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WO2018140827A1 - Micro-organismes rapporteurs et leurs utilisations - Google Patents

Micro-organismes rapporteurs et leurs utilisations Download PDF

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Publication number
WO2018140827A1
WO2018140827A1 PCT/US2018/015602 US2018015602W WO2018140827A1 WO 2018140827 A1 WO2018140827 A1 WO 2018140827A1 US 2018015602 W US2018015602 W US 2018015602W WO 2018140827 A1 WO2018140827 A1 WO 2018140827A1
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WO
WIPO (PCT)
Prior art keywords
reporter
acinetobacter
atcc
microorganism
vol
Prior art date
Application number
PCT/US2018/015602
Other languages
English (en)
Inventor
Dante RICCI
Ami Patel
Original Assignee
Achaogen, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Achaogen, Inc. filed Critical Achaogen, Inc.
Publication of WO2018140827A1 publication Critical patent/WO2018140827A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C07K14/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
    • G01N2333/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella or Psychrobacter

Definitions

  • the present disclosure provides in some aspects reporter polynucleotides comprising a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule.
  • the disclosure further relates to reporter vectors, genetically engineered reporter microorganisms, and uses thereof.
  • Multidrug-resistant bacteria have emerged worldwide and are increasing in prevalence, creating a substantial public health concern.
  • the Centers for Disease Control and Prevention attributes at least 23,000 deaths in the U.S. each year to antibiotic-resistant infections, with some infection types associated with mortality rates as high as 50%.
  • infections such as Acinetobacter spp. and Pseudomonas aeruginosa
  • rates of multi-drug resistance in the U.S. have been reported as 63% and 13%, respectively.
  • the continued prevalence of these multidrug-resistant isolates has left clinicians with few treatment options for the patients with life-threatening infections. Addressing this urgent need for new antibiotics to treat multidrug-resistant Gram-negative infections is critical. There is a need in the art for methods of identifying antibiotics specific for pathogenic bacteria. Provided are methods and articles of manufacture that meet such need.
  • reporter polynucleotides comprising a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule, wherein the OM stress- responsive gene is modulated in response to a stress to the outer membrane of the Acinetobacter bacterium.
  • OM outer membrane
  • the Acinetobacter bacterium is a Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii,
  • Acinetobacter brisouii Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter organizerri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii
  • Acinetobacter soli Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
  • the Acinetobacter bacterium is Acinetobacter baumannii.
  • the Acinetobacter bacterium is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA- 1710, ATCC BAA- 1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA- 1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA-1799, ATCC BAA-
  • Acinetobacter bacterium is ATCC 17978, Ab307-0294, AABA041, or AABA046.
  • the stress to the outer membrane of the Acinetobacter bacterium is or is caused by depletion of BamA or treatment with polymyxin B nonapeptide (PMBN). In certain embodiments, the stress to the outer membrane of the Acinetobacter bacterium is or is caused by depletion of BamA and treatment with PMBN.
  • PMBN polymyxin B nonapeptide
  • stress to the outer membrane of Acinetobacter bacterium involves depletion of BamA and depletion of BamA is performed by removing or decreasing an amount of an inducer that controls expression of BamA from a culture or composition containing Acinetobacter bacterium.
  • the inducer is removed or decreased for more than or more than about 5, 10, 15, 20, 30, 45, 60, 75, 90, or 120 minutes.
  • the inducer is removed or decreased for more than or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.
  • the inducer is arabinose.
  • stress to the outer membrane of Acinetobacter bacterium involves treatment with PMBN and the Acinetobacter bacterium is treated with PMBN for less than or less than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, or 120 minutes. In certain embodiments, the Acinetobacter bacterium is treated with PMBN for greater than or greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, or 120 minutes.
  • the OM stress-responsive gene is upregulated or
  • the OM stress-responsive gene is upregulated in response to the stress. In certain embodiments, the OM stress-responsive gene is upregulated in response to the stress at least or about at least 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold or more in response to the stress.
  • the OM stress-responsive gene is A1SJ3012, A1SJ3023, A1S_0027, A1S_0028, A1S_0029, A1S_0030, A1S_0031, A1S_0032, A1S_0033, A1S_0037, A1SJ3040, A1SJ3041, A1S_0044, A1S_0066, A1S_0092, A1S_0093, A1S_0109, A1S_0110, A1S_0112, A1S_0113, A1S_0114, A1S_0115, A1S_0116, A1S_0117, A1S_0118, A1S_0126, A1S_0158, A1S_0170, A1S_0175, A1S_0178, A1S_0189, A1S_0224, A1S_0245, A1S_0256, A
  • A1S_ _0680, A1S_ _0683 A1S_ _0714, A1S_ _0717 A1S_ _0718, A1S_ _0719 A1S_ _0736, A1S_ _0738,
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene is
  • the OM stress-responsive gene is downregulated in response to the stress. In certain embodiments, the OM stress-responsive gene is downregulated in response to the stress at least or about at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more in response to the stress.
  • the OM stress-responsive gene is A1S_0009, A1S_0010,
  • A1S_ _1072 A1S_ _1079 A1S_ _1080, A1S_ _1088 A1S_ .1089, A1S_ .1091 A1S_ . 1092, A1S_ .1093,
  • the OM stress-responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_380
  • the OM stress- responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
  • the regulatory region comprises a contiguous sequence of nucleotides within 500 base pairs upstream or 5' of the open reading frame (ORF) of the OM stress-responsive gene.
  • the contiguous sequence of nucleotides comprises at least or at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400 or more base pairs.
  • the regulatory region comprises a promoter.
  • the regulatory region comprises a sequence to further promote translation of the encoded reporter molecule.
  • the sequence further promoting translation is or comprises a bacterial ribosome binding site.
  • the ribosome binding site is a Shine-Dalgarno sequence.
  • the Shine-Dalgarno sequence is native to the regulatory region of the OM stress-responsive gene.
  • the Shine-Dalgarno sequence is synthetic and/or heterologous to the regulatory region of the OM stress-responsive gene.
  • the Shine- Dalgarno sequence comprises the sequence set forth in SEQ K) NO: 14 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 14. In certain embodiments, the Shine-Dalgarno sequence comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 14.
  • the regulatory region comprises the sequence set forth in any of SEQ ID NOS: 1-13 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 1-13. In certain embodiments, the regulatory region comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 1- 13.
  • the reporter molecule is a fluorescent protein, a luminescent protein, a chromoprotein, or an enzyme.
  • the fluorescent protein is Sirius, SBFP2, Azurite, mAzurite, EBFP2, moxBFP, mKalamal, mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean, mCerulean3, moxCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl, MiCy (Midoriishi-Cyan), iLOV, AcGFPl, sfGFP, moxGFP, mEmerald, EGFP, mEGFP, AzamiGreen, cfSGFP2, ZsGreen, SGFP2, Clover, mClover2, mClover3, EYFP, Topaz, mTopaz, mVenus, mox Venus, SYFP
  • the fluorescent protein is sfGFP, mClover3, or mRuby2. In certain embodiments, the fluorescent protein is sfGFP.
  • the sequence encoding the reporter molecule comprises the sequence set forth in SEQ ID NO: 15, 33, or 40 or a sequence with at least or at least about 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 15, 33, or 40. In certain embodiments, the sequence encoding the reporter molecule comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 15.
  • the reporter polynucleotide comprises the sequence set forth in any of SEQ ID NOs: 16-28 or 34-36 or a sequence with at least or at least about 85%, 90%, 95%, 99%, or more sequence identity to any of SEQ ID NOs: 16-28 or 34-36. In certain embodiments, the reporter polynucleotide comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 16-28.
  • the luminescent protein is North American firefly luciferase, Genji-botaru luciferase, Italian firefly luciferase, Heike luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, Railroad worm luciferase, Renilla luciferase, Rluc8, Green Renilla luciferase, Gaussia luciferase, Gaussia-Dura luciferase, Cypridina luciferase, Vargula luciferase, Metridia luciferase, OLuc, bacterial luciferase
  • the enzyme is
  • chloramphenicol acetyltransferase CAT
  • ⁇ -galactosidase alkaline phosphatase
  • ⁇ - glucuronidase alkaline phosphatase
  • ⁇ -lactamase neomycin phosphotransferase
  • neomycin phosphotransferase or a modified version thereof.
  • reporter vectors comprising any of the reporter polynucleotide described herein.
  • the reporter polynucleotide is a first reporter polynucleotide and the reporter vector further comprises a second reporter polynucleotide that is any of the reporter polynucleotides described herein.
  • the first reporter polynucleotide and the second reporter polynucleotide are different.
  • the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress-responsive genes.
  • the reporter molecule encoded by the first reporter polynucleotide is different from the reporter molecule encoded by the second reporter polynucleotide. In certain embodiments, the reporter molecule encoded by the first reporter polynucleotide and the reporter molecule encoded by the second reporter polynucleotide do not exhibit an overlapping emission and absorption spectra and/or are distinguishably detectable.
  • the reporter vector is capable of being expressed in a host microorganism. In certain embodiments, the reporter vector is capable of being expressed in a Gram-negative bacterium. In certain embodiments, the host microorganism is Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enter obacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
  • the host microorganism is Acinetobacter. In certain embodiments, the host microorganism is Acinetobacter apis, Acinetobacter baumannii,
  • Acinetobacter baylyi Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii,
  • the host microorganism is Acinetobacter baumannii.
  • the host microorganism is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA- 1791, ATCC BAA- 1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799, ATCC BAA-1800, ATCC
  • the reporter polynucleotide is comprised in a backbone vector, and the backbone vector is pACH106, pWH1266, or pET-RA.
  • the reporter polynucleotide is comprised in a backbone vector, and the backbone vector is pACH106, pWH1266, or pET-RA.
  • a nucleotide sequence comprising the reporter polynucleotide, optionally the first reporter polynucleotide and the second reporter
  • polynucleotide is inserted into or replaces a portion of the nucleotide sequence of the backbone vector.
  • a nucleotide sequence comprising the reporter polynucleotide is inserted into or replaces a portion of the nucleotide sequence of the backbone vector.
  • the backbone vector comprises the sequence of nucleotides set forth in SEQ ID NO: 29 and a nucleotide sequence comprising the reporter polynucleotide replaces nucleotides 5,715-7,395 of the backbone vector.
  • the reporter vector comprises the sequence set forth in any of SEQ ID NOs: 30-32 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 30-32. In certain embodiments, the reporter vector comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 30-32.
  • reporter microorganisms comprising any of the reporter polynucleotides or any of the reporter vectors described herein.
  • reporter microorganisms comprising one or more reporter polynucleotide of any described herein or one or more reporter vector of any described herein.
  • the one or more reporter polynucleotides comprise a first reporter polynucleotide and a second reporter polynucleotide that are different.
  • the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress-responsive genes.
  • the reporter molecule encoded by the first reporter polynucleotide is different from the reporter molecule encoded by the second reporter polynucleotide. In certain embodiments, the reporter molecule encoded by the first reporter polynucleotide and the reporter molecule encoded by the second reporter polynucleotide do not exhibit an overlapping emission and absorption spectra and/or are distinguishably detectable. In certain embodiments, the first reporter polynucleotide and second reporter polynucleotide are comprised in the same reporter vector. In certain embodiments, the first reporter polynucleotide and second reporter polynucleotide are comprised in different reporter vectors.
  • the reporter microorganism is a Gram-negative bacterium. In certain embodiments, the reporter microorganism is Acinetobacter, Bdellovibrio,
  • Burkholderia Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
  • the reporter microorganism is Acinetobacter.
  • the reporter microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii,
  • the reporter microorganism is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA- 1790, ATCC BAA- 1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA- 1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA- 1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA- 1878, ATCC BAA-2093, ATCC BAA-747, S
  • each of at least two reporter microorganisms in the plurality comprise a different reporter polynucleotide in which comprises the same regulatory region of an OM-responsive gene but that is operatively linked to a different reporter molecule.
  • the different reporter molecules do not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable.
  • the plurality of reporter microorganisms comprises 2, 3, 4, 5, or more different reporter microorganisms.
  • the at least two reporter microorganisms are derived from the same host strain.
  • each of the at least two reporter microorganisms is derived from a different host strain, optionally wherein each of the at least two reporter microorganisms is derived from a different isolate or subtype of the strain.
  • compositions comprising a reporter microorganism described herein or a plurality of any of the reporter microorganisms described herein.
  • the composition comprises one or more components capable of activating the complement pathway.
  • the composition comprises serum.
  • the serum is human serum, rabbit serum, bovine serum, or mouse serum.
  • the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol), or 25% (vol/vol).
  • the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol), or 15% (vol/vol) to 25% (vol/vol).
  • the composition comprises an agent, optionally a candidate antibacterial agent.
  • the agent is a small molecule compound, a peptide or a protein.
  • the agent is an antibody or antigen-binding fragment thereof.
  • microdroplets comprising a microorganism described herein, a plurality of microorganisms described herein, or a composition described herein.
  • microdroplets comprising a reporter microorganism described herein.
  • the microdroplet comprises an agent, such as a candidate antibacterial agent, including agents known to cause or suspected of causing OM stress to the reporter
  • microorganism and/or that may impact the integrity or biogenesis of the outer membrane of the reporter microorganism.
  • the microdroplet further comprises a cell that produces or secretes an agent.
  • the cell is an antibody-producing cell.
  • the cell is a B cell.
  • the cell is a plasma cell or a plasmablast.
  • the cell is a mammalian cell.
  • the cell is a microorganism.
  • the cell is a fungal or bacterial cell.
  • the agent is a candidate antibacterial agent.
  • the agent is a small molecule compound, a peptide, or a protein.
  • the agent is an antibody or antigen-binding fragment thereof.
  • the agent is a small molecule antibiotic or peptide antibiotic.
  • the microdroplet comprises agarose, carrageenan, alginate, alginate-polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2- pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium polyacrylate, polydimethyl siloxane, cis- polyisoprene, PuramatrixTM, poly- divenylbenzene, polyurethane, polyacrylamide or combinations thereof.
  • agarose carrageenan
  • alginate alginate-polylysine
  • collagen cellulose
  • methylcellulose gelatin
  • gelatin chitosan
  • extracellular matrix extracellular matrix
  • dextran starch
  • inulin heparin
  • hyaluronan fibrin
  • the microdroplet comprises agarose.
  • the microdroplet comprises growth media.
  • the microdroplet comprises serum.
  • the serum is human serum, rabbit serum, bovine serum, or mouse serum.
  • the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol), or 25% (vol/vol).
  • the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol), or 15% (vol/vol) to 25% (vol/vol).
  • compositions comprising a microdroplet described herein or a plurality of microdroplets described herein.
  • kits comprising: a reporter polynucleotide described herein, a reporter vector described herein, a reporter microorganism described herein, a plurality of reporter microorganisms described herein, a composition described herein, or a microdroplet described herein; and instructions for use.
  • the method further comprises (c) determining if there a change in the level of the detectable signal from the reporter molecule(s) compared to in the absence of exposing the reporter microorganism to the condition, wherein a change in the level of the detectable signal indicates the condition causes OM stress to the microorganism.
  • exposing the reporter microorganism to a condition or agent is carried out in suspension, in an array, or in a microdroplet.
  • the condition is treatment with an agent.
  • kits for screening an agent comprising: (a) contacting a reporter microorganism described herein, a plurality of reporter microorganisms described herein, or a composition described herein with an agent; and b) detecting the presence, absence, or level of a detectable signal from the reporter molecule(s).
  • the method further comprises (c) identifying the agent that causes a change in the level of the detectable signal from the reporter molecule compared to in the absence of contacting the reporter microorganism to the agent.
  • kits for screening an agent comprising: (a) contacting an agent with a first reporter microorganism described herein; (b) contacting the agent with at least one additional reporter microorganism described herein, wherein the at least one additional reporter microorganism is not the same as the first reporter microorganism; and (c) detecting the presence, absence, or level of a detectable signal from the reporter molecule from the first and/or at least one additional reporter microorganism.
  • the contacting in (a) and (b) is carried out separately. In certain embodiments, the contacting in (a) and (b) is carried out together.
  • the first microorganism, the at least one additional reporter microorganism, and the agent are encapsulated together in a microdroplet.
  • the first and the at least one additional reporter microorganism comprise a different reporter polynucleotide in which comprises the same regulatory region of an OM-responsive gene but that is operatively linked to a different reporter molecule.
  • the first and the at least one additional microorganism comprise a different reporter polynucleotide in which comprises a different regulatory region of an OM-responsive gene and is operatively linked to a different reporter molecule.
  • the different reporter molecules do not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable.
  • the first and the at least one additional microorganism are derived from the same host strain. In certain embodiments, the first and the at least one additional microorganism are derived from a different host strain, optionally wherein each of the first and the at least one additional microorganism is derived from a different isolate or subtype of the strain. In certain embodiments, the contacting is carried out in suspension, in an array, or in a microdroplet. In certain embodiments, the contacting is carried out for at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours or 3 hours.
  • step (a) is carried out with a plurality of agents, wherein the reporter microorganism is contacted with each of the plurality of agents.
  • kits for screening an agent comprising: (a) encapsulating in a microdroplet: (i) a reporter microorganism described herein, a plurality of reporter
  • microorganisms described herein, or a composition described herein and (ii) a cell, wherein the cell produces an agent; and (b) detecting, in the microdroplet, the presence, absence, or level of a detectable signal from the reporter molecule(s).
  • identifying an agent that modulates an activity or property of a reporter microorganism comprising: (a) encapsulating in a microdroplet: (i) a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein; and (ii) a cell, wherein the cell produces an agent; and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule; thereby identifying an agent that modulates the activity or property of the target microorganism.
  • the method further comprises (c) isolating the cell from the microdroplet in which there is a change in the level of the detectable signal from the reporter molecule compared to in the absence of exposing the reporter microorganism to the agent.
  • the isolating is carried out using a micromanipulator or an automated sorter.
  • the method further comprises identifying the agent produced by the cell.
  • the identifying comprises determining the sequence of the agent, optionally using single cell PCR and nucleic acid sequencing.
  • step (a) is repeated with a plurality of agents.
  • the microorganism if there is a change in the presence, absence, or level of the detectable signal, the microorganism is identified as potentially not being resistant to the agent; and if there is not a change in the presence, absence, or level of the detectable signal, the microorganism is identified as potentially being resistant to the agent.
  • determining the drug resistance of a reporter microorganism comprising: (a) contacting a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein with a drug; and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of contacting the reporter microorganism with the drug, wherein if there is a change in the detectable signal, the reporter microorganism is not resistant to the drug and if there is not a change in the detectable signal, the reporter
  • microorganism is identified as potentially being resistant to the drug.
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is downregulated in response to an outer membrane stress and the level of the detectable signal decreases.
  • the OM stress- responsive gene is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038, A1S_0067,
  • A1S_ _nn A1S_ _1719 A1S_ _1724, A1S_ _1729 A1S_ _1730, A1S_ _1731 A1S_ _1732, A1S_ _1734,
  • A1S_ _3912, A1S_ _3914, or A1S_3915 are A1S_ _3912, A1S_ _3914, or A1S_3915.
  • the OM stress- responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_35
  • the OM stress-responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is upregulated in response to an outer membrane stress and the change in the detectable signal is an increase in the detectable signal.
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is upregulated in response to an outer membrane stress and the level of the detectable signal increases.
  • the OM stress- responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1S_0029, A1S_0030,
  • the OM stress- responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791.
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene is A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, A1S_3791.
  • the OM stress-responsive gene is A1S_0032, A1S_2885, or A1S_2889.
  • the OM stress-responsive gene is A1S_0113 or A1S_1751. In certain embodiments, the OM stress-responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
  • a method of assessing OM stress comprising: (a) contacting a reporter microorganism described herein or a composition described herein with an agent; and (b) identifying a reporter microorganism producing a detectable signal from the reporter molecule, thereby assessing OM stress.
  • step (a) is carried out with a plurality of agents, wherein the reporter microorganism is contacted with each of the plurality of agents.
  • kits for identifying an agent that modulates an activity or property of a microorganism comprising: (a) encapsulating in a microdroplet: (i) a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein; and (ii) a cell, wherein the cell produces an agent; and (b) identifying a reporter microorganism producing a detectable signal from the reporter molecule.
  • a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein with a drug; and (b) identifying a reporter microorganism producing a detectable signal from the reporter molecule, wherein if the reporter microorganism produces a detectable signal, the reporter microorganism is not resistant to the drug and if the reporter microorganism does not produce a detectable signal, the reporter microorganism is identified as potentially being resistant to the drug.
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is downregulated in response to an outer membrane stress.
  • the OM stress- responsive gene is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038, A1S_0067,
  • A1S_ _3912, A1S_ _3914, or A1S_3915 are A1S_ _3912, A1S_ _3914, or A1S_3915.
  • the OM stress- responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_35
  • the OM stress-responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is upregulated in response to an outer membrane stress.
  • the OM stress- responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1S_0029, A1S_0030,
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene is
  • the contacting is carried out in suspension, in an array, or in a microdroplet.
  • the reporter is carried out in suspension, in an array, or in a microdroplet.
  • microorganism of (a) is encapsulated in a microdroplet with a cell.
  • the agent is a candidate antibacterial agent, such as an agent known to cause or suspected of causing OM stress to the reporter microorganism and/or that may impact the integrity or biogenesis of the outer membrane of the reporter microorganism.
  • the agent is a small molecule compound, a peptide or a protein.
  • the agent is an antibiotic.
  • the agent is an antibody or antigen-binding fragment thereof.
  • the agent is an antibody and the cell is an antibody-producing cell.
  • the cell is a microorganism. In certain embodiments, the cell is a fungal or bacterial cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a B cell. In certain embodiments, the cell is a plasma cell or a plasmablast.
  • the cell is obtained from a donor that has been exposed to a target microorganism or an epitope-comprising fragment of a target microorganism or a variant thereof.
  • the donor has been immunized or infected with a target microorganism or an epitope-comprising fragment of a target microorganism or a variant thereof.
  • the donor is an immunized animal or an infected animal.
  • the donor is a mammal or a bird.
  • the donor is a human, a mouse or a chicken.
  • the donor is a human donor who was infected by the target microorganism.
  • the donor is a genetically modified non-human animal that produces partially human or fully human antibodies.
  • the microdroplet is generated by a microfluidics-based method.
  • the microdroplet comprises agarose, carrageenan, alginate, alginate-polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2- pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium polyacrylate, polydimethyl siloxane, cis- polyisoprene, PuramatrixTM, poly- divenylbenzene, polyurethane, polyacrylamide, or combinations thereof.
  • the microdroplet comprises agarose.
  • the method further comprises: (c) isolating the cell from the microdroplet in which there is a change in the level of the detectable signal from the reporter molecule compared to in the absence of exposing the reporter microorganism to the agent.
  • the isolating is carried out using a micromanipulator or an automated sorter.
  • the method further comprises identifying the agent produced by the cell.
  • the identifying comprises determining the sequence of the agent, optionally using single cell PCR and nucleic acid sequencing.
  • the method further comprises: (c) isolating the microdroplet comprising the cell producing the identified agent. In certain embodiments, the method further comprises: (d) isolating polynucleotides encoding the agent, wherein the agent is a polypeptide. In certain embodiments, the method further comprises: (e) determining the sequence of the nucleic acids encoding the polypeptide. In certain embodiments, the method further comprises prior to (a) introducing the reporter polynucleotide or reporter vector described herein into a host microorganism.
  • the host microorganism is a bacterium. In certain embodiments, the host microorganism is a Gram negative bacterium. In certain embodiments, the host microorganism is a proteobacterium. In certain embodiments, the host microorganism is a species of Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter,
  • Escherichia Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
  • the bacterium Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii,
  • Acinetobacter radioresistans Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis,
  • the agent is a drug.
  • the drug is a small molecule compound, a peptide or a protein.
  • the drug is an antibiotic.
  • the drug is an antibody or antigen-binding fragment thereof.
  • the drug is an antibody and the cell is an antibody-producing cell.
  • agents identified by any of the methods described herein are agents identified by any of the methods described herein.
  • Fig. 1 shows growth curves of a modified A. baumannii strain that is depleted for BamA unless exogenous arabinose is added.
  • the growth curves compare growth in the presence or absence of added arabinose for up to 9 hours.
  • Circles indicate the time points at which the cells were back-diluted to maintain cells in an exponential growth phase.
  • Rectangles indicate the time points at which samples were taken for analysis by RNA-Seq for comparing differences in gene expression caused by outer membrane stress induced by BamA depletion as described in subsequent Examples and Figs.
  • Fig. 2A-2C show differential gene expression in A. baumannii with or without BamA depletion as determined by RNA-Seq with counts normalized as fragments per kilobase per million reads (FPKM).
  • Fig. 2A depicts results for replicate transcript datasets from A.
  • Fig. 2B depicts results of replicate transcript datasets from A. baumannii cells with BamA depletion cultured in the absence of added arabinose for 6 hours.
  • Fig. 2C shows differences in gene expression when comparing BamA-depleted (+Ara) and non-BamA-depleted (-Ara) cells.
  • Fig. 3A-3C depicts log fold differences in gene expression in A. baumannii over time in BamA depleted cell cultures (incubated without arabinose; -Ara) compared to non-BamA- depleted cell cultures (incubated in the presence of arabinose; +Ara).
  • Fig. 3 A compares the abundance of gene transcripts after 2 hours +Ara or -Ara.
  • Fig. 3B compares the abundance of transcripts after 4 hours -i-Ara or - Ara.
  • Fig. 3C compares the abundance of transcripts after 6 hours -i-Ara or -Ara.
  • Fig. 4 shows the log fold change in gene expression of exemplary transcripts after 2, 4, and 6 hours of BamA depletion in A. baumannii compared to expression of the transcript in A. baumannii expressing BamA.
  • Fig. 5A-5B show A. baumannii gene transcripts whose expression was increased by at least 10-fold after 6 hrs of BamA depletion.
  • a heatmap reflecting the relative change in expression for each gene transcript at 2, 4, or 6 hours of BamA depletion is shown. Predicted functions for encoded protein products are given on the right.
  • FIG. 6A-6D show A. baumannii gene transcripts whose expression decreased by at least 10-fold after 6 hrs of BamA depletion.
  • a heatmap reflecting the relative change in expression for each gene transcript at 2, 4 or 6 hours of BamA depletion is shown. Predicted functions for encoded protein products are given on the right.
  • FIG. 7A-7D show differences in gene expression when comparing non-PMBN-treated cells to cells treated with PMBN for various concentrations and times.
  • FIG. 7A shows gene expression in non-PMBN-treated cells compared to cells treated with 25 ⁇ g/mL for 10 minutes.
  • FIG. 7B shows gene expression in non-PMBN-treated cells compared to cells treated with 250 ⁇ g/mL for 10 minutes.
  • FIG. 7C shows gene expression in non-PMBN-treated cells compared to cells treated with 25 ⁇ g/mL for 30 minutes.
  • FIG. 7D shows gene expression in non-PMBN- treated cells compared to cells treated with or 250 ⁇ g/mL for 30 minutes.
  • Transcripts (genes) whose expression changed by at least 10-fold after treatment with PMBN at the indicated concentration and time are shown in black.
  • Fig. 8 shows the log fold change after treatment of A. baumannii cells with 25 ⁇ g/mL PMBN (gray) or 250 ⁇ g/mL PMBN (black) in exemplary gene transcripts.
  • Fig. 9 shows the backbone vector pACH106 which encodes replication factors and selective markers necessary for autonomous replication in Acinetobacter baumannii and maintenance by antibiotic selection.
  • Fig. 10A shows a histogram of fluorescence intensities induced in P A is_2885-sfGFP reporter cells after treatment with PMBN.
  • Fig. 10B shows fluorescent micrographs of PAIS_28S9- sfGFP reporter cells after treatment in the presence or absence of PMBN.
  • FIG. 11A-11B shows fluorescent FACs histograms of P A is_28S5-sfGFP reporter cells after treatment with PMBN.
  • FIG. 11 A cells were grown in microtiter dishes without shaking or aeration.
  • Fig. 1 IB cells were grown in culture flasks with aeration.
  • Fig. 12A-12B depict fluorescent intensities of A. baumannii P A isj»32-sfGFP and P A is_2889-sfGFP reporter cells that are depleted for BamA, an OM biogenesis factor, unless grown in the presence of arabinose.
  • Fig. 12A depicts mean fluorescent intensities of the reporter cells in the presence or absence of arabinose.
  • Fig. 12B shows a representative flow cytometry fluorescence histogram of PAis_2889-sfGFP reporter cells after growth with or without arabinose for 3 hrs.
  • Fig. 13A-13B show flow cytometry fluorescence histograms of a multi-drug resistant strain of A. baumannii expressing the OM stress transcriptional reporter constructs after culture in the presence of PMBN or vancomycin (vane).
  • Fig. 13A depicts flow cytometry fluorescent histograms of PAis_2885-sfGFP reporter cells.
  • Fig. 13B depicts flow cytometry fluorescent histograms of PAis_2889-sfGFP reporter cells.
  • Fig. 14A-14B show flow cytometry fluorescence histoGrams of A. baumannii reporter cells containing either PAisjxm-sfGFP (Fig. 14A) or PAis_2885-sfGFP (Fig. 14B) after culture in the presence of ACHN-975, an antibiotic that inhibits synthesis of lipopolysaccharide (LPS) by inhibiting the activity of the LpxC enzyme.
  • LPS lipopolysaccharide
  • Fig. 15A shows flow cytometry fluorescence histograms of A. baumannii reporter cells containing PAis_oo32-sfGFP after culture in the presence of ACHN-975, an antibiotic that inhibits synthesis of lipopolysaccharide (LPS) by inhibiting the activity of the LpxC enzyme; colistin, a broad-spectrum antibiotic that binds and perturbs the LPS leaflet of the OM; and EDTA, a chelating agent that disrupts LPS layer by stripping away divalent cations that stabilize the OM by neutralizing the negative charges in LPS.
  • LPS lipopolysaccharide
  • Fig. 15B shows flow cytometry fluorescence histograms of A. baumannii reporter cells containing PAis_2885-sfGFP after culture in the presence of PMBN, EDTA, and EDTA with SDS, a detergent that acts synergistically with EDTA to permeabilize the destabilized OM.
  • Fig. 16 depicts one embodiment of a screening assay for identifying anti-bacterial molecules that induce outer membrane stress.
  • animals are immunized with Acinetobacter cells to generate antibody- secreting B cells, isolated B cells and reporter A. baumannii bacterial cells (containing an outer membrane stress transcriptional reporter construct in which a regulatory region of a gene induced by outer membrane stress is fused to a detectable moiety, such as GFP, as described) are mixed and co-encapsulated to form microparticles using microfluidics, and the microparticles are screened for activated reporter cells expressing a detectable signal (e.g. fluorescence).
  • Fig. 17A-17B show fluorescent micrograph images of A. baumannii reporter cells containing PAis_28S9-sfGFP encapsulated in agarose microparticles after treatment of the reporter cells either with colistin (Fig. 17B) or without colistin (Fig. 17A).
  • Fig. 18A-18C show micrographs of co-encapsulated A. baumannii reporter cells and antibody- secreting murine B cells (plasmablasts) after a one-hour incubation.
  • Fig. 18A is a brightfield image of the co-encapsulated agarose microparticles.
  • Fig. 18B is a fluorescent (FITC) image of the co-encapsulated agarose microparticles.
  • Fig. 18C is an overlay of the brightfield and fluorescent images.
  • Fig. 19A-19D show micrographs of a mixed population of A. baumannii cells expressing inducible mRuby2 or mClover3.
  • Fig. 19A is a brightfield image of the mixed population of A. baumannii cells.
  • Fig. 19B is a fluorescent (RFP) image of the mixed population of A. baumannii cells. Cells expressing mRuby2 can be seen.
  • Fig. 19C is a fluorescent (GFP) image of the mixed population of A. baumannii cells. Cells expressing mClover3 can be seen.
  • Fig. 19D is an overlay of the brightfield and fluorescent images.
  • Fig. 20A depicts mean fluorescent intensities of PAis_oo32-sfGFP, PAis_2889-sfGFP, or PAIS 2885-sfGFP reporter cells in the presence of various concentrations of PMBN.
  • Fig. 20B depicts mean fluorescent intensities of cells expressing P tac -sfGFP (an IPTG-inducible reporter) in the presence of various concentrations of IPTG.
  • Fig. 21A-F shows flow cytometry fluorescence histograms of A. baumannii reporter cells containing PAis_2889-sfGFP after culture without serum (Fig. 21A-C) or with serum (Fig. 21D-F) and in media only (Fig. 21 A, D) or in the presence of an IgG isotype control (Fig. 2 IB, E) or a monoclonal antibody that binds the OM (Fig. 21C, F).
  • the dashed circle highlights a population of fluorescent cells.
  • reporter polynucleotides containing a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule.
  • reporter vectors genetically engineered reporter microorganisms, and uses thereof.
  • the provided reporter polynucleotides, reporter vectors and engineered reporter microorganisms are useful for detecting outer membrane (OM) stress, including in connection with screening a plurality of candidate molecules or agents to identify molecules or agents that induce and/or modulate outer membrane stress and/or for determining or assessing if a microorganism is drug resistant.
  • reporter polynucleotides, reporter vectors and reporter microorganisms can be used in methods for the rapid identification or characterization of agents, including drug or other therapeutic candidates, to address a multitude of infectious disease threats.
  • the term "effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., an enhanced immune response to an antigen, a decrease in tumor growth or metastasis, or a reduction in tumor size.
  • An effective amount can be provided in one or more administrations.
  • Reference to "about” a value or parameter herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. In particular embodiments, reference to about refers to a range within 10% higher or lower than the value or parameter, while in other embodiments, it refers to a range within 5% or 20% higher or lower than the value or parameter. Reference to "about” a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se.
  • modulating means changing, and includes positive modulating, such as “increasing,” “enhancing,” “inducing” or “stimulating,” as well as negative modulating such as “decreasing,” “inhibiting” or “reducing,” typically in a statistically significant or a physiologically significant amount as compared to a control.
  • An “increased,” “stimulated” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no treatment as described herein or by a control treatment, including all integers in between.
  • a “decreased,” “inhibited” or “reduced” amount is typically a “statistically significant” amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount produced by no treatment as described herein or by a control treatment, including all integers in between.
  • p-value which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
  • antibodies and “immunoglobulin” include antibodies or
  • immunoglobulins of any isotype fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single- chain antibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non-antibody protein.
  • Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab')2, as well as bi-functional (i.e. bi- specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl.
  • vector refers to a nucleic acid construct, typically a circular DNA vector, that contains discrete elements that are used to introduce heterologous nucleic acid into cells for either expression of the nucleic acid or replication thereof.
  • the vectors typically remain episomal, but can be designed to effect stable integration of a gene or portion thereof into a chromosome of the genome.
  • vectors contain an origin of replication that allows many copies of the plasmid to be produced in a bacterial or eukaryotic cell without integration of the plasmid into the host cell DNA. Selection and use of such vectors are well known to those of skill in the art.
  • polynucleotide and “nucleic acid molecule” are used interchangeably to refer to a single-stranded and/or double- stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs or derivatives of either RNA or DNA.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the length of a polynucleotide molecule is given herein in terms of nucleotides
  • nucleic acid is also included in the term “nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives. Nucleic acids can encode gene products, such as, for example, polypeptides, regulatory RNAs, microRNAs, siRNAs and functional RNAs. Hence, nucleic acid molecule is meant to include all types and sizes of DNA molecules including cDNA, plasmids or vectors and DNA including modified nucleotides and nucleotide analogs.
  • PNA peptide nucleic acid
  • phosphorothioate DNA phosphorothioate DNA
  • Nucleic acids can encode gene products, such as, for example, polypeptides, regulatory RNAs, microRNAs, siRNAs and functional RNAs.
  • nucleic acid molecule is meant to include all types and sizes of DNA molecules including cDNA, plasmids or vectors and DNA including modified nucleot
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length.
  • Polypeptides may include amino acid residues including natural and/or non-natural amino acid residues.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • the term “gene” refers to any and all discrete coding regions of a host genome, or regions that code for a functional RNA only (e.g. , tRNA, rRNA, regulatory RNAs such as ribozymes etc.) as well as associated non-coding regions and optionally regulatory regions.
  • the term “gene” includes within its scope the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non- coding nucleotide sequences involved in the regulation of expression.
  • the gene can further contain control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals.
  • the gene sequences can be cDNA or genomic DNA or a fragment thereof. The gene can be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
  • an outer membrane (OM) stress-responsive gene is a gene whose expression or activity is modulated (e.g. increased or decreased) in a microorganism (e.g. an Acinetobacter spp. ) in response to a stress condition to the outer membrane of the
  • the stress condition modulates the biogenesis and/or integrity of the OM, including that disrupts the OM; destabilizes the OM, for example, by inhibiting the synthesis of lipopolysaccharide (LPS) or the peptidoglycan cell wall; or permeabilizes the OM.
  • LPS lipopolysaccharide
  • the expression or activity of the gene is increased or decreased by greater than or greater than about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold or more.
  • regulatory sequence or “regulatory region” as used in reference to a specific gene, refers to the coding or non-coding nucleic acid expression control sequences within that gene that are necessary or sufficient to provide for the regulated expression of the coding region of a gene.
  • the term encompasses promoter sequences, regulatory protein binding sites, upstream activator sequences and the like.
  • Specific nucleotides within a regulatory region may serve multiple functions.
  • a specific nucleotide may be part of a promoter and participate in the binding of a transcriptional activator protein.
  • ORF open reading frame
  • operably linked is meant a functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • Percent “identical” or “identity” in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences that are the same or have a specified percentage of nucleic acid residues or amino acid residues, respectively, that are the same, when compared and aligned for maximum similarity, as determined using a sequence comparison algorithm or by visual inspection.
  • Percent sequence identity or “% identity” or “% sequence identity or “% amino acid sequence identity” of a subject amino acid sequence to a reference amino acid sequence means that the subject amino acid sequence is identical (i.e., on an amino acid-by-amino acid basis) by a specified percentage to the reference amino acid sequence over a comparison length when the sequences are optimally aligned.
  • 80% amino acid sequence identity or 80% identity with respect to two amino acid sequences means that 80% of the amino acid residues in two optimally aligned amino acid sequences are identical.
  • reporter molecule refers to a molecule that is directly or indirectly detectable or whose presence is otherwise capable of being measured.
  • reporter molecules include proteins that can emit a detectable signal such as a fluorescence signal, and enzymes that can catalyze a detectable reaction or catalyze formation of a detectable product.
  • Reporter molecules also can include detectable nucleic acids.
  • a reporter molecule is a polypeptide which can be detected when it is expressed in the cell.
  • expression of the detectable reporter may lead to the production of a signal, for example a fluorescent, bio luminescent or colorimetric signal, which can be detected using routine techniques.
  • the signal may be produced directly from the reporter, after expression, or indirectly through a secondary molecule, such as a labelled antibody.
  • reporter cell and “reporter microorganism” are used interchangeably to refer to an engineered microorganism into which an exogenous or heterologous polynucleotide, such as a cDNA or gene, encoding a reporter molecule has been introduced. Therefore, reporter cells are distinguishable from naturally occurring microorganisms which do not contain a recombinantly introduced exogenous polynucleotide. Reporter cells are thus cells having a gene or genes introduced through human intervention and that express an exogenous reporter molecule.
  • heterologous with reference to a polynucleotide or gene refers to a nucleotide sequence that is not native to the organism or a gene contained therein or not normally produced in vivo by an organism, such as bacteria, from which it is expressed.
  • kits are packaged combinations that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof. Kits optionally include instructions for use.
  • a reporter polynucleotide comprising a sequence comprising a regulatory region of an OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a sequence encoding a reporter molecule, wherein the OM stress-responsive gene is modulated in response to a stress to the outer membrane (hereinafter referred to as "OM-stress) of he Acinetobacter spp.
  • the sequence comprising the regulatory region is directly or indirectly linked to the sequence encoding the reporter molecule. In certain embodiments, the sequence comprising the regulatory region is directly linked to the 5' end of the open reading frame (ORF) of the reporter molecule. In certain embodiments, the sequence comprising the regulatory region is indirectly linked to the 5' end of the ORF of the reporter molecule.
  • ORF open reading frame
  • the provided reporter polynucleotides and reporter vectors can be produced or generated by standard DNA techniques. Genes or nucleotide sequences of interest can be obtained by amplification of nucleic acids, such as by polymerase chain reaction methods; generated de novo by synthetic construction (e.g. overlapping PCR and/or oligonucleotide hybridization); or desired sequences can be removed from already existing sources such as commercially available vectors. Synthesized or harvested sequences can be further modified by site directed mutagenesis, PCR, or other methods known to those of skill in the art.
  • Such modifications include, but are not limited to optimize codon usage for a host microorganism, modification of a coding sequence to reduce or enhance a desired activity, and addition of restriction sequences for cloning purposes.
  • codon optimization can be employed to optimize expression in a host microorganism, such as by modification of certain codons to reflect particular codon usage of the reporter polynucleotide containing the reporter molecule to that which is found more frequently in particular species in which it is being expressed.
  • Nucleic acids molecules can be synthesized by methods known to one of skill in the art using synthetic gene synthesis.
  • individual oligonucleotides corresponding to fragments of a polynucleotide sequence vector are synthesized by standard automated methods and mixed together in an annealing or hybridization reaction.
  • synthetic genes are assembled from a large number of short partially overlapping DNA oligonucleotides or fragments, generally each independently about or about at least 20, 30, 40, 50, 60, 70, 80, 90 100, 200, 300, 400, 500 or more nucleotides in length.
  • DNA fragments with overlapping ends can be generated by restriction digest, by PCR or by gene synthesis methods.
  • oligonucleotides can be commercially obtained, such as from Integrated DNA Technologies (Coralville, IA).
  • overlapping segments are allowed to anneal and then are assembled into longer double- stranded DNA, for example, by ligation and/or polymerase extension reactions, either alone or in combination.
  • Single nucleotide "nicks" in the duplex DNA are sealed using ligation, for example with bacteriophage T4 DNA ligase.
  • Such strategies are variously referred to as "assembly PCR,” “splicing by overlap extension,” “polymerase chain assembly” and others.
  • Gibson Assembly methods can be employed.
  • reporter polynucleotide A description of the regulatory region and reporter molecule of the provided reporter polynucleotides is provided below. It is within the level of one of skill in the art to generate and design a reporter polynucleotide, and reporter vectors containing such reporter polynucleotides, as described herein. Exemplary reporter polynucleotides and reporter vectors are provided.
  • the provided reporter polynucleotides contain a regulatory region of an Acinetobacter spp. gene that is responsive to and/or whose expression is modulated in response to stress to the outer membrane (hereinafter called "OM stress-responsive gene").
  • the stress to the outer membrane is caused by altering, mutating, depleting, or removing a component from the OM.
  • the stress to the OM can be caused by genetically altering the Acinetobacter spp. to reduce expression of an OM lipid, polysaccharide, or protein.
  • the OM stress responsive gene is responsive to stress caused by altering, mutating, depleting, or removing BamA, LptD, FhuA, PldA, OmpT, PagP, OstA, OmpA, OmpF, Omp200, Ompl21, Omp71, Ompl l7, OprF, PhoE, OmpC, OmpF, NmpC; PorA, PorB, OprA, OprM, OprN, OprJ, OprB, NspA, PagL, OmpW, OpcA, NalP, NupA, OmpG, FadL, PhoE, LamB, FhaC, SucY, FepA, FecA, BtuB, TolC, Porin P, Porin D, SmeC, SmeF, MepC, SrpC, TtgC, TtgF, AdeC, AdeK, or MexA.
  • the stress to the OM of the Acinetobacter bacterium is or is caused by depletion of BamA.
  • the genetic alteration e.g. deletion
  • an outer membrane gene can be deleted from a microorganism and, instead, can be exogenously expressed under the control of an inducer.
  • the outer membrane gene is expressed only in the presence of the inducer but is not expressed, such as is not expressed on the cell surface, in the absence of the inducer.
  • the inducer is arabinose and the genetic alteration is induced by the addition of an inducer. In some embodiments, the inducer is arabinose.
  • the concentration of the arabinose is about 0.0004 -0.2%, such as about 0.0006%-0.02%, 0.0008- 0.02%, or about 0.001%-0.02%.
  • Acinetobacter spp. is AABA046, which carries a deletion of the bamA gene by replacement with a selective marker conferring resistance to kanamycin and contains an exogenous polynucleotide encoding bamA fused to the araBAD promoter from E. coli which allows regulation of transcription by addition of the inducing sugar arabinose.
  • AABA041 which carries a deletion of the bamA gene by replacement with a selective marker conferring resistance to carbenicillin and contains an exogenous polynucleotide encoding bamA fused to the araBAD promoter from E. coli which allows regulation of transcription by addition of the inducing sugar arabinose.
  • the stress to the outer membrane can be caused by treatment with a molecule or combination of molecules that causes temporary or permanent damage to the OM.
  • the stress to the OM is or is caused by treatment with polymyxin B nonapeptide (PMBN), vancomycin, ACHN-975, colistin,
  • the stress to the OM is or is caused by treatment with PMBN.
  • the concentration of the molecule is a sub-lethal concentration. In certain embodiments the concentration of the molecule is below the MIC of the molecule.
  • the concentration of the molecule is about 0.1 ⁇ g/mL - 1000 ⁇ g/mL, such as about 1 ⁇ g/mL - 750 ⁇ g/mL, 10 ⁇ g/mL - 500 ⁇ g/mL, 25 ⁇ g/mL - 500 ⁇ g/mL, or 25 ⁇ g/mL - 250 ⁇ g/mL. In certain embodiments, the concentration of the molecule is about 0.1 mM- 500 mM, such as about 0.1 mM - 500 mM, 1 mM - 50 mM, or 1 mM - 10 mM.
  • the concentration of the molecule is about 0.1% - 5%, such as about 0.5% - 2.5% or 0.75% - 2%.
  • the OM stress-responsive gene is modulated in response to more than 1, 2, 3, 4, 5, or 6 stresses. In certain embodiments, the OM stress-responsive gene is modulated in response to depletion of BamA and treatment with PMBN.
  • the OM stress-responsive gene is identified using a method that includes a) inducing OM stress in Acinetobacter spp. by exposing or subjecting an
  • Acinetobacter spp. to one or more stress conditions, such as any described above and b) identifying one or more genes that is modulated in response to the OM stress (e.g. using RNA- Seq or other method).
  • A. bacterium genes such as Acinetobacter spp. genes in the presence of one or more stress conditions, such as described above.
  • expression levels can be assessed or determined using any method known to a skilled artisan, such as by using quantitative PCR, microarrays, RNA-Seq, northern blotting, or SAGE.
  • genes whose sequences, or portions or fragments of sequences, have been identified as having been modulated can be identified using a reference sequence of the A. bacterium genome.
  • Exemplary A. bacterium genome sequences are known and readily available online on the world wide web at tigr.org, kegg.jp, or ncbi.nlm.nih.gov/genbank.
  • baumannii species or isolates include GENBANKTM Accession Nos. CP00521.1 or
  • the Acinetobacter spp. is an Acinetobacter apis, Acinetobacter baumannii, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter citri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus,
  • Acinetobacter harbinensis Acinetobacter indicus, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis,
  • Acinetobacter qingfengensis Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
  • the Acinetobacter spp. is Acinetobacter baumannii.
  • the Acinetobacter spp. is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA- 1791, ATCC BAA- 1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799, ATCC B
  • the Acinetobacter spp. is a multi-drug resistant bacterium.
  • the OM stress-responsive gene is one that is upregulated or downregulated in response to the stress to the outer membrane of Acinetobacter spp.
  • the OM stress-responsive gene is downregulated in response to the stress.
  • the OM stress-responsive gene is downregulated by at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold or more in response to the stress compared to the expression level of the gene in the absence of the stress condition.
  • the OM stress-responsive gene such as an OM-stress responsive gene that is downregulated in response to OM stress, is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038, A1S_0067, A1S_0070, A1S_0071, A1S_0073, A1S_0076, A1SJ3077, A1SJ3079, A1S_0087, A1S_0090, A1S_0091, A1S_0095, A1S_0096, A1S_0097, A1S_0098, A1S_0099, A1S_0103, A1S_0104, A1S_0105, A1S_0106, A1S_0107, A1S_0108, A1S_0109, A1S_0121, A1S_0128, A1S_0129, A1S_0141
  • the OM stress-responsive gene such as an OM-stress responsive gene that is downregulated in response to OM stress, is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_
  • such an OM stress- responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of the gene described herein.
  • the OM stress- responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
  • the OM stress-responsive gene is one that is upregulated in response to the stress to the outer membrane of Acinetobacter spp. In some embodiments, the OM stress-responsive gene is upregulated in response to the stress by at least or at least about 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold or more in response to the stress compared to the expression level of the gene in the absence of the stress condition.
  • the OM stress-responsive gene such as an OM-stress responsive gene that is upregulated in response to OM stress, is A1S_0012, A1S_0023,
  • A1S_ _3 16 A1S_ _3725, A1S_ _3726, A1S_ _3727, A1S_ _3728, A1S_ _3736, A1S_ _3738, A1S_ _3739,
  • the OM stress-responsive gene such as an OM-stress responsive gene that is upregulated in response to OM stress, is A1S_0032, A1S_0033, A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791.
  • A1S_0032, A1S_0033 A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791.
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene is A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or A1S_3791.
  • the OM stress- responsive gene is A1S_0032, A1S_2885, or A1S_2889.
  • the OM stress-responsive gene is A1S_0113 or A1S_1751. In certain embodiments, the OM stress- responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of the genes described herein.
  • the OM stress- responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1SJ3032, A1S_0033, A1S_0113, A1S_0189, A1SJ3516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791.
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, AIS_3791. In some embodiments, the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0032, A1S_2885, or A1S_2889.
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0113 or
  • the OM stress-responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
  • the provided reporter polynucleotides contain a regulatory region or portion thereof of an OM stress-responsive gene, such as any described above, operatively linked to a reporter molecule.
  • the regulatory region or portion thereof comprises a sequence upstream or 5' of the open reading frame (ORF) of the OM stress- responsive gene, such as any described above.
  • ORF open reading frame
  • the sequence of the regulatory region or portion thereof is sufficient to provide for regulated expression of the coding region of the reporter molecule operatively linked thereto, such as upon induction or in the presence of an OM stress condition.
  • the regulatory region is or comprises a native promoter of the OM-responsive gene.
  • One of skill in the art can identify a regulatory region through standard techniques. For example, one could identify a regulatory region by fusing a putative regulatory region or sequence upstream of an OM stress-responsive gene to a sequence encoding a reporter molecule, introducing the construct using standard techniques into an Acinetobacter spp., inducing the putative regulatory region or upstream sequence by causing OM stress, and determining if the reporter molecule is induced. Putative regulatory regions can often be shortened or lengthened without influencing activity or inducibility. One of skill in the art can systematically test the effect of removing nucleotides from putative regulatory region sequence to determine what putative regulatory elements are critical or required for the OM stress-responsive behavior.
  • the regulatory region comprises a sequence to further promote translation of the encoded reporter molecule.
  • the sequence to further promote translation of the encoded reporter molecule is directly linked to the 5' end of the open reading frame (ORF) of the reporter molecule.
  • the sequence to further promote translation of the encoded reporter molecule is indirectly linked to the 5' end of the ORF of the reporter molecule.
  • the sequence further promoting translation is or comprises a bacterial ribosome binding site.
  • the ribosome binding site is a Shine-Dalgarno (SD) sequence, which is a sequence usually found in natural prokaryotic genes 5' of the ATG translation start codon that acts as a binding site on the mRNA molecule for the ribosome, thereby facilitating translation of the mRNA.
  • the regulatory region is one that contains a SD sequence in which, when operatively linked to the sequence encoding the reporter molecule, such SD sequence is about 2 to about 15 nucleotides 5' of the ATG of the sequence encoding the reporter molecule, such as about 5 to about 7 nucleotides upstream of the ATG.
  • the SD sequence contained in the regulatory region can be any variant of the consensus sequence AGGAGG that retains the characteristic of facilitating translation of the sequence encoding the reporter molecule.
  • the SD is from about 3 nucleotides to about 9 nucleotides in length.
  • the Shine- Dalgarno sequence is native to the regulatory region of the OM stress-responsive gene.
  • the Shine-Dalgarno sequence is synthetic and/or heterologous to the regulatory region of the OM stress-responsive gene.
  • the Shine- Dalgarno sequence comprises a sequence with at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14.
  • the regulatory region such as the promoter, is modified in its sequence compared to the native sequence of an OM stress-responsive gene, such as any described above.
  • the regulatory region is modified or altered in its sequence, for example to alter the configuration of regulatory binding regions, such as for transcription factors and/or can be made to have specific regions deleted.
  • Such mutational and/or deletional analysis can be rationally or empirically performed and the resulting constructs tested by one of skill in the art.
  • the various constructs containing a modified regulatory region can be tested in a construct in which expression of an operatively linked reporter molecule (e.g. GFP) can be used to determine the activity of the modified regulatory region under different conditions, such as one or more different OM stress conditions. In some cases, this approach can be used to identify the smallest region capable of conferring or regulating expression of an operatively linked reporter molecule.
  • an operatively linked reporter molecule e.g. GFP
  • the regulatory region includes a contiguous sequence of nucleotides within or within about 1000, 750, 500, 450, 400, 350, 300, or 250 base pairs upstream or 5' of the open reading frame (ORF) of the OM stress-responsive gene. In certain embodiments, the regulatory region comprises a contiguous sequence of nucleotides within about 500 base pairs upstream or 5' of the ORF of the OM stress-responsive gene. In certain embodiments, the regulatory region comprises a contiguous sequence of nucleotides within about 250 base pairs upstream or 5' of the ORF of the OM stress-responsive gene.
  • the contiguous sequence of nucleotides comprises at least or at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400 or more base pairs including any ranges between these values.
  • the regulatory region comprises a sequence with at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 1-13.
  • the regulatory region is or comprises the sequence of nucleotides set forth in any of SEQ ID NOs: 1-13.
  • the regulatory region does not only contain the promoter sequence of the A1S_2889 OM-responsive stress gene. In some embodiments, the regulatory region does not contain a regulatory region of the A1S_2889 OM-responsive stress gene. In some embodiments, the regulatory region does not include the sequence set forth in SEQ ID NO: 11.
  • the regulatory region of an OM stress-responsive gene is operatively linked to a sequence encoding a reporter molecule.
  • Reporter molecules include molecules that express or that are capable of expressing or producing a detectable signal that is assayable or can be detected.
  • the extent of detection of the assayable product indicates the presence, absence or quantity of the reporter molecule which, in turn, indicates the presence, absence, or degree of an OM-stress on a reporter microorganism bearing the reporter molecule.
  • the reporter molecule is any that is capable of producing a detectable signal when expressed in a reporter microorganism, such as Acinetobacter spp. (e.g. Acinetobacter baumannii).
  • Such signals including a fluorescent, bio luminescent or colorimetric signal, can be detected using routine techniques.
  • the reporter molecule is a fluorescent protein, a luminescent protein, a chromoprotein, or an enzyme.
  • the reporter molecule is a fluorescent protein.
  • the fluorescent protein is a red, green, blue, or yellow fluorescent protein.
  • the fluorescent protein is Sirius, SBFP2, Azurite, mAzurite, EBFP2, moxBFP, mKalamal, mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean, mCerulean3, moxCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl, MiCy (Midoriishi-Cyan), iLOV, AcGFPl, sfGFP, moxGFP, mEmerald, EGFP, mEGFP, AzamiGreen, cfSGFP2, ZsGreen, SGFP2, Clover, mClover2, mClover3, EYFP, Topaz, mTopaz, mVenus, mox Venus,
  • the fluorescent protein is sfGFP, mClover3, mRuby2, Ypet, mCerulean, or mTagBFP2. In some embodiments, the fluorescent protein is sfGFP, mClover3, or mRuby2. In some embodiments, the fluorescent protein is sfGFP.
  • Suitable fluorescent reporters are available commercially (Clontech Labs Inc. USA, Evrogen Moscow, RU; MBL Int MA USA; Addgene Inc. MA USA).
  • the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 15, 33, or 40.
  • the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 15.
  • the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 33.
  • the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 40.
  • the reporter molecule is a luminescent protein.
  • the luminescent protein is North American firefly luciferase, Genji-botaru luciferase, Italian firefly luciferase, Heike luciferase, East European firefly luciferase,
  • the reporter molecule is a chromoprotein.
  • the chromoprotein comprises a heme, riboflavin, or retinal.
  • the chromoprotein is a hemoglobin, hemocyanin, chlorophyll, cytochrome, carotenoid, flavoprotein, or rhodopsin.
  • the reporter molecule is an enzyme.
  • the enzyme is one whose expression in a cell can be detected with a live cell assay (i.e. an assay which does not require cell fixation or lysis).
  • the enzyme is chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase, alkaline phosphatase, ⁇ - glucuronidase, ⁇ -lactamase, neomycin phosphotransferase, or a modified version thereof.
  • the reporter molecule is a gene necessary for the production of essential metabolites such as tryptophan, leucine, uracil, histidine, or methionine.
  • the provided reporter polynucleotides are comprised within a vector (hereinafter called a "reporter vector").
  • a vector hereinafter called a "reporter vector”
  • Suitable vectors can be used for any of the embodiments described herein.
  • a suitable vector can be used that is capable of replicating in a microorganism to which it is introduced.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences for driving transcription of the coding nucleotide sequence, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate, for expression in a bacterial cell as described herein.
  • the vector comprises a reporter polynucleotide described herein. In some embodiments, the vector comprises a first reporter polynucleotide described herein and a second reporter polynucleotide described herein. In certain embodiments, the vector comprises (i) a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an Acinetobacter species (spp).
  • spp Acinetobacter species
  • a second reporter polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a second sequence encoding a second reporter molecule, wherein the first and second OM stress-responsive genes are modulated in response to a stress to the outer membrane (OM-stress) of the Acinetobacter spp.
  • the first and second reporter polynucleotides share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second reporter polynucleotides are the same. In certain embodiments, the first and second reporter polynucleotides are different.
  • the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress- responsive genes.
  • the first OM stress-responsive gene and the second OM stress-responsive gene are different.
  • operatively linking a reporter molecule to different regulatory regions that are both responsive to OM stress can minimize false positives.
  • the different regulatory regions may be responsive to different types of stress, such that the presence of a detectable signal induced from a microorganism carrying both reporter polynucleotides, or a first microorganism comprising a first reporter polynucleotide and a second microorganism comprising a second reporter polynucleotide, can assess different parameters or features of outer membrane stress.
  • an agent is identified as causing or inducing OM stress only if a change in a detectable signal by or from both reporter molecules is observed.
  • the different regulatory regions are each operatively linked to the same reporter molecule. In some embodiments, the different regulatory regions are each operatively linked to different reporter molecules.
  • the first and second OM stress-responsive genes are the same or are functional variants. In certain embodiments, the first and second OM stress- responsive genes share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second OM stress-responsive genes are the same. In certain embodiments, the first and second OM stress-responsive genes are different.
  • the first and second sequences comprising the regulatory regions are the same or are functional variants. In certain embodiments, the first and second sequences comprising the regulatory regions share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences comprising the regulatory regions are the same. In certain embodiments, the first and second sequences comprising the regulatory regions are different.
  • the reporter molecule encoded by the first reporter polynucleotide, i.e. first reporter molecule, and the reporter molecule encoded by the second reporter polynucleotide, i.e. second reporter molecule are different.
  • the first reporter molecule and the second reporter molecule are different.
  • the first reporter molecule and the second reporter molecule do not exhibit an overlapping emission and absorption spectra. Reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected.
  • the use of several reporters can provide a multiplexed system to simultaneously measure or assess the presence or absence of a microorganism to one or more stress condition or potential stress condition and/or ensure that a response to a condition, e.g. caused by a physical condition or by an agent, is real or not likely to be a false positive.
  • the first and second reporter molecule is independently sfGFP and mRuby2.
  • the first and second sequences encoding the first and second reporter molecules are the same or are functional variants. In certain embodiments, the first and second sequences encoding the reporter molecules share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same. In certain
  • the first and second sequences encoding the first and second reporter molecules are different. In certain embodiments, the first and second reporter molecules are the same.
  • the first reporter vector includes a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a first sequence encoding a first reporter molecule.
  • the second reporter vector includes a second reporter polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp).
  • the first and second reporter vector can be introduced into the same microorganism. In some embodiments, the first and second reporter vector can be introduced into different microorganisms.
  • reporter vectors are designed to contain certain components which optimize gene expression for certain microorganisms.
  • optimization components include, but are not limited to, origins of replication, promoters, and enhancers.
  • the vector contains an origin of replication in the IncQ incompatibility group that is proficient for replication in a broad host range, including in Acinetobacter spp.
  • the plasmid is a low-copy plasmid.
  • the vector is a high-copy plasmid.
  • the reporter vector contains a selective marker.
  • selectable markers include, but are not limited to, antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/or nucleic acids that confer a metabolic advantage, such as a nutritional advantage on the reporter microorganism.
  • the provided reporter vector is capable of being expressed in any microorganism or progeny thereof that can be used to heterologously express genes.
  • the reporter vector is capable of being expressed in any microorganisms described herein.
  • the reporter vector is capable of being expressed in a Gram- negative bacterium. In certain embodiments, the reporter vector is capable of being expressed in a multi-drug resistant bacterium. In certain embodiments, the vector is capable of being expressed in Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia. In particular embodiments, the reporter vector is capable of being expressed in Acinetobacter. In certain embodiments, the reporter vector is capable of being expressed in Acinetobacter apis,
  • Acinetobacter baumannii Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii,
  • Acinetobacter brisouii Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter organizerri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii
  • Acinetobacter soli Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
  • the reporter vector is capable of being expressed in
  • the reporter vector is capable of being expressed in ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA-1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA- 1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA- 1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA
  • the reporter polynucleotide as described herein is comprised in a backbone vector.
  • a nucleotide sequence comprising the reporter polynucleotide is inserted into or replaces a portion of the nucleotide sequence of the backbone vector.
  • Backbone vectors that can be expressed in specific microorganism are known in the art, for example pWH1266, or pET-RA (GenBank: M36473.1 and HM219006.1) are known to be capable of expression in Acinetobacter.
  • the backbone vector is pACH106 (SEQ ID NO: 29), pWH1266, or pET-RA.
  • the backbone vector has sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:29.
  • the backbone vector comprises the sequence of nucleotides set forth in SEQ ID NO: 29 and a nucleotide sequence comprising the reporter polynucleotide replaces nucleotides 5,715-7,395 of the backbone vector.
  • the provided reporter vector comprises the sequence of nucleotides set forth in SEQ ID NO:29 in which nucleotides 5,715-7,395 thereof are replaced with a nucleotide sequence comprising a reporter polynucleotide comprising any of SEQ ID NOS: 1- 13, or a reporter polynucleotide with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS: l-13; or a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of such reporter vectors.
  • the provided reporter vector comprises the sequence of nucleotides set forth in SEQ ID NO:29 in which nucleotides 5,715-7,395 thereof are replaced with a nucleotide sequence comprising a reporter polynucleotide comprising any of SEQ ID NOS: 16-28 or 34-36, or a reporter polynucleotide with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS: 16-28 or 34-36; or a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of such reporter vectors.
  • the provided reporter vector comprises the sequence of nucleotides set forth in SEQ ID NO:29 in which nucleotides 5,715-7,395 thereof are replaced with a nucleotide sequence comprising any of SEQ ID NOS: 1-13, or a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of such reporter vectors.
  • the reporter vector is or comprises a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 30-32 or 37-39.
  • the reporter vector is or comprises the sequences set forth in any of SEQ ID NOS: 30-32 or 37-39.
  • the reporter vector is or comprises a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 30-32.
  • the reporter vector is or comprises the sequences set forth in any of SEQ ID NOS: 30-32. In some embodiments, the reporter vector is or comprises a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 37-39. In some embodiments, the reporter vector is or comprises the sequences set forth in any of SEQ ID NOS: 37-39.
  • reporter microorganisms comprising any one or more reporter polynucleotide described herein or any one or more reporter vector described herein.
  • the reporter microorganism comprises 1, 2, 3, 4 or more reporter polynucleotides as described herein and/or 1, 2, 3, 4 or more reporter vectors as described herein.
  • the reporter microorganism is a microorganism that is modified by introduction of one or more heterologous or exogenous reporter polynucleotides or reporter vectors as described into a host microorganism.
  • the provided reporter microorganisms include a host microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein.
  • the host microorganism comprises a first reporter polynucleotide and a second reporter polynucleotide.
  • the reporter polynucleotide is integrated into the genome of the host microorganism.
  • one or more reporter polynucleotides present in the provided reporter microorganisms includes as least two different reporter polynucleotides, i.e. a first reporter polynucleotide and a second reporter polynucleotide, as described.
  • the first and second reporter polynucleotides are comprised in or are part of the same reporter vector.
  • the first and second reporter polynucleotides are comprised in or are part of different reporter vectors.
  • a reporter microorganism as provided herein can include two or more reporter vectors in which, for example, at least one reporter vector is a first reporter vector comprising the first reporter polynucleotide and at least one reporter vector is a second reporter vector comprising the second reporter polynucleotide.
  • the reporter microorganism comprises more than one reporter polynucleotide described herein. In some embodiments, the reporter microorganism comprises a first reporter polynucleotide described herein and a second reporter polynucleotide described herein. In some embodiments, the reporter microorganism comprises more than one reporter vector described herein. In some embodiments, the reporter microorganism comprises a first reporter vector comprising a first reporter polynucleotide described herein and a second vector comprising a second reporter polynucleotide described herein. In certain embodiments, multiple copies of the same polynucleotide increase the signal from the reporter microorganism.
  • the reporter microorganism comprises a first reporter polynucleotide described herein and a second reporter polynucleotide described herein.
  • the reporter microorganism comprises (i) a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a first sequence encoding a first reporter molecule, and (ii) a second reporter polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a second sequence encoding a second reporter molecule, wherein the first and second OM stress-responsive genes are modulated in response to a stress to the outer membrane (OM-stress) of the Acinetobacter spp.
  • OM-stress outer membrane
  • a reporter vector comprises the first reporter polynucleotide and the second reporter polynucleotide.
  • a first reporter vector comprises a first reporter polynucleotide and a second reporter vector comprises the second reporter polynucleotide.
  • the first and second vectors are the same or are functional variants. In certain embodiments, multiple copies of the same vector increases signal from the reporter molecule. In certain embodiments, the first and second vectors share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second vectors are the same. In certain embodiments, the first and second vectors are different.
  • the first and second reporter polynucleotides are the same or functional variants. In certain embodiments, the first and second reporter polynucleotides share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second reporter polynucleotides are the same. In certain embodiments,
  • the first and second reporter polynucleotides are different. In certain embodiments, the first and second reporter polynucleotides are different.
  • different reporter polynucleotides allows for assaying different genes or strains in the same assay.
  • the first and second OM stress-responsive genes are the same or are functional variants.
  • the first and second OM stress-responsive genes share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the first and second OM stress-responsive genes are the same.
  • the first and second OM stress-responsive genes are different.
  • different OM stress-responsive genes allow for the identification of broader spectrum or more potent agents.
  • different OM stress- responsive genes allow for identification of more selective agents.
  • first OM stress-responsive gene was responsive to PMBN but not BamA depletion and the second OM stress-responsive gene was responsive to BamA depletion but not PMBN, different agents may be identified as including the first and/or second OM stress-responsive gene.
  • the reporter microorganism includes at least two different reporter polynucleotides as described in which the regulatory region of a first reporter polynucleotide and the regulatory region of a second reporter polynucleotide are from different OM stress-responsive genes.
  • the first OM stress-responsive gene and the second OM stress-responsive gene are different.
  • such reporter microorganisms are capable of modulating a detectable signal in response to at least two different stress conditions, e.g. a physical condition or by an agent.
  • the ability of a single reporter microorganism to produce or modulate a detectable signal in response to one or more stress condition or potential stress condition can provide a multiplexed system to simultaneously measure or assess the susceptibility or resistance of a microorganism to one or more stress condition or potential stress condition and/or ensure that a response to a condition, e.g. caused by a physical condition or by an agent, is real or is not likely to be a false positive.
  • the different regulatory regions are each operatively linked to the same reporter molecule, such that the reporter microorganism is capable of producing a single detectable signal.
  • the different regulatory regions are each operatively linked to different reporter molecules, such that the reporter microorganism is capable of producing different detectable signals, e.g. induced by the different regulatory regions.
  • the first and second sequences comprising the regulatory regions are the same or functional variants. In certain embodiments, the first and second sequences comprising the regulatory regions share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences comprising the regulatory regions are the same.
  • the reporter microorganism is capable of producing at least two different detectable signals.
  • the reporter microorganism contains at least two reporter polynucleotides as described, in which, for example, a regulatory region of the first OM-responsive stress gene is operatively linked to a first reporter molecule and the regulatory region of the second OM-responsive gene is operatively linked to a second reporter molecule that is different from the first reporter molecule.
  • the first reporter molecule and the second reporter molecule do not exhibit an overlapping emission and absorption spectra. Reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected.
  • the reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected.
  • the reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected.
  • microorganism is capable of producing a detectable signal from a first and second reporter molecule that is independently sfGFP and mRuby2.
  • the first and second sequences encoding the first and second reporter molecules are the same or functional variants. In certain embodiments, the first and second sequences encoding the reporter molecules share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same. In certain embodiments,
  • the first and second sequences encoding the first and second reporter molecules are different. In certain embodiments, the first and second reporter molecules are the same.
  • each of the plurality of reporter microorganisms includes any one or more reporter polynucleotides as described or any one or more reporter vectors as described.
  • each of the plurality of reporter microorganisms includes a reporter polynucleotide that includes the same regulatory region of an OM-responsive gene.
  • each of the plurality of microorganisms includes the same reporter
  • each of the plurality of microorganisms includes a reporter polynucleotide containing the same regulatory region of the same OM responsive gene operatively linked to the sequence encoding a reporter molecule.
  • each of the plurality of microorganisms includes a different reporter polynucleotide.
  • each of the plurality of reporter microorganisms is capable of producing a different detectable signal, whereby each contains a reporter polynucleotide that includes the same regulatory region of an OM-responsive gene, but that is operatively linked to a sequence encoding different reporter molecules.
  • the different reporter molecules do not exhibit overlapping emission and absorption spectra.
  • Reporter molecules capable of producing different, such as non-overlapping signals can be separately or distinguishably detected.
  • a plurality of microorganisms may be useful for comparing the affect or impact of various outer membrane stress conditions capable of engaging a common regulatory region in different microorganisms.
  • the plurality of microorganisms include microorganisms across different strains, isolates or subtypes, such as any as described.
  • each of the plurality of reporter microorganisms includes a different reporter polynucleotide that includes a regulatory region of a different OM-responsive gene.
  • the microorganisms are of the same strain, isolate or subtype of a microorganism, e.g. bacteria.
  • the plurality of microorganisms represents different strains, isolate or subtype of a microorganism, e.g. bacteria.
  • such a plurality of reporter microorganisms can be screened together in response to the same stress condition, e.g. physical or via an agent.
  • the plurality of microorganisms can be co- encapsulated in a gel microdroplet as described.
  • a change in a detectable signal in or from each of the plurality of microorganisms in response to the same stress condition can indicate that the microorganisms are universally responsive or susceptible to the outer membrane stress condition and/or that there is one or more feature (e.g. conserved targeting of an outer membrane protein) that is similar or the same to each of the plurality of microorganisms rendering each similarly susceptible to the outer membrane stress.
  • microorganisms in response to the same stress condition can indicate that susceptibility to the stress condition is specific to one or more microorganism, such as one or more strain, isolate or subtype.
  • the vectors may be used to transfect and transform a host microorganism. Any procedure able to introduce the genetic material into the host microorganism for modulation (e.g. increase or decrease) of expression of the reporter molecule in the presence of OM-stress can be employed.
  • such methods include calcium phosphate transfection, electroporation, retroviral mediated transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasmid vectors, viral vectors and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host
  • Exemplary host microorganisms, and thus the resulting reporter microorganisms include yeast, bacteria, and archaea. Bacteria cells, including Gram positive or Gram negative bacteria can be used to express any of the reporter polynucleotides described above.
  • the host or reporter microorganism is a multi-drug resistant bacterium.
  • the host or reporter microorganism is a Gram-negative bacterium. Non-limiting examples include strains of Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia,
  • reporter polynucleotides or reporter vectors described above can be expressed in Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae,
  • Pseudomonas aeruginosa Salmonella typhimurium, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholera, or Yersinia pestis.
  • the host microorganism, and hence the resulting reporter microorganism is Acinetobacter.
  • the host or reporter microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter organizerri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii
  • the host or reporter microorganism is Acinetobacter baumannii.
  • the host microorganism is or is derived from ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA-1605, ATCC BAA-1709, ATCC BAA- 1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA- 1794, ATCC BAA- 1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799, ATCC B
  • the host microorganism is ATCC 17978, AABA041, AABA046, or Ab307-0294.
  • the reporter microorganism is derived from ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA- 1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA- 1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799
  • the reporter microorganism is derived from ATCC 17978, AABA041, AABA046, or Ab307-0294.
  • the transformed reporter strains may be selected utilizing a selectable marker present on the vector (e.g. a drug resistance gene such as a Kanamycin resistance gene) and cultured by a variety of means known to those of skill in the art (see, e.g. Good et al. Clin. Chest Med. 10: 315-322 (1984), Heifets Ann. Rev. Respir. Dis. , 137: 1217-1222 (1988), and Sommers et al. in Color Atlas and Textbook of Diagnostic Microbiology, Third .Edition, J.B. Lippincott Co., Philadelphia, PA (1988)).
  • a selectable marker present on the vector e.g. a drug resistance gene such as a Kanamycin resistance gene
  • microdroplets comprising a reporter microorganism or a plurality of reporter microorganisms as described herein.
  • the microdroplet comprises (i) an agent, such as a candidate agent, which, in some cases, can be an agent- producing cell, e.g. an antibody-producing cell and (ii) a reporter microorganism or a plurality of reporter microorganisms as provided herein.
  • the microdroplet may comprise multiple copies of reporter microorganisms, such as multiple copies of the reporter microorganism or multiple copies and/or agent or agent-producing cell.
  • the microdroplets may contain a single agent or agent-producing cell (e.g. antibody-producing cell) and multiple reporter
  • the average number of reporter microorganisms per microdroplet can be between about 5 and about 500, such as about 10 and about 250, about 50 and about 200, about 50 and about 150, about 50 and about 100, or about 80 and about 120, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200.
  • the number of reporter microorganisms per microdroplet may be lower on average for microorganisms that are larger in cell size, e.g., a fungus or a parasite.
  • the agent can be any agent as described herein.
  • the agent is a candidate antibacterial agent.
  • the agent is a small molecule, a peptide, or a protein, such as an antibody or antigen-binding fragment thereof.
  • the agent is a small molecule antibiotic or peptide antibiotic.
  • the peptide is a fragment of a larger protein.
  • the peptide comprises about 50 or fewer amino acids, such as about 45, 40, 35, 30, 25, 20, or fewer amino acids.
  • the agent is bound to a solid support, such as a bead.
  • a cell comprises the agent.
  • a cell produces or secretes the agent.
  • the cell is an antibody-producing cell.
  • the cell is a B cell, a plasma cell, or a plasmablast.
  • the cell is a mammalian cell or a microorganism, such as a fungal or bacterial cell.
  • the microdroplet comprises (i) an agent, such as a candidate agent, which, in some cases, can be an agent-producing cell, e.g. an antibody-producing cell; (ii) a first reporter microorganism as provided herein; and (iii) a second reporter microorganism as provided herein.
  • an agent such as a candidate agent
  • an agent-producing cell e.g. an antibody-producing cell
  • a first reporter microorganism as provided herein
  • a second reporter microorganism as provided herein.
  • the microdroplet comprises (i) an agent, such as a candidate agent, which, in some cases, can be an agent-producing cell, e.g. an antibody-producing cell; (ii) a first reporter microorganism comprising a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an
  • Acinetobacter species operably linked to a first sequence encoding a first reporter molecule; and (iii) a second reporter microorganism comprising a second reporter
  • polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a second sequence encoding a second reporter molecule, wherein the first and second OM stress-responsive genes are modulated in response to a stress to the outer membrane (OM-stress) of the Acinetobacter spp.
  • spp Acinetobacter species
  • the first reporter microorganism and the second reporter microorganism are clonal. In certain embodiments, the first reporter microorganism and the second reporter microorganism are not clonal. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host strain. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from different host strains. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from different host species. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host species.
  • the first reporter microorganism and the second reporter microorganism are derived from the same host strain and are not clonal. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host species and are derived from different host strains. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host species and are not clonal.
  • a first vector comprises a first reporter polynucleotide and a second vector comprises the second reporter polynucleotide.
  • the first and second vectors share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the first and second vectors are the same. In certain embodiments, the first and second vectors are different.
  • the first and second reporter polynucleotides share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second reporter polynucleotides are the same. In certain embodiments, the first and second reporter polynucleotides are different.
  • the first and second OM stress-responsive genes share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second OM stress-responsive genes are the same. In certain embodiments, the first and second OM stress-responsive genes are different.
  • the first and second sequences comprising the regulatory regions share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences comprising the regulatory regions are the same. In certain embodiments, the first and second sequences comprising the regulatory regions are different.
  • the first and second sequences encoding the reporter molecules share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are different. In certain embodiments, the first and second reporter molecules are the same. In certain embodiments, the first and second reporter molecules are different.
  • the first reporter microorganism and the second reporter microorganism are derived from different host strains and the first and second reporter polynucleotides are different. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from different host strains; the first and second reporter molecules are different; and the first and second OM stress-responsive genes are the same.
  • the first reporter microorganism and the second reporter microorganism are derived from the same host strain and the first and second reporter polynucleotides are different. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host strain; the first and second reporter molecules are different; and the first and second OM stress-responsive genes are different.
  • the microdroplet comprises a polymer matrix and/or a gel matrix.
  • microdroplet comprises agarose, carrageenan, alginate, alginate -polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2- pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium
  • the microdroplet comprises a polymer matrix, which may be e.g., agarose, carrageenan, alginate, alginate-polylysine, collagen, a plant-derived gum, cellulose or a derivatives thereof (e.g., methylcellulose), gelatin, chitosan or an extracellular matrix (ECM), as described by Kleinman (U.S. Pat. No. 4,829,000), or combinations thereof.
  • a polymer matrix which may be e.g., agarose, carrageenan, alginate, alginate-polylysine, collagen, a plant-derived gum, cellulose or a derivatives thereof (e.g., methylcellulose), gelatin, chitosan or an extracellular matrix (ECM), as described by Kleinman (U.S. Pat. No. 4,829,000), or combinations thereof.
  • Synthetic hydrogels that may be used in the microdroplet include but are not limited to polyvinyl alcohol, block copolymer of ethylene- vinylalcohol, sodium polystyrene sulfonate, vinyl-methyl-tribenzyl ammonium chloride and polyphosphazene.
  • microdroplet comprises agarose.
  • the agarose is low gelling temperature agarose, such as an ultra-low gelling temperature agarose.
  • the low gelling temperature agarose allows for the agarose to stay liquid at lower temperatures, e.g., temperatures that permit viability of the agent-producing cells (e.g. antibody-producing cell) and the reporter microorganism and thereby allow live cell and reporter microorganism encapsulation.
  • the gelling temperature of the agarose used in encapsulation is such that the temperature of liquid agarose does not adversely affect viability of the agent-producing cells (e.g.
  • the agarose has a gelling temperature of lower than about 35°C, such as about 30°C, about 25°C, about 20°C, about 15°C, about 10°C or about 5°C.
  • the agarose is an ultra- low gelling temperature agarose, such as those with a gelling temperature of lower than about 20°C, about 15°C, about 10°C or about 5°C.
  • the agarose has a gelling temperature of between about 5°C and about 30°C, about 5°C and about 20°C, about 5°C and about 15°C, about 8°C and about 17°C or about 5°C and about 10°C, such as about 8°C and about 17°C.
  • the microdroplet comprises growth media and/or is surrounded by a non-aqueous environment.
  • the non-aqueous environment In some embodiments, the non-aqueous
  • the environment includes an oil.
  • the oil is gas permeable.
  • the microdroplet comprises serum.
  • the serum is human, bovine, rabbit, or mouse serum.
  • the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol) or 25% (vol/vol).
  • the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol) or 15% (vol/vol) to 25% (vol/vol).
  • populations of microdroplets comprising reporter
  • the microdroplets on average, comprise one or fewer agent or agent-producing cell (e.g. antibody-producing cells).
  • agent or agent-producing cell e.g. antibody-producing cells
  • the average ratio of candidate agents or candidate agent-producing cell (e.g. antibody-producing cell) per microdroplet is less than or less than about 1. In some embodiments, the average ratio of candidate agents or agent-producing cell (e.g.
  • antibody-producing cell) per microdroplet is between about 0.05 and about 1.0, about 0.05 and about 0.5, about 0.05 and about 0.25, about 0.05 and about 0.1, about 0.1 and about 1.0, about 0.1 and about 0.5, about 0.1 and about 0.25, about 0.25 and about 1.0, about 0.25 and about 0.5 or 0.5 and about 1.0, each inclusive.
  • t the average ratio of agent or agent-producing cell (e.g. antibody-producing cells) per microdroplet is or is about 0.1.
  • compositions comprising a reporter polynucleotide, a reporter vector, a reporter microorganism or a microdroplet as described herein.
  • the composition further comprises an excipient.
  • Acceptable excipients are known in the art, such as, for example buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins;
  • the composition comprises media.
  • the composition comprises an agent or a combination of agents as described herein.
  • the composition comprises one or more components capable of activating the complement pathway.
  • the composition comprises serum, such as human, bovine, rabbit, or mouse serum.
  • the composition comprises a plurality of the reporter
  • polynucleotides polynucleotides, reporter vectors, reporter microorganisms or microdroplets as described herein.
  • kits include instructions for use in the provided methods.
  • kits include one or more containers containing one component, and the component is a reporter polynucleotide, a reporter vector, a reporter microorganism, or a composition described herein.
  • the kit further comprises instructions for use in accordance with any of the methods described herein.
  • kits are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Kits may optionally provide additional components such as buffers and interpretative information. In some embodiments, the components of the kits are contained in one or more containers.
  • the kit contains a reporter microorganism.
  • the reporter microorganism may be in a form suitable for culture or for direct use in an assay, such as a screening assay as described.
  • the kits may contain other materials suitable for the practice of the assays.
  • the kits may also contain buffers, media for culture of the reporter strains, and other components for performing the assay or culturing or maintaining the cells.
  • kits provide a reporter vector suitable for the production of reporter microorganisms.
  • the kits contain various reagents to facilitate the production of reporter microorganisms. Exemplary of such reagents include, but are not limited to, the pathogen to be transformed, culture media, buffers, drugs for selection of transformants, and components for producing a reporter microorganism.
  • the kits are in suitable packaging. Suitable packaging include, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
  • reporter microorganisms containing the reporter polynucleotides or reporter vectors can be used in any desired method in which a change (e.g. increase or decrease) in a signal from the reporter molecule provides a readout of a property or activity of a microorganism, including any property or activity that is or may be or is suspected of being modulated in a response to a condition or in the presence of one or more agents compared to in the absence of the condition or the one or more agents.
  • provided herein are methods of assessing outer membrane (OM) stress, methods for identifying an agent that modulates an activity or property of a microorganism, methods of screening agents, including candidate antibacterial agents, and methods of determining the drug resistance of a microorganism. Also provided herein are agents identified by any method described herein.
  • OM outer membrane
  • the provided methods include contacting a reporter microorganism (i) to a condition that is known to cause or suspected of causing OM stress to the reporter microorganism and/or (ii) with one or more agents, including candidate agents, that may impact the integrity or biogenesis of the outer membrane of the reporter microorganism.
  • following the contacting the reporter microorganism can be assessed or monitored for a change in a detectable signal from the reporter molecule contained therein compared to in the absence of the condition or in the absence of contacting the reporter microorganism with the one or more agents or candidate agents.
  • the provided methods include: (a) contacting or exposing the reporter microorganism or a microorganism containing the reporter polynucleotide or reporter vector provided herein with a condition that is known to cause or suspected of causing stress to the outer membrane and (b) determining if there is a change in a detectable signal from the reporter molecule compared to in the absence of the conditions.
  • such methods can be used to assess if the outer membrane of the reporter microorganism is stressed and/or if the integrity or biogenesis of the outer membrane of the reporter microorganism is being impacted in the presence of the condition.
  • the condition can be a physical condition.
  • the condition is caused by the addition of an exogenous agent, such as one or more candidate agents, to which the reporter microorganism is exposed.
  • the provided methods include: (a) contacting the reporter microorganism or a microorganism containing the reporter polynucleotide or reporter vector provided herein with an agent and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of contacting with the agent.
  • such methods can be used to identify an agent that causes stress to the outer membrane of a microorganism and/or impacts or modulates the integrity or biogenesis of the outer membrane of a microorganism.
  • the agent is a biological molecule or drug, such as a small molecule compound, peptide, or polypeptide or protein.
  • the agent is produced or secreted from a cell, in which case the reporter microorganism is contacted with an agent-producing cell.
  • the method can be carried out a plurality of agents, such as a plurality or library of candidate agents or a plurality of agent-producing cells, such that the reporter microorganism is contacted with each of the plurality of agents.
  • the reporter microorganism is contacted with each of the plurality of agents.
  • the reporter microorganism is contacted with each of the plurality of agents.
  • microorganism is separately contacted with each of the plurality of agents, such as each of the plurality of agent-producing cells.
  • the provided methods include (a) contacting the reporter microorganism or a microorganism containing the reporter polynucleotide or reporter vector provided herein with a drug and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of contacting with the drug.
  • such methods can be used to identify if a microorganism is resistant to the drug.
  • the microorganism if there is a change in the detectable signal, the microorganism is not resistant to the drug and if there is not a change in the detectable signal, the microorganism is identified as potentially being resistant to the drug.
  • the drug is a small molecule antibiotic or peptide antibiotic. In some embodiments, the drug is known.
  • the change in signal is a decrease in signal. In some embodiments, the change in signal is an increase in signal. [0243] In some embodiments, expression of the reporter molecule and/or presence of a signal therefrom is not detected or present in the reporter microorganism or is detected or present at only a low background level when cultured under conditions that optimize or maintain the health of the bacteria and/or under conditions that do not impact the integrity or biogenesis of the outer membrane.
  • expression of the reporter molecule and/or presence of a signal therefrom is induced or increased by OM- stress of the Acinetobacter spp., such as in the presence of an OM stress condition and/or in the presence of one or more agent that causes outer membrane stress and/or impacts the integrity or biogenesis of the outer membrane.
  • expression of the reporter molecule and/or presence of a signal therefrom is increased at least or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 or more fold.
  • the increased expression is compared to a reporter microorganism that was not contacted with the agent.
  • the increased expression is compared to the reporter microorganism prior to contact with the agent.
  • the reporter molecule is not expressed unless the reporter microorganism is contacted with the agent.
  • the regulatory region present in the provided reporter polynucleotides or vectors contained in the reporter microorganism is from or derived from a non-coding regulatory region of an OM stress- responsive gene whose expression is upregulated or increased in the presence of OM stress.
  • the OM stress-responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1S_0029, A1S_0030, A1S_0031, A1S_0032, A1S_0033, A1S_0037, A1S_0040, A1S_0041, A1SJ3044, A1S_0066, A1S_0092, A1S_0093, A1S_0109, A1S_0110, A1S_0112, A1S_0113, A1S_0114, A1S_0115, A1S_0116, A1S_0117, A1S_0118, A1S_0126, A1S_0158, A1S_0170, A1S_0175, A1S_0178, A1S_0189, A1S_0224, A1S_0245, A1S_0256, A1S_02
  • A1S_ _3604 A1S_ _3605 A1S_ _3606, A1S_ _3607 A1S_ _3608, A1S_ _3609 A1S_ _3610, A1S_ _3611,
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791.
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or
  • the OM stress-responsive gene is A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or A1S_3791. In some embodiments, the OM stress- responsive gene is A1S_0032, A1S_2885, or A1S_2889. In certain embodiments, the OM stress-responsive gene is A1S_0113 or A1S_1751. In certain embodiments, the OM stress- responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
  • expression of the reporter molecule and/or presence of a signal therefrom is constitutive in the reporter microorganism when cultured under conditions that optimize or maintain the health of the bacteria and/or that do not impact the integrity or biogenesis of the outer membrane.
  • expression of the reporter molecule and/or presence of a signal therefrom is decreased by OM-stress of the Acinetobacter spp., such as in the presence of an OM stress condition and/or in the presence of one or more agent that causes outer membrane stress and/or impacts the integrity or biogenesis of the outer membrane.
  • the expression of the reporter molecule and/or presence of a signal therefrom is decreased at least or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 or more fold.
  • the decreased expression is compared to a reporter microorganism that was not contacted with the agent.
  • the decreased expression is compared to the reporter microorganism prior to contact with the agent.
  • the regulatory region present in the provided reporter polynucleotides or vectors contained in the reporter microorganism is from or derived from a non-coding regulatory region of an OM stress- responsive gene whose expression is down-regulated in the presence of OM stress.
  • the OM stress-responsive gene is A1SJ3009, A1S_0010, A1SJ3025, A1SJ3027, A1S_0038, A1S_0067, A1S_0070, A1S_0071, A1S_0073, A1S_0076, A1S_0077, A1S_0079, A1SJ3087, A1SJ3090, A1S_0091, A1S_0095, A1S_0096, A1S_0097, A1S_0098, A1S_0099, A1S_ _0103, A1S_ _0104 A1S_ _0105, A1S_ _0106 A1S_ _0107, A1S_ _0108 A1S_ _0109, A1S_ _0121,

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Abstract

L'invention concerne des polynucléotides contenant une région régulatrice d'un gène sensible au stress de la membrane externe (OM) d'une bactérie Acinetobacter liée de manière fonctionnelle à une séquence codant pour une molécule rapporteur. L'invention concerne également des vecteurs rapporteurs et des micro-organismes rapporteurs génétiquement modifiés, ainsi que des procédés et des utilisations de ceux-ci.
PCT/US2018/015602 2017-01-27 2018-01-26 Micro-organismes rapporteurs et leurs utilisations WO2018140827A1 (fr)

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