased expression of traS_R100 in recipient bacteria from the probiotic plasmid pJIMK_ara_traS_R100 with arabinose (0.2%) significantly increased the inhibitory effect of R100 plasmid transfer to recipient bacteria (Table 4). This result suggests that high levels of traS exclusion protein in recipient bacteria can significantly increase the level of protection from acquisition of the target plasmid (R100).

[0238] Table 4. High-level expression of exclusion genes in recipient bacteria can significantly increase protection from acquisition of the corresponding plasmid (R100)

[0239]

[0240] To improve the expression of all three traS variants from a single probiotic plasmid to deliver a high level of protection against acquisition by most IncF plasmids, three traS variants were synthesized using the araBAD promoter ( Figure 6 ) and cloned into pBAD33_Gm to construct pJIMK_ara_traS_3 ( Figure 7). More specifically, the triple traS construct includes three traS variants: (i) a consensus RBS with a traS_R100 sequence, (ii) traS_F with its native promoter and RBS, and (iii) traS_SLT with an araBAD promoter and a consensus RBS, wherein HindIII and XbaI restriction sites flank the three traS variant sequences (SEQ ID NO: 8). The triple traS construct was then restriction digested, purified, and cloned into the HindIII+XbaI sites of pBAB33_Gm to construct pJIMK_ara_traS_3 ( Figure 7 ).

[0241] In this probiotic plasmid, traS_R100 and traS_SLT are directly regulated by the araBAD promoter, while traS_F is under the control of the natural promoter and the araBAD promoter. The probiotic plasmid pJIMK_ara_traS_3 was then transferred into the BW25113Rf strain, which was used as a recipient in the conjugation transfer of different IncF plasmids from E. coli J53 (Table 5). The recipient bacteria were induced with 0.2% arabinose for one hour before mixing the donor and recipient for mating experiments. Surprisingly, it was found that inducing the traS_3 gene with arabinose significantly increased the conjugation transfer inhibition of all three different IncF plasmids tested (Table 5, Table 5). Figure 8 ). This suggests that increasing the expression of exclusion genes from probiotic plasmids in bacteria can improve protection from acquisition of any target plasmid.

[0242] Table 5. High-level expression of multiple entry-exclusion systems from probiotic plasmids IncF plasmid variants can significantly increase the protection of bacteria from acquiring several different IncF plasmid variants

[0243]

[0244] Example 4: Conjugated probiotic plasmids can protect bacteria from invasion by IncM, IncL, IncC and IncA plasmid types in vitro.

[0245] In this example, the inventors designed and constructed Includes Exc protein sequences from various Inc plasmid types conjugated probiotic plasmids and evaluated those probiotic plasmids that protected bacteria from multiple incompatible types of AMR plasmids The ability to invade.

[0246] Analysis of Exc protein sequences of IncM, L, C, and A plasmid types

[0247] The present inventors analyzed the Exc protein sequences of IncM, IncL, IncC and IncA plasmid types available in Genbank.

[0248] Based on this analysis, the inventors identified that all IncC plasmids in GenBank carry a single Exc protein type. The Exc of IncA is slightly different from the Exc of IncC plasmids (see Fig. 9 A), but recent experimental studies have identified that despite some amino acid sequence changes, both Exc proteins belong to the same Exc group and demonstrated that IncC Exc can effectively exclude both IncC group plasmids and IncA group plasmids (Humbert et al., 2019).

[0249] IncL plasmids are also represented primarily by a single Exc type (see Fig. 9 In this regard, 95% (233 / 249) of IncL plasmids in GenBank carried this unique type of Exc protein, while 5% (16 / 249) of IncExc had 1-2 aa changes relative to the dominant Exc type. This suggests that all Exc variants in IncL plasmids are closely related to each other and that the dominant variant (IncL_Exc_1) can exclude all IncL plasmids.

[0250] IncM plasmids carry two major types of Exc proteins (see Fig. 9 C), which differs in only two amino acids and collectively represents >99% of IncM plasmids in GenBank.

[0251] The inventors predicted promoter regions in IncL, M and C plasmids associated with these major Exc variants.

[0252] Construction of conjugated probiotic plasmids to protect bacteria from invasion by IncM, L, C, and A plasmid types

[0253] The inventors previously identified the in vivo conjugation-efficient IncM plasmid pJIBE401. In this study, the conjugation-efficient IncM plasmid backbone of pJIBE401 was used as the backbone for constructing probiotic plasmids to protect bacteria from invasion by four different plasmid types. The multidrug resistance region (MRR) of approximately 28.0 kb carrying multiple antibiotic resistance genes and transposable elements was deleted from the IncM plasmid pJIBE401 and replaced with a tetracycline resistance gene (tetA) to generate a plasmid backbone named pJIMK45 (Kamruzzaman et al., 2017).

[0254] The inventors used pJIMK45 as the backbone of their conjugated probiotic plasmid. This backbone already contained the exc gene of the IncM plasmid type. The inventors then introduced the exc genes from IncL and IncC plasmids to generate a conjugated probiotic plasmid (PB1). Briefly, the exc gene sequences from IncL and IncC plasmids, as well as their native promoters and ribosome binding sites, were combined with the fosA3 gene and flanked on both sides by 60 bp sequences homologous to the regions flanking the tetA gene in pJIMK45 (the synthetic sequence was named "gBlock"; Fig.10 ). gBlock was commercially synthesized and introduced into the pJIMK45 backbone plasmid using an allelic exchange method based on homologous recombination, in which the tetA gene was replaced. To insert gBlock, the E. coli UB5201Rf strain carrying pJIMK45 was transformed with the λ Red recombinase plasmid pKM200 (chloramphenicol resistance) and selected on tetracycline plus chloramphenicol plates at 30°C. Electrocompetent cells were prepared from UB5201Rf (pJIMK45+pKM200) and transformed with gBlock of PCR-amplified excL-excC-fosA3 by electroporation. Transformants were selected on fosfomycin resistance plates. The sensitivity of the selected transformants to tetracycline was then confirmed, and the insertion event was confirmed by PCR and Sanger sequencing.

[0255] The resulting conjugated probiotic plasmid, named PB1, has a complete pemIK toxin-antitoxin system to ensure its stable maintenance in the bacterial population. To destabilize it, the pemK toxin gene was deleted by replacing it with the tetA gene. The resulting unstable probiotic plasmid was named "PB1.1". In the absence of selection pressure, the conjugated probiotic plasmid PB1.1 was lost from the bacteria. The physical map and nucleotide sequence of the probiotic plasmid PB1.1 are respectively Fig.11 and 12 The PB1.1 sequence is shown in SEQ ID NO:13.

[0256] The ability of the conjugated probiotic plasmid PB1.1 to prevent IncM, IncL, and IncC plasmids from invading bacteria.

[0257] The ability of the probiotic plasmid PB1.1 to prevent the invasion of bacteria by AMR plasmids from the IncM, IncL, and IncC groups was then tested using a standard liquid mating assay.

[0258] Briefly, donor and recipient bacteria were grown overnight in 10 mL LB-Lennox broth medium with appropriate antibiotics. The cultures were washed twice with an equal volume of sterile saline solution (0.85% NaCl) and resuspended in 10 mL LB-Lennox broth. The OD of each culture was calculated. 600 The donor culture of 1mL is mixed and put into 15mL falcon tube.Mating mixture is incubated at 37 ℃ for 20 hours, does not shake.Mating is finished by vortexing mating mixture for 10 seconds.Then the mating mixture is diluted with saline continuously, and it is plated on the LB-Lennox agar plate containing sodium azide (100mg / mL) and cefotaxime (cefotaxime, 8mg / mL), to select transconjugant.The conjugation frequency is the sum of transconjugant and the ratio of the quantity of the recipient bacteria added in the conjugation mixture.The intestinal bacteria NH78Rf with IncM plasmid pJIBE401, IncL plasmid pJIE1335 or IncC plasmid pEc158 is used as donor bacteria, and the J53Az of sodium azide resistant bacteria J53Az and the probiotic plasmid PB1.1 is used as the recipient of each plasmid transfer.

[0259] After 20 h of liquid mating experiments, antibiotic resistance IncM, IncL, and IncC plasmids could be readily transferred to the recipient bacterium J53Az, but after the same mating time, the transfer of these plasmids to J53Az(pPB1.1) was significantly reduced ( Fig.13 ). During mating experiments, the conjugated probiotic plasmid protected >99.9% of the recipient bacterial population from invasion by a different set of AMR plasmids. This study demonstrates that a single probiotic plasmid, PB1.1, in the recipient bacteria can significantly prevent the acquisition of the IncM, IncL, and IncC plasmids tested.

[0260] The characteristics of the bacterial strains and plasmids used in this experiment are shown in Table 6.

[0261] Table 6. Characteristics of bacterial strains and plasmids

[0262]

[0263]

[0264] Example 5: Probiotic plasmid PB1.1 protects the mouse microbiota from the acquisition of different AMR plasmids.

[0265] In this example, the inventors evaluated the ability of the conjugated probiotic plasmid PB1.1 to protect the mouse gut microbiota from the acquisition of different AMR plasmid types described in Table 6 (including pJIBE401, pJIE1335, and pEc158) during incidental coprophagous exposure to infected feces.

[0266] Test mice were colonized with the probiotic plasmid PB1.1, while control mice were fed a normal chow diet without any supplements. All mice (i.e., test and control mice) were exposed to mice colonized with three different common AMR plasmid types. Within the experiment, all mice were housed in groups of three mice as biological replicates.

[0267] Sodium azide-resistant E. coli J53Az carrying the probiotic plasmid PB1.1 and rifampicin-resistant E. coli NH78Rf carrying different AMR plasmids were provided to mice in 8% sucrose water.

[0268] Test and control mice were co-housed with AMR colonized mice for 8 hours daily and then separated for 16 hours in 8% sucrose water (with or without PB1.1). AMR plasmid colonized mice were co-housed with probiotic plasmid colonized mice or control mice for 96 hours (4 days) for this experiment.

[0269] Fresh feces were collected and examined on ChromAgar plates containing specific antibiotics (CTX 8 μg / mL supplementation for AMR plasmids, or fosfomycin 200 μg / mL for probiotic plasmids) to measure colonization, using serial dilutions for quantification.

[0270] AMR plasmids appeared in different mice in the non-colonized control group after a total of 48 hours of exposure to infected mice, with all control mice becoming AMR-colonized within 96 hours of exposure (equivalent to 4 days). In contrast, AMR plasmids were not detected in any of the mice that received the probiotic plasmid until 80 hours after the initial exposure ( Fig.14 ). One mouse from the IncC group showed very low levels of AMR plasmid infection in the probiotic group after 96 hours of exposure. These data indicate that there is strong protection against the acquisition and colonization of AMR plasmids after long periods of co-housing with mice that are highly colonized with AMR plasmids.

[0271] PCR performed at 96 hours revealed AMR plasmid DNA in all mice (data not shown), confirming that the AMR plasmid was taken up. These data suggest that in those mice that received overnight administration of the probiotic plasmid therapy, the ingested AMR plasmid was unable to colonize to levels that were easily detected by standard culture methods, even after regular exposure and ingestion of common AMR plasmid types.

[0272] As a test of protection, probiotic-colonized mice (AMR culture negative but PCR positive) and control mice (AMR culture positive after exposure) were treated with cefotaxime (CTX) antibiotic 120 hours after exposure to AMR-colonized mice to select for mouse intestinal bacterial populations carrying AMR plasmids. Fresh feces were collected and examined on ChromAgar plates using serial dilutions of CTX antibiotic.

[0273] High levels of AMR (CTX-resistant) bacteria were detected in untreated control mice ( Fig.15 In contrast, AMR bacteria were detected only in one probiotic plasmid-treated mouse exposed to the IncMAMR plasmid group and in one mouse exposed to the IncCAMR plasmid group, and at very low levels. Since PCR evidence of AMR plasmid DNA was found in all mice, this strongly supports that the probiotic plasmid protects the intestinal environment from AMR plasmids.

[0274] All publications discussed and / or cited herein are incorporated in their entirety.

[0275] References

[0276] Agyekum, A., Fajardo-Lubian, A., Ai, X., Ginn, AN, Zong, Z., Guo, X., et al. (2016). Predictability of Phenotype in Relation to Common beta-Lactam Resistance Mechanisms in Escherichia coli and Klebsiella pneumoniae. J Clin Microbiol 54, 1243-50.

[0277] De La Cruz, F. and Grinsted, J. (1982). Genetic and molecular characterization of Tn21, a multiple resistance transposon from R100.1. J Bacteriol 151, 222-28.

[0278] Espedido, BA, Partridge, SR and Iredell, JR (2008). Bla in Enterobacteriaceae isolates from Australia. IMP-4 Expression in different genetic backgrounds (bla IMP-4 in different genetic contexts in Enterobacteriaceae isolates from Australia). Antimicrobial Agents Chemother 52, 2984-7.

[0279] Fajardo-Lubian, A., Ben Zakour, NL, Agyekum, A., Qi, Q., & Iredell, JR (2019). Host adaptation and convergent evolution increases antibiotic resistance without loss of virulence in a major human pathogen. PLoS Pathogens, 15, e1007218.

[0280] Humbert, M., Huguet, KT, Coulombe, F. and Burrus, V. (2019). Entry Exclusion of Conjugative Plasmids of the IncA, IncC, and Related Untyped Incompatibility Groups. Journal of Bacteria, 201.

[0281] Kamruzzaman, M., Shoma, S., Thomas, C.M., Partridge, S.R., and Iredell, J.R. (2017). Plasmid interference for curing antibiotic resistance plasmids in vivo. PLoS One, 12, e0172913.

[0282] Matsumura, Y., Peirano, G. and Pitout, J. D. (2018). Complete genome sequence of Escherichia coli J53, an azide-resistant laboratory strain used for conjugation experiments. Genome Announc 6.

[0283] Murphy, KC and Campellone, KG (2003). Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol Biol 4, 11.

[0284] Papagiannitsis, C. C., Kutilova, I., Medvecky, M., Hrabak, J., and Dolejska, M. (2017). Characterization of the Complete Nucleotide Sequences of IncA / C. Antimicrobial Agents and Chemotherapy 61.

Claims

1. A recombinant plasmid comprising entry exclusion systems (EES) from a plurality of plasmid variants, wherein the plasmid variants comprise genes conferring a trait of interest.

2. The recombinant plasmid according to claim 1, wherein the trait of interest is an undesirable trait in a bacterial population.

3. The recombinant plasmid of claim 2, wherein the trait of interest is antimicrobial resistance (AMR), bacterial virulence or heavy metal resistance.

4. The recombinant plasmid of claim 1, wherein the recombinant conjugated plasmid comprises EES from at least three plasmid variants, wherein the at least three plasmid variants comprise genes conferring the trait of interest.

5. The recombinant plasmid according to any one of claims 1 to 4, wherein the EES are from different incompatibility groups.

6. The recombinant plasmid according to any one of claims 1 to 4, wherein the EES are from the same incompatibility group.

7. The recombinant plasmid according to any one of claims 1 to 6, wherein one or more of the EES are from a plasmid variant selected from the following incompatibility groups (Inc): IncF, IncI, IncA, IncC, IncL, IncM, IncX, IncP, IncN and IncH.

8. The recombinant plasmid according to claim 7, wherein one or more of the EESs are from a plasmid variant of incompatibility group F (IncF).

9. The recombinant plasmid according to claim 8, comprising one or more of the following: EES comprising a polynucleotide sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 1 (traS_F variant); EES comprising a polynucleotide sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 2 (traS-R100 variant); and / or An EES comprising a polynucleotide sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 3 (traS_SLT variant).

10. The recombinant plasmid according to claim 8 or 9, comprising one or more of the following: EES, comprising the polynucleotide sequence shown in SEQ ID NO: 1 (traS_F variant); EES comprising the polynucleotide sequence shown in SEQ ID NO: 2 (traS_R100 variant); and / or EES, said EES comprising the polynucleotide sequence shown in SEQ ID NO: 3 (traS_SLT variant).

11. The recombinant plasmid according to any one of claims 8 to 12, comprising the traS_F variant EES, the traS_R100 variant EES and the traS_SLT variant EES.

12. The recombinant plasmid according to any one of claims 1 to 10, comprising one or more EES from the following plasmid variants: incompatibility group L (IncL), incompatibility group C (IncC), incompatibility group M (IncM), incompatibility group A (IncA), and any combination thereof.

13. The recombinant plasmid according to claim 12, comprising one or more of the following: EES comprising a polynucleotide sequence at least 80% identical to the sequence shown in SEQ ID NO:9 (excL variant); EES comprising a polynucleotide sequence at least 80% identical to the sequence shown in SEQ ID NO: 10 (excC variant); EES comprising a polynucleotide sequence at least 80% identical to the sequence shown in SEQ ID NO: 11 (excM variant): and / or An EES comprising a polynucleotide sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 12 (excA variant).

14. The recombinant plasmid according to claim 12 or 13, comprising one or more of the following: EES, comprising the polynucleotide sequence shown in SEQ ID NO: 9 (excL variant); EES, comprising the polynucleotide sequence shown in SEQ ID NO: 10 (excC variant); EES comprising the polynucleotide sequence shown in SEQ ID NO: 11 (excM variant); and / or EES comprising the polynucleotide sequence shown in SEQ ID NO: 12 (excA variant).

15. The recombinant plasmid according to any one of claims 1 to 14, wherein one or more of the EES is operably linked to a constitutive promoter, optionally wherein the constitutive promoter is selected from a promoter originating in the EES or a strong integron promoter (PcS).

16. The recombinant plasmid according to any one of claims 1 to 14, wherein one or more of the EES is operably linked to an inducible promoter, optionally wherein the inducible promoter is an L-arabinose inducible promoter.

17. The recombinant plasmid according to any one of claims 1 to 16, wherein each EES is operably linked to a separate promoter.

18. The recombinant plasmid according to any one of claims 1 to 16, wherein the one or more EES are operably linked to the same promoter.

19. The recombinant plasmid according to claims 1 to 18, further comprising a plurality of plasmid replication incompatibility (Inc) control regions (inc-control) and / or selectable markers or genes.

20. The recombinant conjugated plasmid according to claim 19, wherein: The Inc control region is from a plasmid variant selected from the following Inc group: IncF, IncI, IncA, IncC, IncL, IncM, IncX, IncP, IncN and IncH; The Inc control region and the EES are from the same Inc group; One or more of the Inc control regions are derived from a plasmid variant of IncF; One or more of the Inc control regions are derived from plasmid variants of IncA, IncC, IncL and / or IncM; and / or The selectable marker or gene is an antibiotic resistance gene.

21. A bacterial cell comprising the recombinant plasmid according to any one of claims 1 to 20.

22. The bacterial cell of claim 21, wherein the bacteria is a probiotic.

23. The bacterial cell of claim 22, wherein the probiotic is selected from the group comprising, but not limited to, Escherichia, Klebsiella, other members of the Enterobacteriaceae, Lactobacillus, Bifidobacteria, Streptococcus, Lactococcus, Enterococcus, Propionibacterium, Faecalibacterium, Pediococcus, Ruminococcus, Megasphaera, Bacillus, and suitable members of the Pseudomonadaceae and Vibrionaceae families.

24. A composition comprising one or more of the living bacterial cells according to any one of claims 21 to 23.

25. The composition of claim 24, wherein the live bacterial cells are freeze-dried and / or formulated in a food or beverage product.

26. A method for preventing or reducing the acquisition and / or spread of plasmids conferring a trait of interest in a bacterial population, the method comprising introducing into the population one or more bacterial cells according to any one of claims 21 to 23 or a composition according to claim 24 or 25 comprising the one or more bacterial cells.

27. The method of claim 26, wherein the trait of interest is: (i) antimicrobial resistance (AMR), and one or more of the bacterial cells introduced into the population comprises a recombinant conjugated plasmid according to any one of claims 1 to 30 comprising an entry exclusion system (EES) from a plurality of AMR-conferring plasmid variants; or (ii) bacterial virulence, and one or more of the bacterial cells introduced into the population comprises a recombinant conjugated plasmid according to any one of claims 1 to 20, wherein the recombinant conjugated plasmid comprises an entry exclusion system (EES) from a plurality of plasmid variants conferring bacterial virulence.

28. The method of claim 26 or 27, wherein the bacterial population is an intestinal bacterial population and the method comprises administering the one or more bacterial cells or a composition comprising the one or more bacterial cells to a subject in need thereof.

29. The method of claim 28, wherein the subject is a human or a non-human animal.

30. The method of claim 26 or 27, wherein: (i) the bacterial population is a population of bacteria that colonize plants, and the method comprises contacting the plant or soil in which the plant is growing with the one or more bacterial cells or a composition comprising the one or more bacterial cells; or (ii) the bacterial population is present in an environment selected from the group consisting of a healthcare environment, a soil environment, an environment including a water source, an environment including wastewater, an environment including industrial waste, an environment including agricultural waste, an environment including sewage and / or an environment including biosolids, and the method comprises introducing the one or more bacterial cells or a composition comprising the one or more bacterial cells into the environment.

31. A food product or beverage comprising one or more bacterial cells according to any one of claims 21 to 23 or a composition according to claim 24 or 25.

32. The food product of claim 31 , wherein the food product is a dairy product.

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