Genetically engineered bacterial reporter strain and a method of preparing the same there of

Abstract

The present invention relates to a the genetically engineered multidrug-resistant reporter V. cholera strain BLF-1Vc which has been constructed and laboratory tested for rapid and robust screening of natural and synthetic molecules for inhibition of enzymes involved in β- lactams, aminoglycosides and macrolide antibiotic resistance and determine its antibiotic 5 potentiating, synergistic or novel antibacterial property. The reporter strain disclosed herein can also be used for testing novel drugs and antimicrobial agents for its efficacy against multidrug resistant clinical isolates. The present invention also discloses a method for preparing a multidrug-resistant reporter microorganism and a kit for detecting antibiotic resistance or screening antimicrobial compounds, comprising the genetically engineered 0 multidrug-resistant reporter V. cholera strain BLF-1Vc.

Description

GENETICALLY ENGINEERED BACTERIAL REPORTER STRAIN AND A METHOD OF PREPARING THE SAME THERE OFFIELD OF THE INVENTION

[0001] The present invention broadly relates to the field of Biotechnology and genetic engineering. More particularly, the present invention relates to a genetically engineered highly sensitive multidrug-resistant reporter microorganism beneficial for rapid and robust screening of natural and synthetic molecules for its antibiotic potentiating, synergistic and novel antibacterial property. The present invention also relates to the method of constructing the genetically engineered multidrug-resistant reporter microorganism.BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Widespread antimicrobial resistance (AMR) in health care and the community has drastically reduced the effectiveness of all antibiotics routinely used in antimicrobial therapy. Rising antibiotic resistance in bacterial pathogens is mostly due to the vertical acquisition of AMR genes encoding drug-inactivating or drug-modifying enzymes, or the accumulation of spontaneous mutations in the bacterial genome that synthesize target molecules for an antibiotic.

[0004] Currently, the predominant AMR encountered globally is the widespread prevalence of mobile beta-lactamases, aminoglycoside modifying enzymes (AMEs), 16S RMTAses, and the plasmid-mediated colistin resistance. This severely limits the use of beta-lactams, aminoglycosides, and the drug of last resort, colistin. The resistance crisis is further complicated by the co-occurrence nature of these resistance genes in the pathogen genomes, resulting in multi-drug (MDR) and extensive drug-resistant (XDR) phenotypes.

[0005] In a recent study conducted by the inventors, 203 clinical Gram-negative pathogens isolated from different specimen types such as respiratory tract (sputum and ET tube), urine, blood, tissue, stool, pus, and gall bladder tissue were collected from 5 different sites over a period of 5 years and assessed for their resistance profile both phenotypically and genotypically. It was identified that A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae were the four most common pathogens harboring various genes against differentantimicrobials. The study revealed the presence of various P-lactam resistant encoding genes such as ESBLs (WOQXA, blarsM, b / ocrx-M-is) and carbapenemases (blaoxA, W«NDM) among the pathogens.

[0006] In the case of genes conferring aminoglycoside resistance, the overall prevalence of aminoglycoside modifying enzymes (AMEs) was higher as compared to the 16S rRNA methyltransferases (16S RMTases). Among AMEs, aph(3’) aadA were the most prevalent. Additionally, an increasing prevalence of macrolide resistance coded by mphE. msrE. mphA. ermB among Enterobacterales and A. baumannii was noted. Though plasmid-mediated colistin resistance was identified to be very low in the studies from India, it is a looming threat as its prevalence is very high in the neighboring Asian and Western countries. It was also identified that these functional resistance genes highly limited the use of major antibiotic classes in various clinically relevant pathogens.

[0007] Hence, discovery of potential antibiotic adjuvants is highly warranted to continue the use of existing antibiotics. For the same, it is very necessary to have a robust phenotypic whole-cell based screening assay which includes the development of promising antibiotic adjuvants which to circumvent the acquired antimicrobial resistance determinants which majorly codes for drug-inactivating or drug -modifying enzymes. Though there are antibiotic adjuvants for p-lactam antibiotics, adjuvants for aminoglycosides and macrolides have not been developed to restore their efficacy against resistant pathogens.OBJECTIVE OF THE INVENTION

[0008] An objective of the present invention is to provide a genetically engineered multidrugresistant reporter microorganism having potency to identify antibiotic potentiators against five different antibiotic classes including p-lactam, aminoglycoside, macrolide, peptidal antibiotics and chloramphenicol.

[0009] In another objective, the present invention provides a multidrug-resistant reporter microorganism which can be used for rapid and robust screening of natural and synthetic molecules for inhibition of enzymes involved in P-lactams, aminoglycosides and macrolide antibiotic resistance and determine its antibiotic potentiating, synergistic or novel antibacterial property.

[0010] Another objective of the present invention is to provide a multidrug-resistant reporter microorganism which is beneficial for testing novel drugs and antimicrobial agents for its efficacy against multidrug resistant clinical isolates.

[0011] Another objective of the present invention is to provide a method of preparing and constructing a multidrug-resistant reporter microorganism.

[0012] Yet another objective of the present invention is to provide a method where resistance genes are integrated into the genome of the multidrug-resistant reporter microorganism and does not require any antibiotic pressure for the maintenance of the resistance functions.SUMMARY OF THE INVENTION

[0013] The present invention generally relates to a genetically engineered multidrug resistant reporter microorganism wherein the same was laboratory tested for rapid and robust screening of natural and synthetic molecules for inhibition of enzymes involved in [3-lactams, aminoglycosides and macrolide antibiotic resistance and determine its antibiotic potentiating, synergistic or novel antibacterial property.

[0014] In an aspect, the present invention relates to a genetically engineered multidrugresistant reporter microorganism comprising a bacterial host genetically modified to confer resistance to multiple classes of antibiotics and to express a reporter gene in response to antibiotic exposure.

[0015] In an aspect, the present invention relates to a genetically engineered multidrug resistant reporter microorganism comprising one or more resistance genes coding for - lactam resistance, macrolide resistance, aminoglycoside resistance, chloramphenicol resistance, zeocin resistance and colistin resistance.

[0016] In another aspect, the present invention relates to a genetically engineered multidrugresistant reporter V. cholera strain BLF-lVc comprising one or more resistance genes coding for p-lactam resistance, macrolide resistance, aminoglycoside resistance, chloramphenicol resistance, zeocin resistance and colistin resistance.

[0017] In an aspect, the one or more resistance genes are selected from OXA-48 carbapenemase ( / OXA-4,S). aminoglycoside-3 "-adenylyltransferase (aadA), Aminoglycoside phosphotransferase 3’ (ap / z3’j, ribosomal protein S12 (rpsL), chloramphenicol acetyltransferase (catB)'. Bleomycin resistance protein (sh-ble). mobilized colistin resistance gene 1 (mcr-I), macrolide -streptogramin resistance gene E (msrE), and Macrolide Phosphotransferase gene E (mphE) genes.

[0018] In an aspect, the genetically engineered multidrug resistant reporter microorganism is resistant to antibiotics selected from the group consisting of ampicillin, erythromycin, zeocin, kanamycin, spectinomycin, streptomycin, chloramphenicol, and colistin.

[0019] In another aspect, the present invention relates to a recombinant integrative vector comprising the drug resistance genes selected from mcr-1, blaoxA- , msrE. and mphE genes isolated from genomes of clinical isolates, wherein the mcr-1 is cloned under a constitutive promoter of said integrative vector, and Z?Z«OXA-4S, msrE, and mphE are cloned under native promoters. In preferred aspect, the recombinant integrative vector is pSB49 vector and the constitutive promoter is htpG.

[0020] In an aspect, the present invention relates to a method for preparing a multidrugresistant reporter microorganism comprising the steps of: i. selecting a bacterial host strain; consisting of JV9 derivative of a sensitive V. cholerae strain N 16961 ii. introducing multiple antibiotic resistance genes for the deletion of relV, relA, spoT and dksA genes of the WT V cholerae strain N 16961 to confer resistance to different classes of antibiotics; iii. inserting a reporter gene downstream of a responsive promoter into the bacterial host strain; wherein mcr-1 was cloned under the constitutive promoter, htpG of the pSB49 vector while the bZaoxA-48 and msrE, mphE were cloned with its native promoter. iv. obtaining the multidrug-resistant reporter microorganism; and v. analyzing gene expression of multidrug-resistant reporter microorganism in response to antibiotic exposure; wherein the MDR reporter strain (BLF-lVc) was tested against eight different antibiotics falling under 5 different antibiotic classes viz ampicillin, erythromycin, zeocin, kanamycin, spectinomycin, streptomycin, chloramphenicol and colistin.

[0021] In a preferred aspect, the present invention relates to a method of constructing the genetically engineered multidrug-resistant reporter V cholerae strain BLF-lVc as claimed in claim 10, comprising the steps of: i) Deleting relV. relA, spoT. and dksA genes from V cholerae strain N 16961 to obtain theV cholerae strain JV9 as host strain; ii) introducing antibiotic resistance genes aadA. aphl'. catBl , and sh-ble in place of the deleted relV, relA, spoT, and dksA genes in the V cholerae strain JV9 by homologous recombination; iii) providing recombinant integrative vector with cloned mcr-1, blaoxA- and msrE, mphE as disclosed herein;iv) cloning the recombinant integrative vector into an initial bacterial host, followed by conjugating the same to an intermediate bacterial host strain to convert the same as a donor strain; and v) conjugating the donor strain with the V. cholerae strain JV9, wherein the recombinant integrative vector from the donor strain is transferred into JV9 and site-specifically integrates at dif locus of the V. cholerae JV9 to obtain the multidrug -resistant reporter microorganism V. cholerae strain BLF-IVc.

[0022] In an aspect, the initial bacterial host is selected from but not limited to E. coll FCV14. Any E.coli cloning strain that are pir+and can support the replication of plasmid harboring or / R6KTcan be used as the intermediate host, like E.coli BW23474 (pir+), E.coli BW20767 (pir-116), E.coli SI 7-1 Xpir, and the like.

[0023] In an aspect, the intermediate bacterial host strain is selected from but not limited to E. coli [3-2163. Any E. coli laboratory strain that possess the appropriate conjugation machinery and selection markers can be used for conjugating the plasmid into the recipient host, like E.coli [3-2155, [3-2150, and the like.

[0024] In another aspect, the present invention relates to a genetically engineered multidrugresistant V. cholerae strain BLF-IVc that can grow at specific concentrations of multiple antibiotics by expressing a reporter resistance gene in response to antibiotic exposure.

[0025] In yet another aspect, the present invention relates to a method for detecting adjuvants that can potentiate existing antibiotics against multidrug resistant microorganisms.

[0026] Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.BRIEF DESCRIPTION OF FIGURESThe accompanying drawings are included to provide a clear understanding of the present invention and a detailed description, and they constitute a part of this complete specification.

[0027] FIG. 1 illustrates Schematic representation of cloning of resistance genes from clinical isolates and construction of reporter strain for developing in- vitro high-throughput screening assay for detection of antibiotic potentiators, synergistic compounds and novel antimicrobials against multidrug resistant clinical pathogens.

[0028] FIG. 2 provides the Growth of reporter strain BLF-IVc in presence of different antibiotics individually and in combination in 96 well plate.

[0029] FIG. 3: (A and B) Growth of reporter strain BLF-IVc (JV-MDR) in presence of different antibiotics individually in test tubes. Antibiotic concentration used to grow BLF-IVc are spectinomycin (40pg / mL), kanamycin (40pg / mL), chloramphenicol (5pg / mL), ampicillin (30pg / mL), erythromycin (Ipg / mL), and zeocin (25pg / mL). V. cholerae N 16961 or N16961 derivative JV9 is used as the negative control as per the antibiotics tested. (C) Growth of reporter strain BLF-lVc (JV-MDR) in presence of different antibiotics in combination using flask for better growth conditions.

[0030] FIG. 4 provides Screening of Selleckchem Natural product Library (n=803) using BLF-lVc to detect Erythromycin adjuvants. (A) Primary screening of the compound library using BLF-lVc grown in erythromycin (Ipg / mL) detects 26 compounds portraying >35% inhibition of the reporter strain. (B) Secondary screening of compound library using BLF- lVc grown in presence of erythromycin (Ipg / mL) and without the antibiotic detects 1 compound portraying >10% inhibition of the reporter strain.DETAILED DESCRIPTION OF THE INVENTION

[0031] The following is a full description of the disclosure's embodiments. The embodiments are described in such a way that the disclosure is clearly communicated. The level of detail provided, on the other hand, is not meant to limit the expected variations of embodiments; rather, it is designed to include all modifications, equivalents, and alternatives that come within the spirit and scope of the current disclosure as defined by the attached claims. Unless the context indicates otherwise, the term "comprise" and variants such as "comprises" and "comprising" throughout the specification are to be read in an open, inclusive meaning, that is, as "including, but not limited to."

[0032] When "one embodiment" or "an embodiment" is used in this specification, it signifies that a particular feature, structure, or characteristic described in conjunction with the embodiment is present in at least one embodiment. As a result, the expressions "in one embodiment" and "in an embodiment" that appear throughout this specification do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, the specific features, structures, or qualities may be combined in any way that is appropriate.

[0033] Unless the content clearly demands otherwise, the singular terms "a," "an," and "the" include plural referents in this specification and the appended claims. Unless the content explicitly mandates differently, the term "or" is normally used in its broad definition, which includes "and / or."

[0034] All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certainembodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0035] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0036] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0037] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description that follows, and the embodiments described herein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0038] It should also be appreciated that the present invention can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.

[0039] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0040] The term "Vibrio cholerae” or “V. cholerae" according to the present invention means, but not limited to, refers to Comma-shaped, gram-negative bacteria.

[0041] The term “Adjuvant / adjuvant component” according to the present invention means, but not limited to, refers to an adjuvant or an adjuvant component in the broadest sense is typically a (e.g., pharmacological or immunological) agent or composition that may modify,e.g., enhance, the efficacy of other agents, such as a drug or vaccine. Conventionally the term refers in the context of the invention to a compound or composition that serves as a carrier or auxiliary substance for immunogens and / or other pharmaceutically active compounds. It is to be interpreted in a broad sense and refers to a broad spectrum of substances that are able to increase the immunogenicity of anti-gens incorporated into or co-administered with an adjuvant in question. In the context of the present invention an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present invention. Typically, “adjuvant” or “adjuvant component” has the same meaning and can be used mutually. Adjuvants may be divided, e.g., into immuno-potentiators, antigenic delivery systems or even combinations thereof. The term “adjuvant” is typically understood not to comprise agents which confer immunity by themselves. An adjuvant assists the immune system unspecifically to enhance the antigen-specific immune response by e.g., promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response. Furthermore, an adjuvant may preferably e.g., modulate the antigen-specific immune response by e.g., shifting the dominating Th2-based antigen specific response to a more Th 1 -based antigen specific response or vice versa and / or by inducing of mucosal immune responses and / or increased IgA titers. Accordingly, an adjuvant may favourably modulate cytokine ex-pression / secretion, antigen presentation, type of immune response etc.

[0042] Advantages of adjuvants include the enhancement of the immunogenicity of antigens, modification of the nature of the immune response, the reduction of the antigen amount needed for a successful immunization, the reduction of the frequency of booster immunizations needed and an improved immune response in elderly and immunocompromised vaccines. These may be co-administered by any route, e.g., intramuscularly, subcutaneous, IV or intradermal injections.

[0043] The term “microorganism” or “recombinant microorganism” according to the present invention refers to a microorganism, e.g., bacterial or viral cell, or bacteria or virus, that has been genetically modified from its native state. Thus, a “recombinant bacterial cell” or “recombinant bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, a recombinant bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Recombinant bacterial cells disclosed herein may comprise exogenous nucleotide sequenceson plasmids. Alternatively, recombinant bacterial cells may comprise exogenous nucleotide sequences stably incorporated into their chromosome.

[0044] In a general embodiment, the present invention discloses a genetically engineered multidrug resistant reporter microorganism wherein said microorganism was constructed and laboratory tested for rapid and robust screening of natural and synthetic molecules for inhibition of enzymes involved in [3-lactams, aminoglycosides and macrolide antibiotic resistance and determine its antibiotic potentiating, synergistic or novel antibacterial property. The reporter strain can be also used for testing novel drugs and antimicrobial agents for its efficacy against multidrug resistant clinical isolates.

[0045] In an embodiment, the present invention relates to a multidrug-resistant reporter microorganism comprising a bacterial host genetically modified to confer resistance to multiple classes of antibiotics and to express a reporter gene in response to antibiotic exposure.

[0046] In an embodiment of the present invention, the genetically engineered multidrug resistant reporter microorganism comprising one or more resistance genes coding for [3- lactam resistance, macrolide resistance, aminoglycoside resistance, chloramphenicol resistance, zeocin resistance, and colistin resistance.

[0047] In yet another embodiment, the microorganism is a bacterial host is selected from but not limited to Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Caulobacter, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus, Listeria, Mycobacterium, Saccharomyces, Salmonella, Staphylococcus, Streptococcus, Vibrio, Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve UCC2003, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium acetobutylicum, Clostridium butyricum, Clostridium butyricum M-55, Clostridium cochlearum, Clostridium felsineum, Clostridium histolyticum, Clostridium multifermentans, Clostridium novyi-NT, Clostridium paraputrificum, Clostridium pasteureanum, Clostridium pectinovorum, Clostridium perfringens, Clostridium roseum, Clostridium sporogenes, Clostridium tertium, Clostridium tetani, Clostridium tyrobutyricum, Corynebacterium parvum, Escherichia coli MG1655, Escherichia coli Nissle 1917, Listeria monocytogenes, Mycobacterium bovis, Salmonella choleraesuis, Salmonella typhimurium, and Vibrio cholera. In certain embodiments, the genetically engineered bacteria are selected from the group consisting of Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei,Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Oxalobacter formigenes bacterial cell. Saccharomyces boulardii. In certain embodiments, the genetically engineered bacteria are selected from Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Escherichia coli, Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, and Lactococcus lactis. Preferbaly, the bacterial host selected from the genus Vibrionaceae. For example, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus . More preferably, Vibrio cholerae (V. cholerae).

[0048] In a preferred embodiment, the present invention provides a genetically engineered multidrug-resistant reporter V. cholerae strain BLF-lVc comprising one or more resistance genes coding for p-lactam resistance, macrolide resistance, aminoglycoside resistance, chloramphenicol resistance, zeocin resistance and colistin resistance.

[0049] In an embodiment of the present invention, the one or more resistance genes selected from OXA-48 carbapenemase (bZaoxA-4s), aminoglycoside-3 "-adenylyltransferase (aadA), Aminoglycoside phosphotransferase 3’ (aphV). ribosomal protein S12 (rpsL). chloramphenicol acetyltransferase (catB), Bleomycin resistance protein (sh-ble). mobilized colistin resistance gene 1 (mcr-I), macrolide-streptogramin resistance gene E (msrE). and Macrolide Phosphotransferase gene E (mphE) genes.

[0050] In still another embodiment, the antibiotic resistance genes are integrated into the bacterial chromosome within the bacterial host.

[0051] In an embodiment of the present invention, the genetically engineered multidrug resistant reporter microorganism exhibits resistance to one or more antibiotics selected from but not limited to ampicillin, erythromycin, zeocin, kanamycin, spectinomycin, streptomycin, chloramphenicol, and colistin.

[0052] In an embodiment of the present invention, the genetically engineered multidrug resistant reporter microorganism is resistant to ampicillin (30pg / mL), erythromycin (Ipg / mL), zeocin (25pg / mL), kanamycin (40pg / mL), spectinomycin (40pg / mL), streptomycin (500pg / mL), chloramphenicol (5pg / mL) and colistin (50pg / mL) when used individually.

[0053] In an embodiment of the present invention, the genetically engineered multidrug resistant reporter microorganism is resistant to ampicillin (20pg / mL), erythromycin (Ipg / mL), zeocin (lOpg / mL), kanamycin (20pg / mL), spectinomycin (20pg / mL),streptomycin (lOOpg / mL), chloramphenicol (3pg / mL) and colistin (20pg / mL) when used in combination.

[0054] In a preferred embodiment, the present invention provides a genetically engineered multidrug-resistant reporter V. cholerae strain BLF-lVc comprising one or more of the resistance genes selected from OXA-48 carbapenemase (bZaoxA-4s), aminoglycoside-3 "- adenylyltransferase (aadA), Aminoglycoside phosphotransferase 3’ (ap / z3’), ribosomal protein S12 (rpsE), chloramphenicol acetyltransferase (catB). Bleomycin resistance protein (sh-ble), mobilized colistin resistance gene 1 (mcr-1), macrolide-streptogramin resistance gene E (msrE), and Macrolide Phosphotransferase gene E (mphE) genes.

[0055] In some embodiments, the source of the strain BLF-lVc is wild-type (WT) V. cholerae strain N 16961, wherein the relV, relA, spoT and dksA genes of the WT V. cholerae strain N 16961 are deleted by homologous recombination and replacing them with aadA. aph3’, catBl and sh-ble respectively to generate JV9 strain. Further, three other antibiotic resistance genes, mcr-1, blaoxA- and msrE, mphE were introduced into JV9 with the help of an integrative vector to obtain the strain BLF-lVc.

[0056] In an embodiment, the present invention provides a recombinant integrative vector comprising the drug resistance genes selected from mcr-1, blaoxA-ts, msrE, and mphE genes isolated from genomes of clinical isolates, wherein the mcr-1 is cloned under a constitutive promoter of said integrative vector, and bZaoxA-48, msrE, and mphE are cloned under native promoters.

[0057] In an embodiment, the “Constitutive promoter” refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and / or to which it is operably linked. Constitutive promoters and variants are well known in the art and include, but are not limited to, Ptac promoter, BBa_J23100, a constitutive Escherichia coli os promoter (e.g., an osmY promoter), a constitutive Escherichia coli o32 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli o70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E. coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa Kl 19000; BBa Kl 19001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter(BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter(BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter(BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtilis oA promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PliaG (BBa_K823000), PlepA (BBa_K823002), Pveg (BBa_K823003)), aconstitutive Bacillus subtilis oB promoter (e.g., promoter etc (BBa_K143010), promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g., Pspv2 from Salmonella (BBa_K 112706), Pspv from Salmonella (BBa_K 112707)), a bacteriophage T7 promoter (e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa Kl 13010; BBa Kl 13011; BBa Kl 13012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g., SP6 promoter (BBa_J64998)). Preferably, the constitutive promoter used in the present invention is htpG promoter.

[0058] In an embodiment of the present invention, the integrative vector is a plasmid selected from but not limited to pSB49, pCR5, pEc70 / 2, pEcl92, pKAS32, pGP704, pCVD442, pWM91, pCMF2, pYB93, pTL61T, pBAD33, pNQ705, pBR322-based integrative constructs, and pLAFR3. Preferably, pSB49.

[0059] In an embodiment, the present invention relates to a method for preparing a multidrug-resistant reporter microorganism, designated BLF-lVc comprising the steps of: i. selecting a bacterial host strain; consisting of JV9, a derivative of a sensitive V. cholerae strain N 16961 ii. introducing multiple antibiotic resistance genes selected from relV. relA, spoT and dksA genes of the WT V. cholerae strain N 16961 into the bacterial host strain to confer resistance to different classes of antibiotics; iii. inserting a reporter gene downstream of a responsive promoter into the bacterial host strain; wherein mcr-1 was cloned under the constitutive promoter, htpG of the pSB49 vector while the bZfloxA-48 and msrE, and mphE was cloned with its native promoter. iv. obtaining the multidrug-resistant reporter microorganism; and v. analyzing gene expression of multidrug-resistant reporter microorganism in response to antibiotic exposure or environmental stressors; wherein the MDR reporter strain (BLF-lVc) was tested against eight different antibiotics falling under 5 different antibiotic classes viz ampicillin, erythromycin, zeocin, kanamycin, spectinomycin, streptomycin, chloramphenicol and colistin.

[0060] In a preferred embodiment, the present invention relates to a method of constructing the genetically engineered multidrug-resistant reporter V. cholerae strain BLF-lVc as claimed in claim 10, comprising the steps of: i) providing V. cholerae strain N 16961 and deleting relV. relA. spoT. and dksA genes to obtain the V. cholerae strain JV9 as host strain;ii) introducing antibiotic resistance genes aadA. aph3'. catBl , and sh-ble in place of the deleted relV, relA. spoT. and dksA genes in the V. cholerae strain JV9 by homologous recombination; iii) providing recombinant integrative vector as disclosed herein; iv) cloning the recombinant integrative vector into an initial bacterial host, followed by conjugating the same to an intermediate bacterial host strain to convert the same as a donor strain; and v) conjugating the donor strain with the V. cholera strain JV9, wherein the recombinant integrative vector from the donor strain site-specifically integrates at dif locus of the V. cholera strain JV9 to obtain the multidrug -resistant reporter microorganism V. cholera strain BLF-IVc.

[0061] In an embodiment, the initial bacterial host is selected from but not limited to E. coll FCV14, E.coli BW23474 (pir+), E.coli BW20767 (pir-116), E.coli S17-1 Xpir, and the like. Preferably FCV14.

[0062] In an embodiment, the intermediate bacterial host strain is selected from but not limited to E. coll [3-2163, E.coli [3-2155, E.coli [3-2150, and the like. Preferably E.coli [3-2163.

[0063] In an embodiment, the antibiotic resistance genes may also be introduced into the bacterial host strain through plasmid transformation, transduction, or conjugation.

[0064] In another embodiment, said method further comprising characterizing the phenotypic and genotypic properties of the genetically engineered multidrug-resistant reporter microorganism, including its growth rate, antibiotic resistance profde, and stability of genetic constructs.

[0065] In another embodiment, the present invention provides a method for developing a genetically engineered multidrug-resistant V. cholerae strain BLF-IVc that can grow at specific concentrations of multiple antibiotics by expressing a reporter resistance gene in response to antibiotic exposure.

[0066] In still another embodiment, the present invention discloses a genetically engineered multidrug-resistant reporter V. cholerae strain BLF-IVc that can be used to screen adjuvants like antibiotic potentiators, synergistic molecules or novel antibacterials.

[0067] In yet another embodiment, the present invention provides a method for detecting adjuvants that can potentiate existing antibiotics against multidrug resistant microorganisms.

[0068] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scopethereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions, or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.EXAMPLES

[0069] The present invention is further explained in the form of the following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.Example 1: Development of the reporter strain.

[0070] A genetically defined sensitive bacterial strain, named JV9 which is a ppGpp°Ac / G4 derivative of a sensitive V. cholerae strain N 16961 is used for the development of the reporter strain. The V. cholerae strain N 16961 was identified to be ten times more sensitive to antibiotics than the conventional E. coll ATCC 25922 strain used in the laboratory. The ppGpp°A<7GA derivative of N 16961 named JV9 has high membrane permeability and lower efflux pump activity than the wild type V. cholerae strains that further help in better sensitivity of the assay.Example 2: Deletion and replacement of Antibiotic Resistance Genes

[0071] The relV, relA, spoT and dksA genes of the WT V. cholerae strain N 16961 was deleted by homologous recombination replacing it with aadA. aph3’, catBl and sh-ble respectively to generate JV9 strain. Three other antibiotic resistance genes, mcr-1, blaoxA- and msrE, mphE were introduced into JV9 with the help of the integrative vector pSB49 which integrates site specifically at the 'dif loci of the bacterial (JV9) genome. The pSB49 vector also has a constitutive promoter htpG. All the resistance genes were amplified from the genomes of clinical isolates (FIG. 1).

[0072] The mcr-1 was cloned under the constitutive promoter, htpG of the pSB49 vector while the b / floxA-48 and msrE, mphE was cloned with its native promoter. The recombinant vector with the cloned antibiotic resistance genes was selected using cloning bacterial host FCV14 and then conjugated into the JV9 strain using intermediate bacterial host strain [3- 2163 which is an auxotrophic mutant which require externally supplemented diaminopimelic acid (DAP) for its growth.

[0073] After conjugation, the transconjugants were selected on media without diaminopimelic acid (DAP) and antibiotic ampicillin antibiotic selection. The transconjugantswere then confirmed by resistance gene specific PCR and growth of the strain in specific antibiotic media.

[0074] Three antibiotic resistance genes, mcr-1, blaoxA- and msrE, mphE were amplified from the genomes of different clinical isolates. The amplified products are digested with restriction enzymes and ligated in-vitro. The desired three gene ligated product (mcrl- blaoxA-48 -msrEmphE) is amplified, digested and ligated into similarly digested integrative vector pSB49. The recombinant vector with the cloned antibiotic resistance genes was selected using cloning bacterial host FCV14 and then conjugated into the JV9 strain using intermediate bacterial host strain [3-2163, an auxotrophic mutant. The JV9 strain already had aadA. aph3’, catBl and sh-ble antibiotic resistant cassettes inserted during the genome engineering for relV. relA, spoT and dksA genes knockout.Example 2: multi-drug resistance of the reporter strain.

[0075] The ability of the genetically engineered multi-drug resistance strains in different antibiotics individually and in combination was tested by growing the strain in antibiotic containing MHB broth after determining the MIC of the specific antibiotics by broth microdilution assay.Methodology

[0076] In brief, overnight grown reporter strain in MHB medium was diluted at 1: 100 in fresh MHB and cultured at 37°C with shaking at 180rpm to an optical density of 0.5(2 x 108CFU / ml) measured at 600 nm. Then, the culture was diluted at 1: 1000 in fresh MHB (2 x 105CFU / ml) and 100 pL of this diluted culture was added into each well of a 96-well microtiter polystyrene tray. A series of 2-fold dilutions of the particular antibiotic was prepared in this 96-well plate. The mixtures were incubated at 37°C for 16-18h. MIC was defined as the lowest antibiotic concentration that inhibited visible bacteria growth.Result

[0077] Genetically engineered multidrug resistant reporter microorganism is resistant to ampicillin (30pg / mL), erythromycin (Ipg / mL), zeocin (25pg / mL), kanamycin (40pg / mL), spectinomycin (40pg / mL), streptomycin (500pg / mL), chloramphenicol (5pg / mL) and colistin (50pg / mL) when used individually and concentrations ampicillin (20pg / mL), erythromycin (Ipg / mL), zeocin (lOpg / mL), kanamycin (20pg / mL), spectinomycin (20pg / mL), streptomycin (lOOpg / mL), chloramphenicol (3pg / mL) and colistin (20pg / mL) when used in combination.

[0078] Antibiotic concentration used to grow BLF-lVc are zeocin (25pg / mL), kanamycin (40pg / mL), spectinomycin (40pg / mL), chloramphenicol (5pg / mL), erythromycin (Ipg / mL),ampicillin (30pg / mL), streptomycin (500pg / mL), and colistin (50pg / mL). BLF-lVc Abs8 is the reporter strain when grown in combination of 8 antibiotics at concentration of ampicillin (20pg / mL), erythromycin (Ipg / mL), zeocin (lOpg / mL), kanamycin (20pg / mL), spectinomycin (20pg / mL), streptomycin (lOOpg / mL), chloramphenicol (3pg / mL) and colistin (20pg / mL) when used in combination. BLF-lVc Abs3 is the reporter strain when grown in combination of 3 antibiotics at concentration of ampicillin (30pg / mL), kanamycin (40pg / mL) and chloramphenicol (5pg / mL). Wild type V. cholerae strain N 16961 was used as the negative control (FIG. 2).

[0079] Growth of reporter strain BLF-lVc (JV-MDR) in presence of different antibiotics individually in test tubes (FIG. 3 A-C). Growth of reporter strain BLF-lVc (JV-MDR) in presence of different antibiotics in combination using flask for better growth conditions. V. cholerae N16961 is used as the negative control. JV-MDR (1) is the reporter strain when grown in combination of 8 antibiotics at concentration of ampicillin (20pg / mL), erythromycin (Ipg / mL), zeocin (lOpg / mL), kanamycin (20pg / mL), spectinomycin (20pg / mL), streptomycin (lOOpg / mL), chloramphenicol (3pg / mL) and colistin (20pg / mL). JV-MDR (2) is the reporter strain when grown in combination of 3 antibiotics at concentration of ampicillin (30pg / mL), kanamycin (40pg / mL) and chloramphenicol (5pg / mL).Example 2: Screening of antibiotic adjuvants and detection of antibiotic potentiators, synergistic compounds and novel antimicrobial agents the reporter strain.Primary ScreeningOvernight grown reporter strain in MHB medium were diluted at 1: 100 in fresh MHB and cultured at 37°C with shaking at 180rpm to an optical density of 0.5 (2 x 108CFU / ml) measured at 600nm. The reporter strain was then diluted at 1: 1000 in fresh MHB to achieve an initial inoculum of 2 x 105CFU / ml and 145pL of this diluted culture with test antibiotic was added into each well of a 96-well microtiter polystyrene tray. 5pL from 300pM stock solutions of test compound dissolved in appropriate solvents was added to each separate well of a 96-well plate. The controls of screening include the reporter strain cultured without antibiotic or compound, reporter strain culture with test antibiotic, reporter strain culture with both test antibiotic and the solvent in which the compound is dissolved, reporter strain culture with sensitive antibiotic and blank media control. The screening was done in triplicates. The mixtures were incubated at 37°C and after 16-18 hours the optical density at 600 nm (OD600) of the plate was determined using a spectrophotometer. The % inhibition was calculated as (PCAb-TestAb / PCAb-Neg)* 100 where PC is reporter strain culture with theantibiotic and solvent, Test is reporter strain culture with antibiotic and the compound and Neg is the MHB alone.Secondary screeningThe compounds from primary screening were further assessed through secondary screening. Following the same protocol for bacterial inoculum preparation as previously described. Each well contained reporter strain culture with test antibiotics, and an additional set contained only reporter strain culture. To these wells, 5pL from 300pM stock solutions of each compound was added. The mixtures were incubated at 37°C and after 16-18 hours, OD600 of the plate was determined using a spectrophotometer. Potentiating or synergistic compounds that displayed minimal or no antimicrobial activity and inhibited bacteria growth in combination with test antibiotics, were identified for further consideration. The secondary screening was done with and without antibiotic and % inhibition was calculated separately for with antibiotic (Ab) as (PCAb-TestAb / PCAb-Neg)* 100 and without antibiotic (w / o Ab) as (PCw / oAb-Testw / oAb / pcw / oAb-Neg)* 100 and then the final % inhibition was calculated as (% inhibition with Ab) - (% inhibition without Ab).ResultThe genetically engineered multi-drug resistant reporter strain BLF-lVc was used to screen the commercially available natural product library of 803 compounds from Selleckchem to identify erythromycin adjuvants (FIG. 4A and B). The primary screening was performed by growing the reporter strain at erythromycin (Ipg / mL) and testing the compounds which identified 26 compounds portraying % inhibition (>35) of the reporter strain. Secondary screening of compound library using BLF-lVc was performed in the presence of erythromycin (Ipg / mL) and absence of the antibiotic which identified 1 compound (Methoxysalicylic acid) portraying >10% inhibition of the reporter strain. Methoxysalicylic Acid is known to serve as a precursor for the synthesis of drugs targeting inflammatory conditions, analgesics, and potentially anti-cancer agents. Its phenolic structure enables it to engage in hydrogen bonding and other intermolecular interactions, contributing to its bioactivity.

[0080] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.ADVANTAGES OF THE PRESENT INVENTION

[0081] The current strategy of antibiotic adjuvant discovery heavily relies on initial in-silico screening followed by in-vitro assays to determine its synergistic or potentiating abilities. In many cases, in-silico screening gives false positive hits and the expected activity might not be identified in the in-vitro screening. The reporter strain BLF-lVc developed in the present study could be used for a more accurate and specific high-throughput in-vitro screening of compounds / molecules with antibacterial, synergistic or potentiating activity.

[0082] In most of the existing assay systems, the functional genes cloned and introduced into the bacteria are in the plasmid and require antibiotic pressure for the maintenance of the plasmid. The plasmid could be lost / cured off if the selection pressure is weaned. In the present reporter strain, BLF-lVc the recombinant vector is stably expressed as it integrates site specifically at the 'dif site of the bacterial genome preventing curing off of the resistance allele.

[0083] Use of clinical isolates for the screening of potential antibiotic adjuvants or antimicrobials may limit the sensitivity of the screening as the efficacy of the compound / molecule is dependent on multiple functions like membrane permeability and efflux pump activity. The reporter strain in the present study is developed by the genetic engineering of a sensitive bacteria with defined genomic background that has higher membrane permeability and lower efflux pump activity than the canonical laboratory strains thus promising better sensitivity of the assay. The activity of the compound detected will be via acting against the resistance function.

[0084] The MDR reporter strain could be used as an alternative for multidrug resistant clinical isolates to test the efficacy of novel drugs for its resistance enzyme inhibiting ability.

Claims

We Claim:

1. A genetically engineered multidrug resistant reporter microorganism comprising one or more resistance genes coding for p-lactam resistance, macrolide resistance, aminoglycoside resistance, chloramphenicol resistance, zeocin resistance and colistin resistance.

2. The genetically engineered multidrug resistant reporter microorganisms as claimed in claim 1 are bacteria.

3. The genetically engineered multidrug resistant reporter microorganisms as claimed in 2, are derivatives of Vibrio cholerae (V. cholerae).

4. The genetically engineered multidrug resistant reporter microorganism as claimed in claim 3 is V. choleraei strain BLF-IVc.

5. The genetically engineered multidrug resistant reporter microorganism as claimed in anyone of claims 1-4, wherein the one or more resistance genes are selected from OXA-48 carbapenemase (Z?Z«OXA-4.S). aminoglycoside-3 "-adenylyltransferase (aadA), Aminoglycoside phosphotransferase 3’ (ap / z3’), ribosomal protein S12 (rpsL), chloramphenicol acetyltransferase (catB)'. Bleomycin resistance protein (sh-ble). mobilized colistin resistance gene 1 (mcr-I), macrolide-streptogramin resistance gene E (msrE), and Macrolide Phosphotransferase gene E (mphE) genes.

6. The genetically engineered multidrug resistant reporter microorganism as claimed in anyone of claims 1-5 is resistant to antibiotics selected from the group consisting of ampicillin, erythromycin, zeocin, kanamycin, spectinomycin, streptomycin, chloramphenicol, and colistin.

7. A recombinant integrative vector comprising the drug resistance genes selected from mcr- 1, blaoxA-K, msrE, and mphE genes isolated from genomes of clinical isolates, wherein the mcr-1 is cloned under a constitutive promoter, and bZaoxA-48, msrE, and mphE are cloned under native promoters of said integrative vector.

8. The recombinant integrative vector as claimed in claim 7 is pSB49 vector.

9. The recombinant integrative vector as claimed in claim 7, wherein the constitutive promoter is htpG.

10. A genetically engineered multidrug -resistant reporter V. cholera strain BLF-IVc comprising resistance genes coding for p-lactam resistance, macrolide resistance, aminoglycoside resistance, chloramphenicol resistance, zeocin resistance and colistin resistance.

11. A method of constructing the genetically engineered multidrug-resistant reporter V. cholera strain BLF-lVc as claimed in claim 10, comprising the steps of: i) providing V. cholerae strain N 16961 and deleting relV. relA. spoT. and dksA genes to obtain the V. cholerae strain JV9 as host strain; ii) introducing antibiotic resistance genes aadA. aph3'. catBl , and sh-ble in place of the deleted reTV, relA. spoT. and dksA genes in the V. cholerae strain JV9 by homologous recombination; iii) providing recombinant integrative vector as claimed in anyone of claims 7-9; iv) cloning the recombinant integrative vector into an initial bacterial host, followed by conjugating the same to an intermediate bacterial host strain to convert the same as a donor strain; and v) conjugating the donor strain with the V. cholera strain JV9, wherein the recombinant integrative vector from the donor strain site-specifically integrates at dif locus of the V. cholera strain JV9 to obtain the multidrug -resistant reporter microorganism V. cholera strain BLF-lVc.

12. The method as claimed in claim 11, wherein the initial bacterial host is E. coll FCV14.

13. The method as claimed in claim 11, wherein the intermediate bacterial host strain is E. coll P-2163.

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