(12) Patent Application Publication (10) Pub. No.: US 2017/0211121 A1 Stern Et Al

Home , Antitrichomonal agent, Artemotil, Arterolane, Chlorproguanil, Coccidiostat, Difetarsone

US 20170211121A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0211121 A1 Stern et al. (43) Pub. Date: Jul. 27, 2017

(54) METHODS FOR RAPID ANTIBOTC filed on Feb. 23, 2016, provisional application No. SUSCEPTIBILITY TESTING 62/326,545, filed on Apr. 22, 2016, provisional ap plication No. 62/338,376, filed on May 18, 2016, (71) Applicant: SeLux Diagnostics, Inc., Cambridge, provisional application No. 62/370,579, filed on Aug. MA (US) 3, 2016, provisional application No. 62/383, 198, filed on Sep. 2, 2016. (72) Inventors: Eric Stern, Cambridge, MA (US); Aleksandar Vacic, Cambridge, MA (US); Benjamin Spears, Cambridge, Publication Classification MA (US); Kelly Flentie, Cambridge, (51) Int. C. MA (US); Alec Flyer, Cambridge, MA CI2O 1/18 (2006.01) (US) CI2O 1/02 (2006.01) (52) U.S. C. (21) Appl. No.: 15/464,240 CPC ...... CI2O 1/18 (2013.01); C12O I/025 (2013.01); G0IN 24.58/00 (2013.01); G0IN (22) Filed: Mar. 20, 2017 2333/195 (2013.01); G0IN 2500/10 (2013.01) Related U.S. Application Data (63) Continuation of application No. PCT/US 17/14343, (57) ABSTRACT filed on Jan. 20, 2017. The present invention relates, in part, to methods and kits for (60) Provisional application No. 62/281,698, filed on Jan. rapidly determining antimicrobial Susceptibility of microor 21, 2016, provisional application No. 62/298,821, ganisms.

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METHODS FOR RAPID ANTIBOTC include signaling agents (e.g., Europium compounds) that SUSCEPTIBILITY TESTING are bound to microorganisms non-specifically rather than specifically (e.g., via chemically conserved groups or bio RELATED APPLICATIONS chemically conserved binding sites on microorganisms), thereby expanding the generalization of the present inven 0001. This application is a continuation of International tion to any microorganism and allowing onset of an appro Patent Application No. PCT/US 17/14343 filed Jan. 20, priate treatment without first needing to identify the particu 2017. PCT/US 17/14343 designates the United States and lar infectious microorganism. Also, the present invention claims priority to and benefit of U.S. Provisional Patent permits signal amplification Such that microbes may be Application No. 62/281,698, filed Jan. 21, 2016; U.S. Pro rapidly detected at lower concentrations, e.g., from a dilute visional Patent Application No. 62/298,821, filed Feb. 23, culture of microorganisms or via a patient’s biological 2016; U.S. Provisional Patent Application No. 62/326,545, sample. Additionally, the present invention may use Euro filed Apr. 22, 2016; U.S. Provisional Patent Application No. pium formulations as chemical moiety, thereby expanding 62/338,376, filed May 18, 2016; U.S. Provisional Patent the dynamic range of the methods and allowing for more Application No. 62/370,579, filed Aug. 3, 2016; and U.S. accurate determinations from a range of microbial samples. Provisional Patent Application No. 62/383, 198, filed Sep. 2, Finally, the present invention is compatible with existing 2016. The contents of the aforementioned patent applica equipment, thereby enabling rapid adoption in current clini tions are incorporated herein by reference in their entireties. cal laboratories. Accordingly, the present invention, in a greatly reduced amount of time and expense, relative to BACKGROUND OF THE INVENTION standard methods, can provide a patient with an appropriate 0002 Antimicrobial-resistant microbial infections are treatment regimen, i.e., a specific antimicrobial and at a associated with poor clinical outcomes including increased particular dosage. Thus, the present invention will improve morbidity, mortality, and healthcare costs among infected patient outcomes, lower hospital costs, and help reduce patients. The prevalence of these organisms in Such facilities further evolution of antimicrobial resistant microorganisms; in the United States has steadily increased over the last 30 thus, the present invention represents a significant break years. Phenotypic antimicrobial susceptibility testing (AST) through in the AST field. of microorganisms is critical for informing physicians of 0005. An aspect of the present invention is a method for appropriate therapeutic regimens. Using current methods, determining antimicrobial Susceptibility of microorganisms. AST determination typically requires a minimum of eight The method includes steps of incubating a liquid suspension hours, rendering it an overnight process due to shift work in of microorganisms in the presence of an antimicrobial and a many clinical microbiology laboratories. While awaiting a signaling agent, which is capable of binding to a surface of determination from current AST methods, patients are often the microorganisms, under conditions that promote growth administered broad-spectrum antimicrobials which often of the microorganisms; separating the microorganisms have significant detrimental effects on patient health and/or bound by the signaling agent from the unbound signaling contribute to the growing antimicrobial resistance epidemic. agent; and determining signal levels associated with the Furthermore, this time delay to getting accurate antimicro microorganisms as compared to one or more controls. bial treatment information increases patient stays in hospi 0006 Another aspect of the present invention is a method tals, thereby increasing costs and inconvenience to the for determining antimicrobial Susceptibility of microorgan patient. isms. The method includes steps of incubating a liquid 0003. Accordingly, a need exists for a method that rapidly Suspension of microorganisms in the presence of an antimi determines antimicrobial susceptibility of a microbial infec crobial under conditions that promote growth of the micro tion. The method described here is further advantageous in organisms; adding a signaling agent capable of binding to a that it addresses this need in a cost-effective manner because Surface of the microorganisms; separating the microorgan it is compatible with existing assay hardware components. isms bound by the signaling agent from the unbound sig naling agent; and determining signal levels associated with SUMMARY OF THE INVENTION the microorganisms as compared to one or more controls. 0004. The present invention permits rapid determination 0007. Yet another aspect of the present invention is a of antibiotic susceptibility of microbial infections. The method for determining antimicrobial susceptibility of invention is based in part upon the Surprising discovery of microorganisms. The method includes steps of incubating a non-specific Surface binding assays that provide accurate liquid Suspension of microorganisms in a cartridge including and rapid Antimicrobial Susceptibility Testing (AST) deter a plurality of chambers, each chamber containing one or minations in fewer than twelve hours—and, specifically, more antimicrobials, under conditions that promote growth under four hours. The present invention (“Fast-AST) pro of the microorganisms; adding a signaling agent, which is vides accurate results that are consistent with results capable of binding to a surface of the microorganisms, to the obtained using the Clinical Laboratory Standards Institute plurality of chambers; removing unbound signaling agent; (CLSI) reference methods when tested with multiple anti and determining signaling levels in the plurality of chambers microbials and on a plurality of microorganisms; however, as compared to one or more controls. the present invention takes significantly less time to obtain 0008. An aspect of the present invention is a method for results than the CLSI methods. Moreover, the present inven determining antimicrobial Susceptibility of microorganisms. tion accurately differentiates an antimicrobials MIC for The method includes incubating microorganisms in the clinically-relevant microbial strains that are resistant to one presence of an antimicrobial and a signaling agent, which or more antimicrobials and the antimicrobials MIC for includes a signal amplifier and one or more chemical moi strains of the same microorganism that are sensitive to the eties capable of binding non-specifically to a surface of the antimicrobials. Furthermore, the present invention may microorganisms, under conditions that promote growth of US 2017/0211121 A1 Jul. 27, 2017 the microorganisms; separating the microorganisms bound standard, overnight growth followed by optical density (OD) by the signaling agent from the unbound signaling agent; reading at 600 nm. The data shown for the “fast-AST is and determining signal levels associated with the microor from the five minute point after the start of incubation with ganisms as compared to one or more controls. detection solution and represents the average and standard 0009. Another aspect of the present invention is a method deviations of four wells, with values for each assay type for determining antimicrobial Susceptibility of microorgan relative to an antimicrobial-free control. isms. The method includes incubating microorganisms in the 0020 FIG. 6 is a graph showing a comparison of a MIC presence of an antimicrobial under conditions that promote determination of ceftazidime on Pseudomonas aeruginosa growth of the microorganisms; adding a signaling agent (ATCC strain 27853) using the present invention (“fast including a signal amplifier and one or more chemical AST' technique) and a standard, overnight growth followed moieties capable of binding non-specifically to a surface of by optical density (OD) reading at 600 nm. The data shown the microorganisms; separating the microorganisms bound represents the average and standard deviations of four wells, by the signaling agent from the unbound signaling agent; with values for each assay type relative to an antimicrobial and determining signal levels associated with the microor free control. ganisms as compared to one or more controls. 0021 FIG. 7 is a graph showing a MIC determination 0010 Yet another aspect of the present invention is a kit comparison using the present invention (“fast-AST) for two for determining antimicrobial Susceptibility of microorgan strains of Pseudomonas aeruginosa: a susceptible strain isms. The kit includes a signaling agent capable of binding (ATCC strain 27853) and a resistant strain (ATCC strain to a Surface of the intact microorganisms of interest; a BAA-2 108). The data shown represents the average of four Solution for incubating a sample containing microorgan wells, with values for each assay relative to an antimicro isms; and one or more reagents for generating signals from bial-free control. the signaling agent. 0022 FIG. 8 is a table summarizing data from Example 0011. Any aspect or embodiment described herein can be 2. It compares MIC calls by the “fast-AST' technique with combined with any other aspect or embodiment as disclosed those of the standard overnight ODoo procedure. The data herein. While the disclosure has been described in conjunc shown represents the average of four wells, with values for tion with the detailed description thereof, the foregoing each assay type relative to an antimicrobial-free control. description is intended to illustrate and not limit the scope of 0023 FIG. 9 is a graph showing a comparison of raw the disclosure, which is defined by the scope of the appended optical signal vs. Staphylococcus aureus concentration (in claims. Other aspects, advantages, and modifications are CFU/ml) for two techniques: the present invention (“fast within the scope of the following claims. AST' amplification technique) and the standard optical 0012. The patent and scientific literature referred to density at 600 nm technique (ODoo). herein establishes the knowledge that is available to those 0024 FIG. 10 is a graph showing the MIC results for the with skill in the art. All United States patents and published present invention (the “fast-AST method) compared to the or unpublished United States patent applications cited herein Clinical Laboratory Standards Institute (CLSI) reference are incorporated by reference. All published foreign patents method for seven pathogenic bacterial species. and patent applications cited herein are hereby incorporated 0025 FIG. 11 is a table identifying the bacteria, antimi by reference. All other published references, documents, crobials, and the signaling agent/chemical moiety used in manuscripts and scientific literature cited herein are hereby Example 4. incorporated by reference. (0026 FIG. 12A to FIG. 12C are tables showing repre 0013. Other features and advantages of the invention will sentative SensiTitre R results for S. aureus (FIG. 12A) and be apparent from the Drawings and the following detailed K. pneumonia (FIG. 12B). Data is not presented using the description and claims. present invention for the nitrofurantonin S. aureus experi ment because only two wells were dedicated to this antimi BRIEF DESCRIPTION OF THE DRAWINGS crobial. 0014. The above and further features will be more clearly (0027 FIG. 13 A to FIG. 13C is a graph showing a appreciated from the following detailed description when comparison between MIC results obtained by the present taken in conjunction with the accompanying drawings. The invention and the CLSI reference method for the antimicro drawings however are for illustration purposes only; not for bials oxacillin (FIG. 13A), Vancomycin (FIG. 13B), and limitation. levofloxacin (FIG. 13C) on S. aureus clinical strains. 0015 FIG. 1 is a schematic showing generalized steps of 0028 FIG. 14A to FIG. 14D is a graph showing a the present invention. comparison between MIC results obtained by the present 0016 FIG. 2A to FIG. 2D are graphs and illustrations invention and the CLSI reference method for the antimicro showing key features of aspects of the present invention bials amplicillin (FIG. 14A), ciprofloxacin (FIG. 14B), imi (“fast-AST). penem (FIG. 14C), and gentamicin (FIG. 14D) on E. coli 0017 FIG. 3 is a schematic comparing steps required in clinical strains. currently-used Antimicrobial Susceptibility Testing (AST) 0029 FIG. 15 is a graph showing that the present inven systems and aspects of the present invention. tion (“fast-AST) consistently produces MIC results similar 0018 FIG. 4 is a graph showing the time delay required to those obtained by the CLSI standard reference method to obtain results using a currently-used AST System (i.e., over the course of one month on the same clinical species of BioMeriéux's Vitek2). S. aureus. 0019 FIG. 5 is a graph showing a comparison of a 0030 FIG. 16 to FIG. 23 are graphs comparing sensitives minimum inhibitory concentration (MIC) determination of for a plurality of antimicrobials for a chemically sensitive E. clindamycin on Staphylococcus aureus (ATCC strain 29213) coli strain (“OC 25922) and a clinically-relevant antimi using the present invention (“fast-AST' technique) and a crobial-resistant strain (“Clinical'). The antimicrobials used US 2017/0211121 A1 Jul. 27, 2017

are Imipenem (FIG. 16); Ampicillin (FIG. 17); Ceftazidime 0043 FIG. 40 is a schematic that illustrates a method for (FIG. 18); Gentamicin (FIG. 19); Levofloxacin (FIG. 20): minimizing the interference of filamentous microorganisms Trimethethoprim/Sulfamethoxazole (SXT) (FIG. 21); Cip in AST determinations. rofloxacin (FIG. 22); and Cetriaxone (FIG. 23). 0044 FIG. 41 is a graph showing the result of an assay 0031 FIG. 24 to FIG. 26 are graphs comparing sensitives using signaling agent comprising a fluorescent nanoparticle for a plurality of antimicrobials for a chemically sensitive S. for E. coli and ampicillin-resistant E. coli treated with and aureus (“OC strain 29213) strain. The antimicrobials used without 100 g/ml amplicillin (a concentration well above are Vancomycin (FIG. 24); Penicillin (FIG. 25); and Teico the MIC). planin (FIG. 26). 0045 FIG. 42 is a graph showing the result of an assay 0032 FIG. 27 and FIG. 28 are graphs showing MIC using signaling agent comprising a fluorescent nanoparticle results using a method of the present invention directly on a for E. coli and with varying amplicillin concentrations. Error clinical sample and compared to clinical results obtained bars shows the standard deviation of three replicates. with a Beckman-Coulter MicroScan Walkaway (in which a 0046 FIG. 43 is a graph showing the ability of E. Sub-culturing step is performed prior to overnight growth). coli-functionalized magnetic beads are capable of binding and isolating intact bacterium from a solution. 0033 FIG. 29 is a graph showing the detected fluores 0047 FIG. 44 is a graph showing the number of intact cence (via a signaling agent comprising Europium and use bacteria that are isolated by functionalized magnetic beads of wheat germ agglutinin, which specifically binds gram from Solutions comprising varying amounts of an antimi positive bacteria) of a gram-positive bacterial Solution rela crobial. tive to the concentration of bacteria in the solution. 0048 FIG. 45 includes graphs comparing the number of 0034 FIG. 30 is a graph showing the detected fluores intact bacteria that are isolated by centrifugation versus cence (via a signaling agent comprising Europium and use functionalized magnetic beads from Solutions comprising of Polymixin B, which specifically binds gram negative varying amounts of an antimicrobial (here, Vancomycin bacteria) of a gram-negative bacterial Solution relative to the “VAN). The MIC for VAN for this clinical S. aureus strain concentration of bacteria in the solution. was 8 g/ml by CLSI overnight method. 0035 FIG. 31 are graphs that compare MIC values 0049 FIG. 46A to FIG. 46C show tetra-amino metalor obtained when an antibody-bound Europium formulation is ganic ligand (TAMLR) nanolabel design and performance. used as signaling agent to MIC values obtained when an FIG. 46A is a schematic showing nanolabel constituents. antibody-horse radish peroxidase (HRP) is used as signaling FIG. 46B is a graph showing catalytic comparison of HRP agent. The MIC for SXT for this clinical S. aureus strain was and TAML (inset); the dashed lines are linear best fits for s0.5 lug/ml by CLSI overnight method. each dataset with R2 of 0.997 for HRP and 0.987 for TAML. 0036 FIG. 32 are graphs showing the relative fluores FIG. 46C is a graph showing the TAML nanolabel vs. HRP cence units (RFU) obtained for specific bacterial concentra comparison for C. difficile Toxin A immunoassay. In FIG. tions for two Europium formulations that are non-specifi 46B and FIG. 46C, the signals were normalized to “1” at cally bound to bacterial surfaces. Zero concentration, errors were propagated, and error bars 0037 FIG.33 are graphs showing the RFU obtained for representitl Standard deviation. Experiments were repeated specific bacterial concentrations of two bacterial species for three times in triplicate with similar results. two Europium formulations that are non-specifically bound DEFINITIONS to bacterial surfaces. 0038 FIG. 34 and FIG. 35 are graphs showing the RFU 0050. In order for the present invention to be more obtained for specific bacterial concentrations of various readily understood, certain terms are first defined below. bacterial species for a Europium formulation that is non Additional definitions for the following terms and other specifically bound to bacterial surfaces. terms are set forth throughout the Specification. 0039 FIG. 36A to FIG. 36C are graphs showing the RFU 0051. As used in this Specification and the appended obtained for specific bacterial concentrations of various claims, the singular forms “a,” “an and “the include plural bacterial species for a Europium formulation that is non referents unless the context clearly dictates otherwise. specifically bound to bacterial Surfaces when using various 0.052 Unless specifically stated or obvious from context, washes comprising glutaraldehyde. as used herein, the term 'or' is understood to be inclusive and covers both 'or' and “and”. 0040 FIG. 37 are graphs showing the RFU obtained for 0053. The terms "e.g., and “i.e. as used herein, are used specific bacterial concentrations of E. coli for a Europium merely by way of example, without limitation intended, and formulation that is non-specifically bound to bacterial sur should not be construed as referring only those items explic faces using a two-step process comprising NH2-PEG-Biotin itly enumerated in the specification. followed by streptavidin-europium (Eu-SAv). 0054. The terms “one or more”, “at least one'. “more 004.1 FIG. 38 is a graph showing the RFU obtained for than one', and the like are understood to include but not be specific bacterial concentrations of E. coli for a Europium limited to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, formulation that is non-specifically bound to bacterial sur 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, faces using a two-step process comprising NHS-LC-LC 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, Biotin followed by Eu-SAV. 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 0.042 FIG. 39 is a schematic that illustrates the confound 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, ing effect that filamentous growth has on Volumetric-based 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89,90,91, 92,93, 94, determinations of microorganisms antimicrobial Suscepti 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, bilities. Susceptible bacteria entering filamentous growth 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, may appear falsely resistant due to their increased Volume. 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, US 2017/0211121 A1 Jul. 27, 2017

132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, and/or cell-membrane associated proteins, other anionic 144, 145, 146, 147,148, 149 or 150, 200, 300, 400, 500, 600, phospholipids, and a combination thereof. 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more and 0061 Growth, as in growth of microorganisms, includes any number in between. a proliferation in number, an increase in length, an increase 0055 Conversely, the term “no more than includes each in Volume, and/or an increase in nucleic acid and/or protein value less than the stated value. For example, “no more than content of the microorganisms. 100 nucleotides' includes 100, 99,98, 97,96, 95, 94, 93, 92, 0062 Controls may include antimicrobials for which the 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, microorganism are not susceptible. As examples, if the assay 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, is used to determine the Susceptibility of gram-positive 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, bacteria, then the controls (and the test incubations) may 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, include one or more antimicrobials that target gram-negative 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, bacteria and if the assay is used to determine the Suscepti 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. bility of eukaryotic microorganisms, the control (and the test 0056. The terms “plurality”, “at least two”, “two or incubations) may include one or more antibacterial antimi more”, “at least second, and the like, are understood to crobials. include but not limited to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 0063 A control may be a positive control measured from 12, 13, 14, 15, 16, 17, 18, 1920, 21, 22, 23, 24, 25, 26, 27, microorganisms under otherwise identical conditions but 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, without antimicrobials or with one or more antimicrobials 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, for which the microorganisms are not susceptible. 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 0064. A control may be measured from microorganisms 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90,91, under otherwise identical conditions but without nutrients. 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 0065. A control may be measured from microorganisms 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, under otherwise identical conditions with one or more toxins 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, known to inhibit growth of the microorganisms. 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 0.066 Controls may be historic controls. Here, the test 142,143, 144, 145, 146, 147,148, 149 or 150, 200, 300, 400, incubations may be performed after control incubations have 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or been performed. more and any number in between. 0067. Alternately, controls may be performed in a car 0057 Throughout the specification the word “compris tridge distinct from the cartridge comprising the test incu ing,” or variations such as "comprises' or "comprising.” will bations. be understood to imply the inclusion of a stated element, 0068. By “processed' is meant a step that isolates micro integer or step, or group of elements, integers or steps, but organisms from a biological sample, a step that increases the not the exclusion of any other element, integer or step, or concentration of microorganisms obtained from a biological group of elements, integers or steps. sample, and/or a step that increases the number of micro organisms obtained from a biological sample, e.g., by cul 0058. Unless specifically stated or obvious from context, turing the microorganisms under conditions that promote as used herein, the term “about is understood as within a proliferation of the microorganisms. range of normal tolerance in the art, for example within 2 0069 Compounds of this invention include those standard deviations of the mean. “About can be understood described generally herein, and are further illustrated by the to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, classes, Subclasses, and species disclosed herein. As used 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the stated value. herein, the following definitions shall apply unless otherwise Unless otherwise clear from the context, all numerical indicated. For purposes of this invention, the chemical values provided herein are modified by the term “about'. elements are identified in accordance with the Periodic Table 0059 A surface can be an external surface of cell wall, of the Elements, CAS version, Handbook of Chemistry and cell envelope, plasma membrane, or cell capsule; internal Physics, 75th Ed. Additionally, general principles of organic Surface of cell wall, cell envelope, plasma membrane, or cell chemistry are described in "Organic Chemistry. Thomas capsule; or within a cell wall, cell envelope, plasma mem Sorrell, University Science Books, Sausalito: 1999, and brane, or cell capsule. The Surface may include structures of “March's Advanced Organic Chemistry, 5th Ed., Ed.: the cell projecting extracellularly, including but not limited Smith, M. B. and March, J., John Wiley & Sons, New York: to cilium, pilus, and flagellum. The Surface may include an 2001, the entire contents of which are hereby incorporated organelle. The Surface may include transmembrane proteins, by reference. cell-wall proteins, extracellular proteins, intracellular pro (0070. The term “heterocycle”, “heterocyclyl”, or “het teins, extracellular-associated polysaccharides, intracellular erocyclic” as used herein means nonaromatic, monocyclic, associated polysaccharides, extracellular lipids, intracellular bicyclic, or tricyclic ring systems in which one or more ring lipids, membrane lipids, cell-wall lipids, proteins, polysac members are an independently selected heteroatom. In some charides, and/or lipids integral to or associated with a cell embodiments, the "heterocycle”, “heterocyclyl', or "hetero envelop. The Surface may include a nucleic acid. cyclic' group has three to fourteen ring members in which 0060. The surface may include a biomolecule to which one or more ring members is a heteroatom independently the signaling agent binds or associates. Exemplary biomol selected from oxygen, Sulfur, nitrogen, or phosphorus, and ecules include peptidoglycans, mureins, mannoproteins, each ring in the system contains 3 to 7 ring members. porins, beta-glucans, chitin, glycoproteins, polysaccharides, (0071. The term “heteroatom refers to one or more of lipopolysaccharides, lipooligosaccharides, lipoproteins, oxygen, Sulfur, nitrogen, phosphorus, and silicon (including, endotoxins, lipoteichoic acids, teichoic acids, lipid A, car any oxidized form of nitrogen, Sulfur, phosphorus, or sili bohydrate binding domains, efflux pumps, other cell-wall con; the quaternized form of any basic nitrogen or, a US 2017/0211121 A1 Jul. 27, 2017

substitutable nitrogen of a heterocyclic ring, for example N meric, and geometric (or conformational) mixtures of the (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or present compounds are within the scope of the invention. NR+ (as in N-substituted pyrrolidinyl)). Unless otherwise stated, all tautomeric forms of the com The term “alkoxy’, or “thioalkyl', as used herein, refers to pounds of the invention are within the scope of the inven an alkyl group, as previously defined, attached through an tion. Additionally, unless otherwise stated, structures oxygen (“alkoxy”) or sulfur (“thioalkyl) atom. depicted herein are also meant to include compounds that 0072. The terms “haloalkyl”, “haloalkenyl”, “haloali differ only in the presence of one or more isotopically phatic', and "haloalkoxy' mean alkyl, alkenyl or alkoxy, as enriched atoms. For example, compounds having the present the case may be, Substituted with one or more halogen structures where there is a replacement of hydrogen by atoms. This term includes perfluorinated alkyl groups. Such deuterium or tritium, or a replacement of a carbon by a 13C as CF3 and CF2CF3. or 14C-enriched carbon are within the scope of this inven 0073. The terms “halogen”, “halo', and “hal” mean F, Cl, tion. Such compounds are useful, as a nonlimiting example, Br, or I. as analytical tools or probes in biological assays: 0074 The terms “aryland “ar-”, used alone or as part of 0082 It is to be understood that, when a disclosed com a larger moiety, e.g., “aralkyl”, “aralkoxy’, or “aryloxy pound has at least one chiral center, the present invention alkyl, refer to an optionally substituted C6-14aromatic encompasses one enantiomer of inhibitor free from the hydrocarbon moiety comprising one to three aromatic rings. corresponding optical isomer, racemic mixture of the inhibi For example, the aryl group is a C6-10aryl group (i.e., tor and mixtures enriched in one enantiomer relative to its phenyl and naphthyl). Aryl groups include, without limita corresponding optical isomer. When a mixture is enriched in tion, optionally Substituted phenyl, naphthyl, or anthracenyl. one enantiomer relative to its optical isomers, the mixture The terms “aryl and “ar-, as used herein, also include contains, for example, an enantiomeric excess of at least groups in which an aryl ring is fused to one or more 50%, 75%, 90%, 95% 99% or 99.5%. cycloaliphatic rings to form an optionally Substituted cyclic 0083. Unless otherwise defined, all technical and scien structure such as a tetrahydronaphthyl, indenyl, or indanyl tific terms used herein have the same meaning as commonly ring. The term “aryl' may be used interchangeably with the understood by one of ordinary skill in the art to which this terms 'aryl group', 'aryl ring, and 'aromatic ring. application belongs and as commonly used in the art to 0075. The compounds of this invention can exist in free which this application belongs; Such art is incorporated by form for treatment, or where appropriate, as a pharmaceu reference in its entirety. In the case of conflict, the present tically acceptable salt. Specification, including definitions, will control. 0076. As used herein, the term “aromatic” includes aryl and heteroaryl groups as described generally below and DETAILED DESCRIPTION OF THE herein INVENTION 0077. The term “aliphatic' or “aliphatic group', as used I0084. The present invention permits rapid determination herein, means an optionally Substituted Straight-chain or of antibiotic susceptibility of microbial infections. The branched C1-12 hydrocarbon which is completely saturated invention is based in part upon the Surprising discovery of or which contains one or more units of unsaturation. For non-specific Surface binding assays that provide accurate example, Suitable aliphatic groups include optionally Sub and rapid Antimicrobial Susceptibility Testing (AST) deter stituted linear or branched alkyl, alkenyl, and alkynyl minations in fewer than twelve hours—and, specifically, groups. Unless otherwise specified, in various embodiments, under four hours. The present invention (“fast-AST) pro aliphatic groups have 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 vides accurate results that are consistent with results carbon atoms. It is apparent to a skilled person in the art that obtained using the Clinical Laboratory Standards Institute in some embodiments, the “aliphatic' group described (CLSI) reference methods and when tested with multiple herein can be bivalent. antimicrobials and on a plurality of microorganisms; how 0078. The term “alkyl, used alone or as part of a larger ever, the present invention takes significantly less time to moiety, refers to a saturated, optionally Substituted Straight obtain results than the CLSI methods. Moreover, the present or branched chain hydrocarbon group having 1-12, 1-10, invention accurately differentiates an antimicrobials MIC 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms. for clinically-relevant microbial strains that are resistant to 007.9 The term “alkenyl', used alone or as part of a larger one or more antimicrobials and the antimicrobials MIC for moiety, refers to an optionally Substituted Straight or strains of the same microorganism that are sensitive to the branched chain hydrocarbon group having at least one antimicrobials. Furthermore, the present invention may double bond and having 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 include signaling agents (e.g., Europium compounds) that carbon atoms. are bound to microorganisms non-specifically rather than 0080. The term “alkynyl', used alone or as part of a larger specifically (e.g., via chemically conserved groups or bio moiety, refers to an optionally Substituted Straight or chemically conserved binding sites on microorganisms), branched chain hydrocarbon group having at least one triple thereby expanding the generalization of the present inven bond and having 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon tion to any microorganism and allowing onset of an appro atOmS. priate treatment without first needing to identify the particu 0081. Unless otherwise stated, structures depicted herein lar infectious microorganism. Also, the present invention are also meant to include all isomeric (e.g., enantiomeric, permits signal amplification Such that microbes may be diastereomeric, and geometric (or conformational)) forms of rapidly detected at lower concentrations, e.g., from a dilute the structure; for example, the R and S configurations for culture of microorganisms or via a patient’s biological each asymmetric center, (Z) and (E) double bond isomers, sample. Additionally, the present invention may use Euro and (Z) and (E) conformational isomers. Therefore, single pium formulations as chemical moiety, thereby expanding Stereochemical isomers as well as enantiomeric, diastereo the dynamic range of the methods and allowing for more US 2017/0211121 A1 Jul. 27, 2017

accurate determinations from a range of microbial samples. ment and use of rapid diagnostics for the detection of Finally, the present invention is compatible with existing antibiotic resistant bacteria one of its main goals. equipment, thereby enabling rapid adoption in current clini I0089. The present invention, which generates more rapid cal laboratories. Accordingly, the present invention, in a and accurate AST determinations, may provide actual cost greatly reduced amount of time and expense, relative to benefits of over $2,000 per patient. These value points standard methods, can provide a patient with an appropriate include the more easily quantifiable (reduced length of stay treatment regimen, i.e., a specific antimicrobial and at a and expensive treatments) and the more intangible, difficult particular dosage. Thus, the present invention will improve to value (patient mortality and Societal impacts from patient outcomes, lower hospital costs, and help reduce improved antimicrobial stewardship). Some of these intan further evolution of antimicrobial resistant microorganisms; gible values, such as the value of antimicrobial Stewardship, thus, the present invention represents a significant break may become more quantified as regulatory bodies start to through in the AST field. impose costs on hospitals for not adopting more rigorous 0085 Aspects of the present invention deliver accurate, antimicrobials stewardship programs. In September 2014, low-cost phenotypic AST results by chemically amplifying California Senate Bill 1311 was signed into law, further microorganism Surfaces. This novel approach offers two requiring hospitals to adopt and implement an antimicrobial primary advances over currently-used methods: 1) Quanti Stewardship policy in accordance with guidelines estab fication of microorganism growth by determining relative lished by federal government and professional organiza Surface area, which overcomes limitations of current plat tions, and to establish a physician-Supervised multidisci forms with regard to filamentous growth regimes, as are well plinary antimicrobial stewardship committee with at least known to those skilled in the art; and 2) Microorganism one physician or pharmacist who has undergone specific amplification with optimal sensitivity in the 1x10 to 1x10 training related to stewardship. In June 2016, the Centers for CFU/ml range using standard optical detection equipment. Medicare and Medicaid Systems (CMS) used proposed roles I0086. As disclosed herein (e.g., in the Examples), the promoting antimicrobial Stewardship in hospitals, with present invention has been shown to deliver equivalent many industry experts expecting financial incentives to be results to the gold-standard for a broad range of microor implemented in the coming two years. The present invention ganism species, including all six (Enterococcus faecium, will further the governments and the healthcare industry’s Staphylococcus aureus, Klebsiella pneumoniae, Acineto goals of better antimicrobial stewardship. bacter baumannii, Pseudomonas aeruginosa, and Entero 0090 Generalized steps of aspects of the present inven bacter species) (“ESKAPE) pathogens. Because of the tion are shown in FIG. 1. Images in FIG. 1 show an aspect generality of the present invention, it is flexible, in that it can with distinct process steps; however, aspects of the present be easily and cheaply adapted to new microorganism species invention may be automated. strains and diagnostic tests. (0091 FIG. 2A to FIG. 2D show features of aspects of the 0087. The present invention provides low-cost, pheno present invention. FIG. 2A shows a detection sensitivity typic ASTs from standard microbial colony isolates or from range for three representative pathogens. Dashed lines show direct-from-positive blood samples, in less than 8 hours, Zero-concentration signal levels. FIG. 2B shows the “Croco preferably less than 5 hours. This allows standard clinical dile' (Titertek-Berthold) automated fast-AST prototype microbiology laboratories same-shift, phenotypic AST platform which may be used in the present invention. FIG. results. The below working Examples demonstrate that 2C is a schematic showing anionic bacteria interacting with chemical amplification of microorganism Surfaces produces cationic nanolabels and polymers. The decreased solubility accurate minimum inhibitory concentration (MIC) and of the resulting neutral complexes allows magnetic beads to breakpoint calls in less than four hours. This will shorten bind. FIG. 2D shows data for S. aureus with a SensiTitre(R) current wait times by over twenty hours and will match Gram Positive panel (GPALL3F) showing a bacteriostatic direct-from-positive blood culture MALDI-TOF identifica (clindamycin) and bactericidal (penicillin) antimicrobial tions currently nearing FDA trials, as well as direct-from results relative to the high-growth and “frozen-in-time positive blood culture multiplex PCR identification plat (FIT) controls. forms that have already obtained FDA clearance. This 0092. As is known to those skilled in the art, AST design enables the present invention (“fast-AST' platform) platforms may yield minimum inhibitory concentration to break the traditional speed vs. cost tradeoff. The present (MIC) results and/or qualitative susceptibility results invention is compatible both with standard microplate for (QSRs) for each antimicrobial tested. MICs are commonly mats (e.g., having 6, 12, 24, 48, 96, 384, or 1536 wells) and known to be the lowest concentration of antimicrobial that conventional optical detectors. inhibits microorganism growth and provides physicians with 0088. Identification and antimicrobial susceptibility test dosing information. QSRS may also provide physicians with ing (AST) of the invading pathogen with speed and accuracy similar dosing information but may not provide a numerical allow for timely administration of the most effective thera MIC. AST assays are predominantly configured to test peutic agent. Such treatment ameliorates the infection, multiple antimicrobials in parallel for each obtained biologi decreases length of stay for hospitalized patients, and dimin cal sample. In order to produce MIC or QSR results, dilution ishes the time patients are subject to broad spectrum anti series are required for each antimicrobial. Thus, for liquid microbials, the latter contributing the global epidemic of based ASTs, termed “broth microdilution” by the CLSI, antimicrobial resistance. In contrast, the currently-accepted assays are commonly performed in cartridges and/or over thirty hour wait for microorganism identification and microplates, which enable parallel testing of different anti Susceptibility results necessitates overuse of broad-spectrum microbials at different concentrations. antimicrobials and longer than necessary patient stay. For 0093 Long times to obtain an AST determination result this reason, the Presidential Advisory Council on Combating in incomplete information being delivered to physicians. Antibiotic-Resistant Bacteria recently made the develop These long times often prevent the identification of rates of US 2017/0211121 A1 Jul. 27, 2017

antimicrobial efficacy, or kill kinetics. This additional infor under appropriate conditions, most preferably aerobic, for mation may be important for informing treatment. Current growing bacteria. During this time, the microorganism can ASTs, which are not determined until over six hours (and grOW. generally over twelve hours) after treatment commences, (0097. The broth may be cation-adjusted Mueller Hinton often lose the ability to discern differences between the rate broth and may contain additional Supplements known by of antimicrobial efficacy: an antimicrobial that kills a micro those skilled in the art to be advantageous for microbial organism instantly looks the same after twelve hours as one growth, Such as lysed horse blood, and/or for determining that killed it within four hours. antimicrobial efficacies, such as high sodium chloride con centrations. The microplates may be agitated during this 0094 Table 1 estimates the effects of different treatments growth period, which may be advantageous for dispersing on the number of bacteria after a two-hour incubation. nutrients and/or gas exchange and/or antimicrobials in each Assuming a thirty minute doubling time, untreated controls well and/or decreasing biofilm formation. should increase by sixteen-fold. Treatment groups with a (0098. Within Zero to eight hours of the AST onset (most “potent antimicrobial (defined as one having efficacy preferably Zero to four hours), a known quantity of signaling against the bacteria, for example) above the MIC should agent is added to each well. Adding reagents (including result in minimal microorganism growth and, in the case of signal generators) may be performed by an automated bactericidal antimicrobials, death of the microorganism. instrument or a semi-automated instrument or may be per Thus, fewer bacteria are expected than the starting concen formed manually. tration. Treatment groups with a “potent antimicrobial 0099 Signaling agents (which may be referred to as below its MIC should result in microorganism growth equal “sticky-amps') comprise a moiety capable of binding to a to or lesser than the no-antimicrobial control. Slow-acting microorganism (e.g., an antibody and/or a lectin that bind to antimicrobials, defined in this case as those requiring more a microorganism Surface, a charged moiety and/or a func than two hours to kill the bacteria (e.g., as it the case for tional moiety that non-specifically binds to the microorgan bacteriostatic antimicrobials) will produce a signal between ism Surface) and a chemical moiety capable of providing a the starting concentration and the sixteen-fold increase. signal or contributing to production of a signal (e.g., an enzyme chemiluminophore, and lanthanide chelate). Exem TABLE 1. plary enzymes include horseradish peroxidase, alkaline Potent phosphatase, acetylcholinesterase, glucose oxidase, beta-D- No antimicrobial No galactosidase, beta-lactamase, and a combination thereof. Step antimicrobial at COInc. Step antimicrobial 0100 AS used herein, signal generator may include one Starting bacteria 5 x 10 5 x 10 5 x 10 5 x 10 or more chemical moieties (i.e., "signal generators') conju concentration gated to one or more “microorganism receptors.” Signal Estimated 8 x 10 s8 x 10 <5 x 10° 5 x 10 to bacteria <8 x 10 generators include, but are not limited to, one or more concentration catalysts (including enzymes, metal-oxide nanoparticles, after 2 hours, organometallic catalysts, nanoparticles designed for signal with 30 min. amplification (such as those described in the U.S. Provi doubling time sional Applications to which the present application claims priority and incorporates by reference in their entireties), bacteriophages comprising signal generating elements, fluo 0095. The starting concentration of bacteria of 5x10 rophores (including organic fluorophores, europium, or CFU/ml is given in the American Society for Microbiolo ruthenium(II), rhenium(I), palladium(II), platinum(II)-con gy’s “Manual of Antimicrobial Susceptibility Testing” (C) taining organometallics), and/or colorimetric dyes (includ 2005, with Marie B. Coyle as the coordinating editor, for the ing organic "stains'). Combinations of the above may be broth micro dilution technique. Since each well contains used. Such as nanoparticles, dendrimers, and/or other approx. 100 L, there are approx. 5x10" bacteria per well. nanoscale structures with enzymes, fluorophores, and/or Standard fluorescent dyes begin to be quantifiable at approx. organometallic molecules. 0.1 nM concentrations, which correspond to approximately 0101 The chemical moiety may be conjugated to a 1.2x10" molecules. Thus, for a thirty minute doubling time signaling agent before contacting the signaling agent to a bacteria to be visible after two hours, each individual microorganism, while the signaling agent is initially con bacterium would have to be labeled with 1.5x10' fluorescent tacted to a microorganism, or after the signaling agent has molecules. Practical considerations. Such as fluorescent contacted a microorganism. background and non-specific binding, may increase this 0102. When the signaling agents are added to AST dilu number by orders of magnitude. In order to enable compat tions containing a microorganism, signaling agent receptors ibility with standard optical detectors, it may thus be advan (e.g., moieties that can bind specifically or non-specifically tageous to use a chemical and/or biochemical amplifier that to a microorganism) associate with microorganism surfaces. produces a detectable signal at lower concentrations. Thus, the more intact microorganisms, for example, there 0096. Without wishing to be bound by theory, the present are in solution, the greater the number of signaling agents invention is based in part on the principle of broth micro that will be associated with these bacteria. Consequently, dilution. A culture to be assessed is diluted, most preferably there is an inverse relationship between the number of intact to 1-10x10 CFU/ml, and introduced to wells containing bacteria and the number of signaling agents that are “free” different antimicrobials at different concentrations, such that in solution, as defined by those not bound to intact bacteria. MICs can be determined for an appropriate panel of anti Note that free signaling agents may be bound to soluble microbials. The plate is then introduced into an incubator at microbial components if for example, microorganisms lyse the appropriate temperature, most preferably 31-37°C., and in response to antimicrobial treatment. US 2017/0211121 A1 Jul. 27, 2017

0103) The number of signaling agents that associate with nalized, thus all associations are included. Signaling agents and/or intercalate into microorganism Surfaces is propor designed for intercalation may include, but are not limited tional to the microorganism surface area. Microorganism to, Small hydrophobic molecules, hydrophobic peptides, Surface area is strongly associated with truly resistant micro and/or peptides with alternating hydrophobic and hydro organisms. In particular, in the case of microorganisms that philic regions. Molecules designed for microorganism inter swell or elongate in response to MIC- and Sub-MIC con calation are well-known to those skilled in the art. Signaling centrations of antimicrobials (e.g., filament forming bacte agents may further be specific to one or more types of ria), metabolic and/or Volumetric identifications are known microorganisms. Signaling agents may have multiple recep to give false susceptibility profiles for “rapid AST time tors. These may enhance binding and/or enable simultane points, defined as those less than six hours. To overcome this ous binding to two or more microorganisms, which may limitation, the present invention translates microorganism further serve to “agglutinate' bacteria. Prior to or concur Surface area (rather than Volume) into a measurable signal, rently with the addition of signaling agents it may be most preferably an optical signal. The present methods are advantageous to adjust the solution pH. This may be ben able to accurately determine microorganism resistance pro eficial for enhancing charge-charge interactions between files in less than six hours. microorganisms and signaling agents. The anionic charge of 0104. In order to separate signaling agents associated microorganisms may be increased by titrating the Solution with and/or intercalated into microorganisms from free pH above neutral (more basic). It may thus be beneficial to signaling agents, it may be necessary to perform one or more utilize moieties with one or more fixed, cationic charges. separation and/or competitive binding steps. Such steps 0109. It is noteworthy that the signaling agent may spe include, but are not limited to, centrifugation (e.g., with a cifically bind to a microorganism (e.g., an antibody that g-force >500xg), filtration (e.g., via a filter having pores specifically binds to a microorganism species or a strain of smaller than or equal to 0.45 microns, and preferably smaller microorganism) or my non-specifically binds to a microor than or equal to 0.2 microns), electrophoresis, and/or mag ganism (e.g., by a generic covalent or non-covalent bond netic capture. Such steps are well-known to those skilled in formation and another non-specific chemical association the art. known in the art). 0105. In order to promote signaling agent binding and/or 0110. It is preferred that signaling agents bind native reduce background, it may further be advantageous, before microorganism surfaces. adding signaling agents, to separate microorganisms from 0111 Alternately, chemicals and/or biochemicals which the liquid in which they were suspended during incubation. are capable of associating with signaling agents may be Such separations may include but are not limited to, cen added to the liquid in which the microorganisms are Sus trifugation, filtration, electrophoresis, and/or magnetic cap pended during growth, Such that chemicals and/or biochemi ture cals are incorporated into microorganisms during incuba 0106 When these data are compared across treatment tion. This may serve to enhance signaling agent association groups, microbial resistance profiles may be determined, with microorganisms. In alternative embodiments, the sig using steps similar to currently-used AST determinations. naling agents themselves may be present in the liquid in Additionally, these data may enable determination of rates of which the microorganisms are suspended during incubation antimicrobial efficacy, or kill kinetics. and may be incorporated into microorganisms during 0107 Signaling agents may be added together with growth. microorganisms and/or antimicrobials, such that they are 0112 Preferably the signaling agents comprise an ampli present for the entire AST incubation period. This total fier signal generator, such that the signal from each intact period may be up to twenty-four hours but is preferably microorganism may be amplified beyond the number of within eight hours and more preferably within five hours. signaling agents associated with each microorganism. For Alternatively, signaling agents may be added to microor example, the enzyme horseradish peroxidase (HRP) is ganisms and antimicrobial after a prescribed incubation known to be able to amplify signals >1x10-fold. Thus, if period. This period may be up to twenty-four hours but is one hundred HRP molecules are bound to each microorgan preferably within eight hours and more preferably within ism surface, an amplification of 10 may be achieved. This four hours. may increase the speed with which AST determinations may 0108 Signaling agents are designed to associate with be made by enabling discrimination of microorganism con and/or intercalate in microorganism Surfaces, including centrations that cannot otherwise be differentiated. Use of walls and/or membranes. Signaling agents designed for Europium formulations similarly provides signal amplifica association comprise binding moieties including, but are not tion. limited to, one or more antibodies, lectins, other proteins, 0113 Alternatively, the signaling agents may comprise Small molecules with one or more charged chemical groups, optical dye precursors known to those skilled in the art as Small molecules with one or more functional chemical “membrane dyes' that are designed to greatly increase groups, phages, glycoproteins, peptides, aptamers, charged fluorescence emission upon intercalation into a hydrophobic Small molecules, Small molecules with fixed charges, region, such as a cell membrane. Assays designed with these charged polymers, charged polymers with fixed charges, signaling agents may require microorganisms to be concen hydrophobic Small molecules, charged peptide, charged trated into a smaller Volume, approaching a plane, to pro peptides with fixed charges, peptides with alternating hydro duce Sufficient signals so as to be easily optically measured. philic and hydrophobic regions, and/or small molecule Interfering species may require the use of near-IR fluoro ligands, which may or may not be organometallic com phores. plexes. Molecules designed for microorganism association 0114 Potential separation techniques include, but are not are well-known to those skilled in the art. Signaling agents limited to, filtering (e.g., via a filter having pores Smaller may remain bound to microorganisms and/or may be inter than or equal to 0.45 microns, preferably smaller than or US 2017/0211121 A1 Jul. 27, 2017 equal to 0.2 microns), centrifugation (e.g., with a g-force ganisms. In this case, unless microorganisms are specifically >500xg), electrophoresis, dielectrophoresis, and magnetic lysed, only bound signaling agents will contribute to the capture. These techniques are employed to separate signal signal. ing agents associated with microorganisms, which are stuck I0122. In order to maximize separation efficiency, i.e., in a filter, pelleted in a centrifuge, and/or separated electro minimize the number of free signaling agents remaining, phoretically and/or magnetically, from those free in solution. one or more washing steps may be performed. These may be Free signaling agents pass through a filter ("filtrate'), remain continuous, as in the cases of filtering, magnetic capture, or in Solution after centrifugation or magnetic separation (“Su electrophoresis, and/or discrete, as in the cases of centrifu pernatant”), and/or run separately electrophoretically. Cen gation or magnetic capture. trifugation may be standard, density gradient, or differential 0123. In alternative embodiments, signaling agents may centrifugation. Magnetic separation may require the addition not require washing. This may be the case when “membrane of one or more magnetic particles specifically targeted to dye signaling agents are used. Molecules not intercalated associate with or bind to microorganisms. These may be into microorganism membranes have significantly lower added prior to or concurrently with signaling agent addition. optical activities than intercalated species, thus washing may 0115 Such separation techniques may also isolate micro not be required. organisms that change morphology in response to an anti 0.124 One or more washes may be performed before microbial treatment and may confound a determination. An signaling agents are added to the microorganisms. These example of Such a microorganism is filamentous bacteria washes may, for example, remove interfering species present which initially elongate in response to antimicrobial treat in the liquid in which the microorganisms were suspended ment. This growth regime is known to those skilled in the during incubation. art. Isolating and excluding filamentous bacteria from an 0.125. In embodiments, no wash is performed. assay, using a herein-described separation technique, will 0.126 Signal development may require the addition of a increase the accuracy of the obtained results. “development Solution.” For signaling agents comprising 0116 Microorganism separation may be enhanced catalysts, the development Solution may comprise one or through the association of particles with microorganism more signal precursors that can be converted to an optically species. For example, in the case of magnetic separation, and/or electrically active signaling molecule. For signaling magnetic beads may associate with microorganisms (spe agents comprising encapsulated molecules, such as within cifically or non-specifically). Moieties present on magnetic nanoparticles, the development Solution may comprise one bead surfaces may bind the same surface (or a biomolecule or more reagents to release the encapsulated species. At a thereof) of microorganisms as the singling agent or different specified time after addition of the development solution, a Surface (or a biomolecule thereof). The magnetic beads may colorimetric and/or electrochemical signal may be mea have the same and/or different moieties as the signaling Sured. Such signals include, but are not limited to, absor agents. For example, if a signaling agent comprises an bance, fluorescence, time-resolved fluorescence, chemilumi antibody that binds to E. coli, then a magnetic bead may be neScence, electrochemiluminescence, amperometric, functionalized with the same antibody. In other examples, Voltammetric, impedance, and/or impedance spectroscopy. the signaling agent may include a motif that binds a micro The data may then be compared to determine ASTs and organism and the magnetic bead is functionalized to non MICs, similarly to current AST protocols. specifically bind to microorganisms. I0127. In embodiments, determining signal levels 0117 The one or more binding moieties associated with includes measuring the signal levels associated with intact the magnetic beads may be identical of different to the microorganisms. Alternately or additionally, determining chemical moiety associated with or of the signaling agent. signal levels includes measuring the signal levels not asso 0118. The one or more binding moieties associated with ciated with intact microorganisms. the magnetic beads may bind the microorganisms prior to, I0128. These processes may be performed directly from simultaneously with, or Subsequent to the biding of the cultures, Sub-cultures, positive blood cultures, samples. signaling agent with the microorganism. Treatments to concentrate microorganisms and/or remove 0119 The one or more binding moieties associated with potential interfering species may be performed prior to AST the magnetic beads may associate with one or more poly or prior to signaling agent addition. mers that precipitate microorganisms. The one or more I0129. Signaling agents may also be used with plate-based polymers that precipitate microorganisms may cationic. The methods for AST determination, such as gradient diffusion. one or more polymers that precipitate microorganisms may They may be added simultaneously upon microorganism be poly(ethylene glycol). addition to plates or following a set incubation period. 0120 Magnetic beads, as are known to those skilled in Spatial information for the optical and/or electrical signal is the art, may range in size from 20 nm to 20 microns. important in these cases. With this approach an assay for 0121. After separation, one or more assays to determine intact microorganism-bound signaling agents may be pref the number of signaling agents remaining after microorgan erable in order to retain spatial information. In this case, one ism separation and/or the number of signaling agents or more wash steps may be performed prior to the addition removed during microorganism separation (“free” signaling of the development solution in order to remove free signal agents) may be performed. Performing an assay for free ing agents. signaling agents provides a signal inversely proportional to O130 In embodiments, no wash is performed. microorganism concentration. In this case signaling agents 0131 Alternatively, signaling agents may be designed to associated with microorganisms may be either bound to or be up-taken by bacteria, e.g., which may be achieved internalized by microorganisms. Alternatively, an assay may through the use of bacteriophages. In Such methods, an assay be performed for signaling agents associated with microor for free signaling agents is performed. US 2017/0211121 A1 Jul. 27, 2017

0132 Alternatively, a blot-transfer approach, such as is chambers) that do not contain antimicrobials or one or more standard with nitrocellulose paper, may be used to transfer antimicrobials for which the microorganisms are not Sus bacteria or free signaling agents and a spatial assay then ceptible. performed on the blotted paper. 0.138. In embodiments of an above aspect, binding to a 0.133 Separation step(s) may not be required if the sig Surface of the microorganisms is non-specific, e.g., com naling agent produces a signal upon binding. Alternatively, prising a non-covalent interaction and via forming a cova a separation step-free process may be achieved if the sig lent bond. naling agent becomes Susceptible or resistant to a specific 0.139. In embodiments of an above aspect, the signaling developer solution constituent upon binding. agent may include a chemical and/or biochemical group 0134 Final MIC and/or QSR output data may be inter capable of binding a Surface of the microorganisms, wherein preted by a user directly from the data produced by the the Surface comprises one or more of membranes, walls, assays described herein. Alternatively, these data may be proteins, organelles, saccharides, lipids, cell envelope, and/ processed by one or more algorithms to yield MICs and/or or nucleic acids. QSRs. Reported MIC and/or QSR values may be derived 0140. In embodiments of an above aspect, the signaling from one or more of the assays described herein or may be agent may include a chemical and/or biochemical group derived from one or more of the assays described herein capable of binding a biomolecule of the surface of the together with one or more known assays for microorganism microorganisms, wherein the Surface biomolecule is growth including, but not limited to, metabolic dye indicator selected from peptidoglycans, mureins, mannoproteins, assays, pH indicator assays, nucleic acid assays, and ATP porins, beta-glucans, chitin, glycoproteins, polysaccharides, assayS. lipopolysaccharides, lipooligosaccharides, lipoproteins, endotoxins, lipoteichoic acids, teichoic acids, lipid A, car Methods of the Present Invention bohydrate binding domains, efflux pumps, other cell-wall and/or cell-membrane associated proteins, other anionic 0135 An aspect of the present invention is a method for phospholipids, and a combination thereof. determining antimicrobial Susceptibility of microorganisms. 0.141. In embodiments of an above aspect, the signaling The method includes steps of incubating a liquid Suspension agent may include a signal amplifier and one or more of microorganisms in the presence of an antimicrobial and a chemical moieties capable of binding non-specifically to a signaling agent under conditions that promote growth of the Surface of the microorganisms. microorganisms, wherein the signaling agent is capable of 0142. Another aspect of the present invention is a method binding to a Surface of the microorganisms; separating the for determining antimicrobial Susceptibility of microorgan microorganisms bound by the signaling agent from the isms. The method includes incubating microorganisms in the unbound signaling agent; and determining signal levels presence of an antimicrobial and a signaling agent under associated with the microorganisms as compared to one or conditions that promote growth of the microorganisms, more controls, thereby determining the antimicrobial Sus wherein the signaling agent comprises a signal amplifier and ceptibility of the microorganisms. one or more chemical moieties capable of binding non 0.136 Another aspect of the present invention is a method specifically to a Surface of the microorganisms; separating for determining antimicrobial Susceptibility of microorgan the microorganisms bound by the signaling agent from the isms. The method includes steps of incubating a liquid unbound signaling agent; and determining signal levels Suspension of microorganisms in the presence of an antimi associated with the microorganisms as compared to one or crobial under conditions that promote growth of the micro more controls, thereby determining the antimicrobial Sus organisms; adding a signaling agent capable of binding to a ceptibility of the microorganisms. Surface of the microorganisms; separating the microorgan 0.143 Another aspect of the present invention is a method isms bound by the signaling agent from the unbound sig for determining antimicrobial Susceptibility of microorgan naling agent; and determining signal levels associated with isms. The method includes incubating microorganisms in the the microorganisms as compared to one or more controls, presence of an antimicrobial under conditions that promote thereby determining the antimicrobial susceptibility of the growth of the microorganisms; adding a signaling agent microorganisms. In embodiments, adding the signaling comprising a signal amplifier and one or more chemical agent occurs prior to or during the incubating step or adding moieties capable of binding non-specifically to a surface of the signaling agent occurs after the incubating step. the microorganisms; separating the microorganisms bound 0.137 Another aspect of the present invention is a method by the signaling agent from the unbound signaling agent; for determining antimicrobial Susceptibility of microorgan and determining signal levels associated with the microor isms. The method includes steps of incubating a liquid ganisms as compared to one or more controls, thereby Suspension of microorganisms in a cartridge comprising a determining the antimicrobial susceptibility of the microor plurality of chambers, each chamber containing one or more ganisms. In embodiments, the signaling agent occurs prior antimicrobials, under conditions that promote growth of the to, at the beginning of, or during the incubating step, microorganisms; adding a signaling agent to the plurality of preferably during the incubating step. In embodiments, the chambers, wherein the signaling agent is capable of binding microorganisms are incubated in a liquid Suspension. to a surface of the microorganisms; removing unbound 0144. In embodiments of an above aspect, the liquid signaling agent; and determining signaling levels in the Suspension may be prepared by inoculating a liquid media plurality of chambers as compared to one or more controls, with a microbial isolate grown from a biological sample. thereby determining the Susceptibility of microorganisms to 0145. In embodiments of an above aspect, the liquid the one or more antimicrobials. In embodiments, the car Suspension of microorganisms may be prepared from an tridge further includes one or more control chambers (e.g., unprocessed biological sample, e.g., an unprocessed biologi at least 2, 4, 6, 8, 12, 24, 48, 96, 192, 384, 1536 or more cal sample has not undergone a culturing step. US 2017/0211121 A1 Jul. 27, 2017

0146 In embodiments of an above aspect, the liquid duration of broad-spectrum antimicrobial treatment. Fur Suspension of microorganisms may be prepared from a thermore, the wait is often compounded by one-shift opera cultured or processed biological sample. tion common in many clinical microbiology laboratories. 0147 In embodiments of an above aspect, the biological 0157 Alternatively, the present invention may be used sample is selected from blood, cerebrospinal fluid, urine, directly from positive blood cultures. After the standard step stool, vaginal, sputum, bronchoalveolar lavage, throat, 1 and step 2 of blood draw and incubation/culture, if the nasal/wound Swabs, and a combination thereof. culture is positive, the culture bottle would move directly to 0148. In embodiments of an above aspect, the method a microorganism isolation (step 3) and then into an auto does not involve a step of capturing microorganisms on a mated system (step 4). The present invention can be fully Solid Surface prior to or during incubation. automated, requiring a technician to only load the system 0149. In embodiments of an above aspect, the method with a standard cartridge with microorganism dilutions and does not include a step of growing microorganisms on a then initiate the four-hour “fast-AST process. The lab Solid Surface during or Subsequent to the incubating step. technician would then receive the same standard phenotypic 0150. In embodiments of an above aspect, the incubating results for AST of a minimum inhibitory concentration may include agitating the liquid Suspension of microorgan (“MIC). However, the streamlined process would reduce 1SS. the time to AST by over twenty-four hours in theory, and 0151. In embodiments of an above aspect, the liquid potentially two days in practice, and simplify lab workflow. Suspension of microorganisms may be agitated by means of 0158. The currently used AST systems perform variants mechanical, acoustic, and/or magnetic agitation continu of the Clinical Laboratory Standards Institute (CLSI) broth ously or discretely during the incubating. microdilution procedure. Bacteria are inoculated into mul 0152. In embodiments of an above aspect, the incubating tiple wells in parallel, each of which contains one (or more) occurs at 31-37° C.Comparison of the present invention to antimicrobials at a known concentration and a nutrient currently-used AST systems broth. Wells are inoculated at 5x10 CFU/ml to ensure 0153. The present invention is superior to currently-used bacteria are in the log growth phase, important for detecting AST methods, in part because it provides accurate AST accurate responses to antimicrobials. Microorganism detec results in significantly less time. tion is then performed visually. 0154 Three automated AST systems that are currently 0159. The slow speed of phenotypic AST testing is due, used in clinics are BioMerieux's Vitek2. Beckman Dickin in part, to its reliance on microorganism growth in order to son’s Phoenix, and Beckman-Coulter's MicroScan. A com produce a detectable optical signal. Bacteria cannot be parison between steps in currently-used AST Systems and quantified by optical density measurements below a con the present invention are shown in FIG. 3. The processes centration of ~1x10 CFU/ml, rendering the CLSI starting described in this invention may be performed in at least two concentration invisible for a minimum of eight doubling modes. The first is for standard isolates, with no changes to times. Since differentiation of microorganism growth in the current laboratory workflows. The second is direct from slowest-growing well from the no-growth well is critical for positive blood cultures. minimum inhibitory concentration (MIC) determinations, 0155 For standard isolate processing, the present inven significantly longer times are required. As is known to those tion is compatible with existing clinical laboratory work skilled in the art, some existing platforms overcome these flows and, thus, requires no changes. As shown in “step 8 growth issues by including metabolic probes in the liquid in of FIG. 3, current susceptibility (AST) testing is performed which the microorganisms are suspended during incubation. after colony isolation (step 6) and microorganism concen However, inclusion of these probes may miss growth tration standardization (step 7). In this workflow, the present regimes, such as filamentous growth, and may impact the invention would replace current systems at “step 8.” accuracy of results. Because AST results with the present invention are available 0160 Once the AST-specific steps have commenced, the within a healthcare workers the shift (<5 hours), utility may currently-used AST systems still typically require over eight increase the speed with which patients receive optimized hours to report results for simple, highly-susceptible bacteria therapies (step 9) by up to one day in practice. Automation and require over ten hours for pathogens with complex may be designed to include “step 7 and, potentially, addi resistance profiles or slow growth kinetics, see FIG. 4. tional steps in the workflow. Such automation is known to 0.161 Additionally, the currently-used automated AST those skilled in the art. Note that FIG. 3 illustrates the systems Suffer from two shortcomings that prevent the workflow for blood samples. Many sample types, such as reporting of accurate results in less than six hours: 1) very urine and Swabs, may be streaked directly on plates (step 4). major errors, inaccurate “susceptible' calls for truly-resis In this case, the gram stain may be performed at step 6. tant strains; and 2) major errors, inaccurate “resistant calls 0156 For a blood test, the currently-used AST systems for truly-Susceptible strains. Indeed, these issues have require an obtained blood culture to become detectibly required positive (which takes ten or more hours), followed by a (0162 BioMeriéux and BD to revise their initial four-hour sub-culture step (of at least twelve hours), and then an AST speed claims for the Vitek2(R) and Phoenix(R). test (which requires a minimum of eight hours): this totals 0163 The existence of very major errors is explained in over forty-eight hours, depending on the pathogen, and often part by the metabolic energy expended by the microorgan takes greater than three days in practice. In most workflows, isms in achieving antimicrobial resistance. Resistant micro the identification of the organism happens after the Sub organisms may alter energy expenditures in response to culture step and increasingly done by mass spectrometry. antimicrobials, confounding the results of metabolic probes Since both identification and AST results are required for around the MIC. These may also result from the present of clinicians or pharmacists to prescribe proper targeted anti additives, such as redox indicators, in the growth media. The microbials, this delay to AST results directly extends the prevalence of major errors is due primarily to filamentous US 2017/0211121 A1 Jul. 27, 2017

growth of certain bacteria. This growth regime is a common throat Swabs, vaginal Swabs, urethral Swabs, cervical Swabs, antimicrobial response amongst gram-negative bacteria, in rectal Swabs, lesion Swabs, abscess Swabs, nasopharyngeal particular to cell-wall-acting antimicrobials, such as Swabs, and the like), and any combination thereof. Also B-lactams. Filamentous bacteria continue to replicate their included are bacteria cultures or bacteria isolates, fungal internal contents but do not septate. Thus, again, metabolic cultures or fungal isolates. The ordinary-skilled artisan will probes give erroneous near-MIC results. The removal of also appreciate that isolates, extracts, or materials obtained filamentous bacteria was shown to significantly reduce from any of the above exemplary biological samples are also major errors for the AST method. within the scope of the invention. 0164. Measurements of relative microorganism surface 0168 Microorganisms obtained from a biological sample area, as used in the present invention, overcome the pitfalls may be cultured or otherwise processed as is routinely of metabolic probes for AST. First, since relative surface performed in the art. area is not confounded by shifts in metabolic activity, fast-AST enables rapid, accurate resistance calls. Second, Exemplary Microorganisms Surface area measurements prevent over-resistance calls. In 0169. As used herein, infection is meant to include any contrast to Volumetric measurements obtained with meta infectious agent of a microbial origin, e.g., a bacterium, a bolic probes of the currently-used AST systems, surface area fungal cell, an archaeon, and a protozoan. In preferred measurements enable accurate differentiation between true examples, the infectious agent is a bacterium, e.g., a gram resistance and filamentous growth. As illustrated in the positive bacterium, a gram-negative bacterium, and an atypi schematic of FIG. 39, volumes of resistant and susceptible cal bacteria. The term “antimicrobial resistant microorgan filamentous bacteria are difficult to distinguish. But the lack ism' is a microorganism (e.g., bacterium, fungus, archeaon, of septation creates a filamentous Surface area significantly and protozoan) that is resistant to one or more distinct lower than that of truly resistant bacteria. Thus, by ampli antimicrobials, i.e., anti-bacterial drugs, antifungal drugs, fying each bacteria's Surface area, the present invention is anti-archaea medications, and anti-protozoan drugs. able to accurately call four-hour, B-lactam (ampicillin) MICs 0170 The microorganisms (e.g., a liquid Suspension of for E. coli samples (see, the below Examples). As illustrated microorganisms) may include one strain of microorganism. in FIG. 39, the surface area differential between elongation The microorganisms may include one species of microor and “true' resistance approaches 2/3, which may be detected ganism. The microorganisms may include more than one with an amplified signal. strain of microorganism. The microorganisms may include one order of microorganism. The microorganisms may Patient include one class of microorganism. The microorganisms 0.165. As used herein, the term “patient' (also inter may include one family of microorganism. The microorgan changeably referred to as “host' or “subject') refers to any isms may include one kingdom of microorganism. host that can serve as a source of one or more of the 0171 The microorganisms (e.g., a liquid Suspension of biological samples or specimens as discussed herein. In microorganisms) may include more than one strain of micro certain aspects, the donor will be a vertebrate animal, which organism. The microorganisms may include more than one is intended to denote any animal species (and preferably, a species of microorganism. The microorganisms may include mammalian species such as a human being). In certain more than one genus of microorganism. The microorgan embodiments, a “patient refers to any animal host, includ isms may include more than one order of microorganism. ing but not limited to, human and non-human primates, The microorganisms may include more than one class of avians, reptiles, amphibians, bovines, canines, caprines, microorganism. The microorganisms may include more than cavities, corvines, epines, equines, felines, hircines, lapines, one family of microorganism. The microorganisms may leporines, lupines, Ovines, porcines, racines, Vulpines, and include more than one kingdom of microorganism. the like, including, without limitation, domesticated live 0172. The microorganism may be a bacterium. Examples stock, herding or migratory animals or birds, exotics or of bacterium include and are not limited to Acetobacter Zoological specimens, as well as companion animals, pets, aurantius, Acinetobacter bitumen, Acinetobacter spp., and any animal under the care of a veterinary practitioner. Actinomyces israelii, Actinomyces spp., Aerococcus spp., Agrobacterium radiobacter, Agrobacterium tumefaciens, Biological Samples Anaplasma, Anaplasma phagocytophilum, Azorhizobium caulinodans, Azotobacter vinelandii, Bacillus, Bacillus 0166 The biological sample is any sample that contains anthraces, Bacillus brevis, Bacillus cereus, Bacillus fusifor a microorganism, e.g., a bacterium and a fungal cell. mis, Bacillus licheniformis, Bacillus megaterium, Bacillus 0167 Exemplary biological samples include, but are not mycoides, Bacillus spp., Bacillus Stearothermophilus, Bacil limited to, whole blood, plasma, serum, sputum, urine, stool, lus subtilis, Bacillus Thuringiensis, Bacteroides, Bacte white blood cells, red blood cells, buffy coat, tears, mucus, roides fragilis, Bacteroides gingivalis, Bacteroides melani saliva, semen, vaginal fluids, lymphatic fluid, amniotic fluid, nogenicus (also known as PrevOtella melaninogenica), spinal or cerebrospinal fluid, peritoneal effusions, pleural Bartonella, Bartonella hemselae, Bartonella quintana, Bar effusions, exudates, punctates, epithelial Smears, biopsies, tonella spp., Bordetella, Bordetella bronchiseptica, Borde bone marrow samples, fluids from cysts or abscesses, Syn tella pertussis, Bordetella spp., Borrelia burgdorferi, Bru ovial fluid, vitreous or aqueous humor, eye washes or cella, Brucella abortus, Brucella melitensis, Brucella spp., aspirates, bronchoalveolar lavage, bronchial lavage, or pull Brucella suis, Burkholderia, Burkholderia cepacia, Burk monary lavage, lung aspirates, and organs and tissues, holderia mallei, Burkholderia pseudomalilei, Calymmato including but not limited to, liver, spleen, kidney, lung, bacterium granulomatis, Campylobacter; Campylobacter intestine, brain, heart, muscle, pancreas, and the like, Swabs coli, Campylobacter fetus, Campylobacter jejuni, Campy (including, without limitation, wound Swabs, buccal Swabs, lobacter pylori, Campylobacter spp., Chlamydia, Chla US 2017/0211121 A1 Jul. 27, 2017 mydia spp., Chlamydia trachomatis, Chlamydophila, Chla 0173 The microorganism may be a fungus. Examples of mydophila pneumoniae (previously called Chlamydia fungi include and are not limited to Aspergillus spp., Blas pneumoniae), Chlamydophila psittaci (previously called tomyces spp., Candida spp., Cladosporium, Coccidioides Chlamydia psittaci), Chlamydophila spp., Clostridium, spp., Cryptococcus spp., Exserohilum, fitsarium, Histo Clostridium botulinum, Clostridium difficile, Clostridium plasma spp., Issatchenkia spp., mucOrmycetes, Pneumocys perfingens (previously called Clostridium welchii), tis spp., ringworm, Scedosporium, Sporothrix, and Stachy Clostridium spp., Clostridium tetani, Corynebacterium, botry's spp. Corynebacterium diphtheriae, Corynebacterium fisiiforme, 0.174. The microorganism may be a protozoan. Examples Corynebacterium spp., Coxiella burnetii, Ehrlichia chaf. of protozoan include and are not limited to Entamoeba feensis, Ehrlichia spp., Enterobacter cloacae, Enterobacter histolytica, Plasmodium spp., Giardia lamblia, and Try spp., Enterococcus, Enterococcus avium, Enterococcus panosoma brucei. durans, Enterococcus faecalis, Enterococcus faecium, Exemplary Antimicrobials Enterococcus gallinarum, Enterococcus maloratus, Entero coccus spp., Escherichia coli, Francisella spp., Francisella 0.175 When the microorganism is a bacterium, exem tularensis, Fusobacterium nucleatum, Gardenerella spp., plary antimicrobials include Amikacin, Aminoglycoside, Gardnerella vaginalis, Haemophilius spp., Haemophilus, Aminoglycoside amoxicillin, Aminoglycosides, Amoxicil Haemophilus ducreyi, Haemophilus influenzae, Haemophi lin, Amoxicillin/clavulanate, Ampicillin, Ampicillin/sul lus parainiluenzae, Haemophilus pertussis, Haemophilus bactam, Antitoxin, Arsphenamine, Azithromycin, AZlocillin, vaginalis, Helicobacter pylori, Helicobacter spp., Klebsiella Aztreonam, 3-lactam, Bacitracin, Capreomycin, Carbapen pneumoniae, Klebsiella spp., Lactobacillus, Lactobacillus ems, Carbenicillin, Cefaclor, Cefadroxil, Cefalexin, Cefa lothin, Cefalotin, Cefamandole, Cefazolin, Cefdinir, Cefdi acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, toren, Cefepime, Cefixime, Cefoperazone, Cefotaxime, Lactobacillus spp., Lactococcus lactis, Legionella pneumo Cefoxitin, Cefpodoxime, Cefprozil, Ceftaroline, Ceftaroline phila, Legionella spp., Leptospira spp., Listeria monocyto fosamil, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftobi genes, Listeria spp., Methanobacterium extroquens, Micro prole, Ceftriaxone, Cefuroxime, Cephalosporin, Chloram bacterium multiforme, Micrococcus luteus, Moraxella phenicol, Chloramphenicol(Bs), Ciprofloxacin, Clarithro catarrhalis, Mycobacterium, Mycobacterium avium, Myco mycin, Clindamycin, Clofazimine, Cloxacillin, Colistin, bacterium bovis, Mycobacterium diphtheriae, Mycobacte Co-trimoxazole, Cycloserine, Dalbavancin, Dapsone, Dap rium intracellulare, Mycobacterium leprae, Mycobacterium tomycin, Demeclocycline, Dicloxacillin, Dirithromycin, lepraemurium, Mycobacteriumphlei, Mycobacterium Smeg Doripenem, Doxycycline, Enoxacin, Ertapenem, Erythro matis, Mycobacterium spp., Mycobacterium tuberculosis, mycin, Ethambutol, Ethambutol(Bs), Ethionamide, Flu Mycoplasma, Mycoplasma fermentans, Mycoplasma geni cloxacillin, Fluoroquinolone, Fluoroquinolones, Fosfomy talium, Mycoplasma hominis, Mycoplasma penetrans, cin, Furazolidone, Fusidic acid, Gatifloxacin, Mycoplasma pneumoniae, Mycoplasma spp., Neisseria, Geldanamycin, Gemifloxacin, Gentamicin, Grepafloxacin, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria Herbimycin, Imipenem/Cilastatin, Isoniazid, Kanamycin, spp., Nocardia spp., Pasteurella, Pasteurella multocida, Levofloxacin, Lincomycin, Linezolid, Lomefloxacin, Lorac Pasteurella spp., Pasteurella tularensis, Peptostreptococ arbef, Macrollides, Mafenide, Meropenem, Methicillin, Met cus, Porphyromonas gingivalis, PrevOtella melaninogenica ronidazole, Mezlocillin, Minocycline, Moxifloxacin, Mupi (previously called Bacteroides melaninogenicus), Proteus rocin, Nafcillin, Nafcillin, Nalidixic acid, Neomycin, spp., Pseudomonas aeruginosa, Pseudomonas spp., Rhizo Netilmicin, Nitrofurantoin(Bs), Norfloxacin, Ofloxacin, Ori bium radiobacter; Rickettsia, Rickettsia prowazekii, Rickett tavancin, Oxacillin, Oxytetracycline, Paromomycin, Peni sia psittaci, Rickettsia quintana, Rickettsia rickettsia, Rick cillin, Penicillin G, Penicillin V. Piperacillin, Piperacillin/ ettsia spp., Rickettsia trachomae, Rochalimaea, taZobactam, Platensimycin, Polymyxin B, PosiZolid, Rochalinaea hemselae, Rochalimaea quintana, Rothia den Pyrazinamide, Quinupristin/Dalfopristin, Radezolid, Raxi tocariosa, Salmonella, Salmonella enteritidis, Salmonella bacumab, Rifabutin, Rifampicin, Rifampin, Rifapentine, spp., Salmonella typhi, Salmonella typhimurium, Serratia Rifaximin, Roxithromycin, Silver sulfadiazine, Sparfloxa marcescens, Shigella dysenteriae, Shigella spp., Spirillum cin, Spectinomycin, Spectinomycin (BS), Spiramycin, Strep volutans, Staphylococcus, Staphylococcus aureus, Staphy togramins, Streptomycin, Sulbactam, Sulfacetamide, Sulfa lococcus epidermidis, Staphylococcus spp., Stenotrophomo diazine, Sulfadimethoxine, Sulfamethizole, nas maltophilia, Stenotrophomonas spp., Streptococcus, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisox Streptococcus agalactiae, Streptococcus avium, Streptococ azole, Sulfonamidochrysoidine, Tedizolid, Teicoplanin, cus bovis, Streptococcus Cricetus, Streptococcus faceium, Teixobactin, Telavancin, Telithromycin, Temafloxacin, Streptococcus faecalis, Streptococcus ferus, Streptococcus Temocillin, Tetracycline. Thiamphenicol, ticarcillin, Ticar gallinarum, Streptococcus lactis, Streptococcus minor; cillin/clavulanate, Ticarcillin/clavulanic acid, Tigecycline, Streptococcus mitis, Streptococcus mutans, Streptococcus Tigecycline(Bs), Tinidazole, TMP/SMX, Tobramycin, Tor Oxalis, Streptococcus pneumoniae, Streptococcus pyogenes, eZolid, Trimethoprim(Bs), Trimethoprim-Sulfamethoxazole, Streptococcus rattus, Streptococcus salivarius, Streptococ Troleandomycin, Trovafloxacin, Vancomycin, and generics cus sanguis, Streptococcus sobrinus, Streptococcus spp., thereof or a variant thereof. Treponema, Treponema denticola, Treponema pallidum, (0176 Antimicrobials whose interactions with the micro Treponema spp., Ureaplasma spp., Vibrio, Vibrio cholerae, organism affect and are affected by the negative charges on Vibrio comma, Vibrio parahaemolyticus, Vibrio spp., Vibrio the microorganism surface include: polycationic aminogly vulnificus, viridans streptococci, Wolbachia, Yersinia, Yers cosides, which upon binding the cell surface displace Mg" inia enterocolitica, Yersinia pestis, Yersinia pseudotubercu ions, which bridge lipid membrane components, thereby losis., and Yersinia spp. disrupting the outer membrane and enhancing drug uptake; US 2017/0211121 A1 Jul. 27, 2017

cationic polymyxins (colistin and polymyxin B), whose 0180. Other antimicrobial drugs known in the art may be binding to the microorganism cell is also dependent on the used in the present invention. membrane's negative charge and for which both mutational and plasmid-mediated resistance occurs by reducing mem Liquid Suspensions brane negative charge; and daptomycin, a lipopeptide that 0181. A liquid Suspension of microorganisms may by resembles host innate immune response cationic antimicro agitated using mechanical, acoustic, and/or magnetic agita bial peptides and requires Ca" and phosphatidylglycerol tion. Examples of mechanical agitation include shaking or for its membrane-disrupting mechanism of action and for rocking and/or use of stir bars, stir paddles, stir blades, which resistance can also involve alteration in cell Surface and/or stir propellers or impellers. charge. 0182. The microorganism separation is performed by 0177. When the microorganism is a fungus, exemplary centrifugation (e.g., with a g-force >500xg), magnetic sepa antimicrobials include 5-fluorocytosine, Abafungin, Alba ration, filtration (e.g., via a filter having pores Smaller than conazole, Allylamines, Amphotericin B, Ancobon, Anidula or equal to 0.45 microns, and preferably smaller than or fungin, Azole, Balsam of Peru, Benzoic acid, Bifonazole, equal to 0.2 microns), electrophoresis, dielectrophoresis, Butoconazole, Candicidin, Caspofungin, Ciclopirox, Clotri precipitation, agglutination, or any combination thereof. mazole, Cresemba, Crystal violet, Diflucan, Echinocandins, 0183 The liquid may include a growth media, such as Econazole, Efinaconazole, Epoxiconazole, Fenticonazole, cation-adjusted Mueller Hinton broth. This media may con Filipin, Fluconazole, Flucytosine, Grifulvin V, Griseofulvin, tain one or more additives, known to those skilled in the art Gris-Peg, Haloprogin, Hamycin, Imidazoles, Isavuconazole, to promote microorganism growth, and stability. In addition isavuconazonium, Isoconazole, Itraconazole, Ketoconazole, to different antimicrobials, different test wells may contain Lamisil, Luliconazole, Micafungin, Miconazole, Natamy one or more additives known to improve AST accuracy for cin, Noxafil. Nystatin, Omoconazole, Onmel, Oravig. Oxi specific antimicrobials. For example, additional sodium conazole, Posaconazole, Propiconazole, Ravuconazole, chloride may be added to tests comprising oxacillin and Rimocidin, Sertaconazole, Sporanox, Sulconazole, Terbin additional calcium may be added to tests comprising dap afine, Terconazole. Thiazoles. Thiocarbamate antifungal, tomycin. Tioconazole, Tolnaftate, Triazoles, Undecylenic acid, Vfend, Voriconazole, and generics thereof or a variant Cartridges thereof. 0.184 The type of cartridge is not limited. A cartridge is 0.178 When the microorganism is a protozoan, exem a container that is capable of holding and allowing growth plary antimicrobials include 8-Aminoquinoline, Acetarsol, of a liquid Suspension of microorganisms. Non-limited Agents against amoebozoa, Ailanthone, Amodiaquine, examples of a cartridge include a culture flask, a culture Amphotericin B, Amprolium, Antitrichomonal agent, Aplas dish, a petri dish, a bioassay dish, a culture tube, a test tube, momycin, Arsthinol, Artelinic acid, Artemether, Artemether/ a microfuge tube, a bottle, a microwell plate, a multiwell lumefantrine, Artemisinin, Artemotil, Arterolane, Artesu plate, a microtiter plate, a microplate. The cartridge may nate, Artesunatefamodiaquine, Atovaquone, Atovaquone/ contain one chamber. The cartridge may include a plurality proguanil, AZanidazole, Azithromycin, BenZnidazole, of chambers each chamber being a space capable of holding Broxyquinoline, Buparvaquone, CarbarSone, Carnidazole, a liquid Suspension in physical isolation from another space; Chiniofon, Chloroquine, Chlorproguanil, Chlorproguanil/ an example of a chamber is a well in a multiwall plate. The dapsone, Chlorproguanil/dapsone/artesunate, Chlorquinal cartridge may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, dol, Chromalveolate antiparasitics, Cinchona, Cipargamin, 24, 48, 96, 192, 384, 1536, or more chambers, and any ClaZuril, Clefamide, Clioquinol, Coccidiostat, Codinaeop number of chambers in between. sin, Cotrifazid, Cryptolepine, Cycloguanil, Dehydro emetine. Difetarsone, Dihydroartemisinin, Diloxanide, Optical Device Diminazen, Disulfiram, Doxycycline, Eflornithine, ELQ 0185. Any optical device (e.g., microscope, microplate 300, Emetine, Etofamide, Excavata antiparasitics, Fumagil reader) with a number of varying features is capable of lin, Furazolidone, Glycobiarsol, GNF6702, Halofantrine, detecting a signal is useful in the present invention. For Hydroxychloroquine, Imidocarb, Ipronidazole, Jesuits instance: broad spectrum lamp (e.g., Xenon), narrow spec bark, KAF156, Lumefantrine, Maduramicin, Mefloquine, trum lamps, laser, LED, multi-photon, confocal or total MegaZol, Meglumine antimoniate, Melarsoprol, Mepacrine, internal reflection illumination can be used for excitation. Metronidazole, Miltefosine, Neurolenin B, Nicarbazin, Cameras (single or multiple), single or arrays (1D or 2D) of Nifurtimox, Nimorazole, Nitarsone, Nitidine, Nitrofural, photodiodes, avalanche photodiodes, CMOS or CCD sen Olivacine, Ornidazole, Oroidin, Pamaquine, Paromomycin, sors, Solid-state photomultipliers (e.g. silicon photomultipli Pentamidine, Pentavalent antimonial, Phanquinone, Phena ers), and/or Photomultiplier tube (single or multiple) with midine, Piperaquine, Primaquine, Proguanil, Project 523, either filter-based or grating-based spectral resolution (one Propenidazole, Pyrimethamine, Pyronaridine, Quinfamide, or more spectrally resolved emission wavelengths) are pos Quinine, Ronidazole, Schedula Romana, SCYX-7158, Sec sible on the detection side. nidazole, Semapimod, Sodium Stibogluconate, Spiroin dolone, Sulfadoxine, Sulfadoxine-Pyrimethamine, Sul Kits falene, Suramin, Tafenoquine, Teclozan, Tenonitrozole, 0186 The terms "kits' and “systems, as used herein in Tilbroquinol, Tinidazole, Trimetrexate, Trypanocidal agent, the present invention, are intended to refer to such things as Warburg's tincture, and generics thereof or a variant thereof. combinations of multiple signaling agents with one or more 0179 An antimicrobial may be a drug that operates by a other types of elements or components (e.g., other types of mechanism similar to a herein-recited drug. biochemical reagents, signal detection reagents, controls US 2017/0211121 A1 Jul. 27, 2017

(i.e., positive and negative controls, e.g., chemically sensi 0.198. In embodiments, the one or more binding moieties tive/resistant microorganisms), separation means (e.g., fil may bind to one or more biomolecules specifically. ters and magnetic beads), containers, packages such as 0199. In embodiments, the one or more binding moieties packaging intended for commercial sale, Substrates/car may bind to one or more species-specific biomolecules. tridges to which microorganism suspensions can be cul 0200. In embodiments, the one or more binding moieties tured, processed, or contained, electronic hardware compo may bind to the microorganisms non-specifically, e.g., via a nents, and Software recorded on a non-transitory processor non-covalent interaction and via forming a covalent bond. readable medium). 0201 In embodiments, the kit further includes magnetic 0187. Another aspect of the present invention is a kit for beads to magnetically separate the microorganisms from the determining antimicrobial Susceptibility of microorganisms. Supernatant. The kit includes a signaling agent capable of binding to a 0202 In embodiments, the magnetic beads are associated Surface of the intact microorganisms of interest; a solution with one or more binding moieties that bind to microorgan for incubating a sample containing microorganisms; and one isms. The one or more binding moieties associated with the or more reagents for generating signals from the signaling magnetic beads may identical to those associated with the agent. signaling agents. The one or more binding moieties associ 0188 In embodiments, the signaling agent is associated ated with the magnetic beads may be different than those with one or more binding moieties capable of binding associated with the signaling agents. directly or indirectly to the intact microorganisms of interest. 0203. In embodiments, the magnetic beads have diam 0189 In embodiments, the one or more binding moieties eters ranging between 20 nm to 20 microns. are selected from antibodies, lectins, natural and/or synthetic peptides, synthetic and/or natural ligands, synthetic and/or 0204. In embodiments, the kit further includes one or natural polymers, synthetic and/or natural glycopolymers, more ions or Small molecules to enhance the binding carbohydrade-binding proteins and/or polymers, glycopro between the binding moieties and the microorganism. tein-binding proteins and/or polymers, charged Small mol 0205. In embodiments, the solution comprises <0.15 M ecules, other proteins, bacteriophages, and/or aptamers. salt. 0190. In embodiments, the one or more binding moieties 0206. In embodiments, the kit further includes a micro may be a polyclonal and/or monoclonal antibody. organism binding agent, and wherein the binding moiety 0191 In embodiments, the one or more binding moieties binds to the microorganisms indirectly via the microorgan may be a synthetic and/or natural ligand and/or peptide. The ism binding agent. The binding moiety may be conjugated to ligand and/or peptide may be selected from bis(zinc-dipi streptavidin, neutravidin, or avidin and the microorganism colylamine), TAT peptide, serine proteases, cathelicidins, binding agent may be biotinylated. The binding moiety may cationic dextrins, cationic cyclodextrins, salicylic acid, be an antibody that binds to a species specific Fc domain and lysine, and a combinations thereof. the microorganism binding agent may be an antibody 0.192 In embodiments, the one or more binding moieties capable of binding to the microorganisms with the species may be a synthetic and/or natural polymer and/or glycopo specific Fc domain. lymer. The natural and/or synthetic polymer may be amy 0207. In embodiments, the signaling agent may include lopectin, Poly(N-3-(dimethylamino)propyl methacrylam one or more of a chemiluminophore, a catalyst, or an ide), poly(ethyleneimine), poly-L-lysine, polyI2-(N.N- enzyme. The enzyme may be at least one of horseradish dimethylamino)ethyl methacrylate, and combinations peroxidase, alkaline phosphatase, acetylcholinesterase, glu thereof. The natural and/or synthetic polymer and/or glyco cose oxidase, beta-D-galactosidase, beta-lactamase, and polymer my include moieties including, but not limited to, combinations thereof. The catalyst may be an organometal chitosan, gelatin, dextran, trehalose, cellulose, mannose, lic compound. cationic dextrans and cyclodextrans, or combinations 0208. In embodiments, the signaling agent is provided in thereof including, but not limited to, co-block, graft, and a form of a nanoparticle, e.g., the signaling agent is encap alternating polymers. Sulated within a nanoparticle. The nanoparticle may be 0193 In embodiments, the one or more binding moieties dissociable, which may include a metal oxide; the metal may include a glycoprotein selected from mannose-binding oxide may be or include iron oxide, cesium oxide, and/or lectin, other lectins, annexins, and combinations thereof. cerium oxide. 0194 In embodiments, the one or more binding moieties 0209. In embodiments, Zero, one, or two washes are may include two or more binding moieties. performed prior to determining signal levels. 0.195. In embodiments, the one or more binding moieties 0210. In embodiments, Zero, one, or two washes are may bind directly or indirectly to one or more biomolecules performed prior to addition of the signaling agents. present on the microorganism surface. Exemplary biomol 0211. In embodiments, the kit further includes a devel ecules include peptidoglycans, mureins, mannoproteins, oper reagent to produce a measurable signal. porins, beta-glucans, chitin, glycoproteins, polysaccharides, 0212. In embodiments, the one or more reagents include lipopolysaccharides, lipooligosaccharides, lipoproteins, reagents for a catalytic reaction and a reagent that stops the endotoxins, lipoteichoic acids, teichoic acids, lipid A, car catalytic reaction. bohydrade binding domains, efflux pumps, other cell-wall 0213. In embodiments, the kit further comprises a device and/or cell-membrane associated proteins, other anionic for measuring signal, e.g., an optical and/or electrical signal. phospholipids, and a combination thereof. The optical measurement may be fluorescent, time-resolved 0196. In embodiments, the binding moiety is a nanopar fluorescent, absorbent, and/or luminescent. ticle. 0214. In embodiments, the kit further comprises a mul 0197) In embodiments, the binding moiety is a bacterio tiwell plate, e.g., a 24-well, 96-well, 192-well, or 384-well phage. plate. US 2017/0211121 A1 Jul. 27, 2017

0215. In embodiments, the further contains instructions ments, a signaling agent is present after an incubating step for using the kit to perform a herein-disclosed method. The of a method described herein. kit may additionally contain instructions for performing 0224. In embodiments, binding comprises the formation steps conducted prior to or Subsequent to one or more of a covalent bond. In embodiments, a signaling agent is methods as described herein. capable of binding to the Surface of a microorganism, 0216. In one embodiment, kits are provided which con wherein said binding comprises the formation of a covalent tain the necessary reagents to carry out one or more methods bond. In embodiments, a method as described herein results as described herein or reagents necessary to carry out steps in the formation of a covalent bond between a group on a prior to or Subsequent to one or more methods as described microorganism Surface (e.g., via a reactive group Such as an herein. electrophilic or nucleophilic group as described herein) and a signaling agent as described herein. In embodiments, a Treatment Methods signaling agent has formed a covalent bond to the Surface of a microorganism. 0217. As used herein, the terms “treat,” “treating,” “treat 0225. In embodiments, binding comprises the formation ment, and the like refer to reducing or ameliorating a of a non-covalent interaction. In embodiments, a signaling disorder and/or a symptom associated therewith. It will be agent is capable of binding to the Surface of a microorgan appreciated that, although not precluded, treating a disorder ism, wherein said binding comprises the formation of a or condition does not require that the disorder, condition or non-covalent interaction. In embodiments, a method as symptoms associated therewith be completely eliminated. described herein results in the formation of non-covalent Treating may include a health care professional or diagnostic interaction between a group on a microorganism Surface Scientist making a recommendation to a subject for a desired (e.g., via a reactive group Such as an electrophilic or course of action or treatment regimen, e.g., a prescription. nucleophilic group as described herein) and a signaling 0218. As used herein, the terms “prevent,” “preventing.” agent as described herein. In embodiments, a signaling agent “prevention,” “prophylactic treatment” and the like refer to has formed a non-covalent interaction with the Surface of a reducing the probability of developing a disorder or condi microorganism. tion in a Subject, who does not have, but is at risk of or 0226. In embodiments, a non-covalent interaction com Susceptible to developing a disorder or condition. prises: ionic interaction, ion-ion interaction, dipole-dipole 0219. The term “methods of treating includes methods interaction, ion-dipole interaction, electrostatic interaction, of managing, and when used in connection with the infec London dispersion, van der Waals interaction, hydrogen tions microbial organism or infection, includes the amelio bonding, JU-T interaction, hydrophobic interaction, or any ration, elimination, reduction, prevention, or other relief or combination thereof. In embodiments, a non-covalent inter management from the detrimental effects of the infections action is: ionic interaction, ion-ion interaction, dipole-dipole microbe. interaction, ion-dipole interaction, electrostatic interaction, 0220. As used herein, the terms “drug”, “medication', London dispersion, Van der Waals interaction, hydrogen “therapeutic”, “active agent”, “therapeutic compound', bonding, JU-T interaction, hydrophobic interaction, or any “composition’, or "compound are used interchangeably combination thereof. and refer to any chemical entity, pharmaceutical, drug, 0227. In embodiments, a non-covalent interaction com biological, botanical, and the like that can be used to treat or prises ionic interactions, van der Waals interactions, hydro prevent a disease, illness, condition, or disorder of bodily phobic interactions, JC-T interactions, or hydrogen bonding, function. A drug may comprise both known and potentially or any combination thereof. In embodiments, a non-covalent therapeutic compounds. A drug may be determined to be interaction comprises ionic interaction, Van der Waals inter therapeutic by Screening using the screening known to those action, hydrogen bonding, or JC-T interaction, or any com having ordinary skill in the art. A “known therapeutic bination thereof. compound”, “drug, or “medication” refers to a therapeutic 0228. In embodiments, a signaling agent capable of bind compound that has been shown (e.g., through animal trials ing to a microorganism's Surface comprises a group (e.g., a or prior experience with administration to humans) to be chemical or biochemical group) capable of binding micro effective in such treatment. A “therapeutic regimen” relates organism membranes, walls, proteins, organelles, saccha to a treatment comprising a "drug”, “medication”, “thera rides, lipids, cell envelope, or nucleic acids, or any combi peutic', 'active agent”, “therapeutic compound”, “compo nation thereof. In embodiments, a signaling agent capable of sition’, or “compound as disclosed herein and/or a treat binding to a microorganism’s Surface comprises a chemical ment comprising behavioral modification by the Subject group (e.g., a nucleophilic group or an electrophilic group) and/or a treatment comprising a Surgical means. capable of binding microorganism membranes, walls, pro 0221. An antimicrobial, e.g., an antibiotic, is an agent teins, organelles, saccharides, lipids, cell envelope, or capable of killing a microorganism or inhibiting the growth nucleic acids, or any combination thereof. In embodiments, of a microorganism. a signaling agent capable of binding to a microorganism’s Surface comprises a biochemical group capable of binding Signaling Agents and Chemical Attachment microorganism membranes, walls, proteins, organelles, sac charides, lipids, cell envelope, or nucleic acids, or any 0222. In embodiments, the invention features a signaling combination thereof. agent capable of binding to the Surface of a microorganism. 0229. In embodiments, the surface may include a biomol In embodiments, said binding is non-specific. In embodi ecule to which the signaling agent binds or associates. ments, said binding is specific. Exemplary biomolecules include peptidoglycans, mureins, 0223. In embodiments, a signaling agent is present during mannoproteins, porins, beta-glucans, chitin, glycoproteins, an incubating step of a method described herein. In embodi polysaccharides, lipopolysaccharides, lipooligosaccharides, US 2017/0211121 A1 Jul. 27, 2017 lipoproteins, endotoxins, lipoteichoic acids, teichoic acids, 0244. In embodiments, a linker group L comprises a lipid A, carbohydrate binding domains, efflux pumps, other group (e.g., a chemical or biochemical group) that is capable cell-wall and/or cell-membrane associated proteins, other of forming a covalent bond to a microorganism’s Surface. In anionic phospholipids, and a combination thereof. embodiments, a linker group L comprises a group (e.g., a 0230. In embodiments, a signaling agent capable of bind chemical or biochemical group) that forms a covalent bond ing to a microorganism’s Surface comprises a biochemical to a microorganism’s Surface. group capable of binding microorganism membranes, walls, 0245. In embodiments, a linker group L comprises a proteins, organelles, saccharides, lipids, cell envelope, or group (e.g., a chemical or biochemical group) that is capable nucleic acids, or any combination thereof. of forming one or more non-covalent interactions with a 0231. In embodiments, a signaling agent capable of bind microorganism’s Surface. In embodiments, a linker group L ing to a microorganism's Surface comprises a chemical comprises a group (e.g., a chemical or biochemical group) group (e.g., a nucleophilic or electrophilic functional group) that forms one or more non-covalent interactions with a capable of binding microorganism membranes, walls, pro microorganism’s Surface. teins, organelles, saccharides, lipids, cell envelope, or 0246. In embodiments, a linker group L comprises a nucleic acids, or any combination thereof. In embodiments, chemical moiety 101, wherein said chemical moiety is said chemical group is a nucleophilic functional group. In capable of forming a non-covalent interaction with the embodiments, said chemical group is an electrophilic func Surface of a microorganism. In embodiments, a linker group tional group. L comprises a chemical moiety 101, wherein said chemical 0232. In embodiments, a signaling agent is a biochemical moiety is capable of forming a covalent bond with the signaling agent. In embodiments, a biochemical signaling Surface of a microorganism. In embodiments, a linker group agent comprises a biomolecule Such as an antibody, ligand, L comprises a chemical moiety 101, wherein said chemical protein, aptamer, ss-DNA, ss-RNA, or ss-PNA). moiety forms a non-covalent interaction with the Surface of 0233. In embodiments, a signaling agent is a chemical a microorganism. In embodiments, a linker group L com signaling agent. In embodiments, a chemical signaling agent prises a chemical moiety 101, wherein said chemical moiety is a chemical compound (e.g., a synthetic chemical com forms a covalent bond with the Surface of a microorganism. pound). In embodiments, a chemical signaling agent does 0247. In embodiments, a linker group L comprises a not comprise a biomolecule Such as an antibody, ligand, spacer moiety 102. In embodiments, spacer moiety 102 is protein, aptamer, ss-DNA, ss-RNA, or ss-PNA). covalently attached to chemical moiety 101 and/or to chemi 0234. In embodiments, a signaling agent capable of bind cal moiety 103. In embodiments, spacer moiety 102 is ing to a microorganism’s Surface comprises covalently attached to chemical moiety 101. In embodi 0235 a linker group L; and ments, spacer moiety 102 is covalently attached to chemical 0236 an amplifier group (e.g., an amplifier group 104 moiety 103. In embodiments, spacer moiety 102 is cova that is a chemical or biochemical amplifier). lently attached to chemical moiety 101 and to chemical 0237. In embodiments, an amplifier group is an amplifier moiety 103. In embodiments, spacer moiety 102 forms a group 104, which is a chemical or biochemical amplifier. In non-covalent interaction with chemical moiety 101 and/or embodiments, an amplifier group 104 is a chemical ampli with chemical moiety 103. In embodiments, spacer moiety fier. In embodiments, an amplifier group 104 is a biochemi 102 forms a non-covalent interaction with chemical moiety cal amplifier. 101. In embodiments, spacer moiety 102 forms a non 0238. In embodiments, a signaling agent is a chemical covalent interaction with chemical moiety 103. In embodi compound. In embodiments, a chemical compound com ments, spacer moiety 102 forms a non-covalent interaction prises a chemical amplifier group Such as those described with chemical moiety 101 and with chemical moiety 103. herein). 0248. In embodiments, a linker group L comprises a 0239. In embodiments, a linker group L comprises the chemical moiety 103, wherein said chemical moiety is conserved (Fc) region of an antibody. In embodiments, a capable of forming a covalent bond to an amplifier group linker group L is capable of forming a covalent bond to an (e.g., an amplifier group 104 that is a chemical or biochemi amplifier group (e.g., an amplifier group 104 that is a cal amplifier). In embodiments, a linker group L comprises chemical or biochemical amplifier). a chemical moiety 103, wherein said chemical moiety has 0240. In embodiments, a linker group L forms a covalent formed a covalent bond to an amplifier group (e.g., an bond to a signal amplifier group (e.g., an amplifier group 104 amplifier group 104 that is a chemical or biochemical that is a chemical or biochemical amplifier). amplifier). In embodiments, a linker group L comprises a 0241. In embodiments, a linker group L is capable of chemical moiety 103, wherein said chemical moiety is forming one or more non-covalent interactions to an ampli capable of forming a non-covalent interaction with an ampli fier group (e.g., an amplifier group 104 that is a chemical or fier group (e.g., an amplifier group 104 that is a chemical or biochemical amplifier). biochemical amplifier). In embodiments, a linker group L 0242. In embodiments, a linker group L forms one or comprises a chemical moiety 103, wherein said chemical more non-covalent interactions to an amplifier group (e.g., moiety has formed a non-covalent interaction with an ampli an amplifier group 104 that is a chemical or biochemical fier group (e.g., an amplifier group 104 that is a chemical or amplifier). biochemical amplifier). 0243 In embodiments, a linker group L comprises a 0249. In embodiments, a signaling agent is a chemical group (e.g., a chemical or biochemical group) capable of compound comprising a linker group L that comprises: binding a microorganism surface. In embodiments, a linker 0250 a chemical moiety 101, wherein said chemical group L comprises a group (e.g., a chemical or biochemical moiety is capable of forming a covalent bond or a non group) that binds a microorganism Surface. covalent interaction with the Surface of the microorganisms; US 2017/0211121 A1 Jul. 27, 2017

0251 a spacer moiety 102, wherein spacer moiety is chlorotriazines, epoxides, isocyanates, isothiocyanates, covalently attached to chemical moiety 101 and to chemical organic acids, monomers, polymers, silanes, or silcates, or moiety 103; and any combination thereof. 0252) a chemical moiety 103, wherein said chemical 0270. In embodiments, agents that promote coupling moiety has formed or can form a covalent bond to an include a carbodiimide, a phosphonium salt, or an ammo amplifier group 104 that is a chemical or biochemical nium salt, or any combination thereof. amplifier. 0271 In embodiments agents that promote coupling 0253. In embodiments, a signaling agent is a chemical include glutaraldehyde, N-(3-dimethylaminopropyl)-N'-eth compound comprising a linker group L that comprises: ylcarbonate (EDC), (1-bis(dimethylamino)methylene)-1H 0254 a chemical moiety 101, wherein said chemical 1, 2,3-triazolo 4,5-b]pyridinium 3-oxid hexafluorophos moiety is capable of forming a covalent bond or a non phate) (HATU), (O-benzotriazol-1-yl-N,N.N N covalent interaction with the Surface of a microorganism; -tetramethyluronium hexafluorophosphate) (HBTU), N-hy 0255 a spacer moiety 102, wherein spacer moiety is droxysuccinimide (NHS), N,N'-dicyclohexylcarbodiimide covalently attached to chemical moiety 101 and to chemical (DCC), diisopropylcarbodiimide (DIC), hydroxy-3,4-di moiety 103; and hydro-4-oxo-1,2,3-benzotriazine (HOOBt), hydroxybenzo 0256 a chemical moiety 103, wherein said chemical triazole (HOBT), 1-hydroxy-7-azabenzotriazole (HOAt), moiety has formed or can form a non-covalent interaction (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide with an amplifier group 104 that is a chemical or biochemi (EDAC), 4-(N,N-dimethylamino)pyridine (DMAP), benzo cal amplifier. triazol-1-yloxy-tris(dimethylamino)- phosphonium 0257. In embodiments, a linker group comprises one hexafluorophosphate (BOP), benzotriazol-1-yloxy-tripyrro chemical moiety 101. In embodiments, a linker group com lidino-phosphonium hexafluorophosphate (PyBOP), bromo prises more than one chemical moiety 101 (e.g., a linker tripyrrolidino-phosphonium hexafluorophosphate (Py group comprises 1, 2, 3, 4, 5, or 6 chemical moieties 101). BrOP), 7-aza-benzotriazol-1-yloxy 0258. In embodiments, a linker group comprises one tripyrrolidinophosphonium hexafluorophosphate (PyAOP), spacer moiety 102. In embodiments, a linker group com ethylcyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)- prises more than one spacer moiety 102 (e.g., a linker group phosphonium hexafluorophosphate (PyOxim), 3-(diethoxy comprises 1, 2, 3, 4, 5, or 6 spacer moieties 102). phosphoryloxy)-1,2,3-benzod triazin-4(3H)-one 0259. In embodiments, a linker group comprises one (DEPBT), 2-(6-chloro-1H-benzotriazol-1-yl)- N.N.N',N'-te chemical moiety 103. In embodiments, a linker group com tramethylaminium hexafluorophosphate (HCTU), N-(5- prises more than one chemical moiety 103 (e.g., a linker chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino group comprises 1, 2, 3, 4, 5, or 6 chemical moieties 103). uronium hexafluorophosphate N-oxide (HDMC), 1-1- 0260. In embodiments, a linker group comprises: one (cyano-2-ethoxy-2-oxoethylideneaminooxy)- chemical moiety 101, one spacer moiety 102, and one dimethylamino-morpholino-uronium hexafluorophosphate chemical moiety 103. In embodiments, a linker group con (COMU), 2-(1-oxy-pyridin-2-yl)-1,1,3,3-tetramethylisoth sists of one chemical moiety 101, one spacer moiety 102. iouronium tetrafluoroborate (TOTT), tetramethylfluorofor and one chemical moiety 103. mamidinium hexafluorophosphate (TFFH), N-Ethoxycarbo 0261. In embodiments, a linker group has the structure of nyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ), substructure (I): 2-propanephosphonic acid anhydride (PPA), triphosgene, 1,1-carbonyldiimidazole (CDI), (6-nitrobenzotriazol-1-yl) -101-102-103-, (I) oxytris(pyrrolidino)phosphonium hexafluorophosphate 0262 wherein (PyNOP), 6-(trifluoromethyl)benzotriazol-1-yl)oxytris 0263) “101 represents a chemical moiety 101; (pyrroli-dino)phosphonium hexafluorophosphate (PyFOP), 0264) “102 represents a spacer moiety 102; and 4-nitro-6-(trifluoromethyl)benzotriazol-1-yl)oxytris(pyr 0265 “103 represents a chemical moiety 103. rolidino)phosphonium hexafluorophosphate (PyNFOP), (6- 0266. In embodiments, a chemical moiety 101 is capable nitrobenzo-triazol-1-yOoxytris(dimethyl-amino)phospho of forming a covalent bond with the surface of a microor nium hexafluorophosphate (NOP), 1-B- ganism. naphthalenesulfonyloxy benzotriazole (NSBt), 1-3- 0267 In embodiments, a chemical moiety 101 is capable naphthalenesulfonyloxy-6-nitrobenzotriazole (N-NSBt), of forming a covalent bond with the surface of a microor tetramethylfluoroformamidinium hexafluorophosphate ganism in the presence of one or more agents that promote (TFFH), bis(tetramethylene) fluoroformamidinium coupling (also referred to herein as coupling agents). hexafluorophosphate (BTFFH), 1,3-dimethyl-2-fluoro-4, 0268. In embodiments, agents that promote coupling 5-dihydro-1H-imidazolium hexafluorophosphate (DFIH), include glutaraldehyde, formaldehyde, paraformaldehyde, Cyanuric chloride (CC), or 2,4-dichloro-6-methoxy-1,3,5- 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), triazine (DCMT), and 2-chloro-4,6-dimethoxy-1,3,5-triaz N,N'-dicyclohexylcarbodiimide (DCC), N-cyclohexyl-N'- ine (CDMT), or any combination thereof. (2-morpholinoethyl)carbodiimide-methyl-p-toluenesul 0272. In embodiments, agents that promote coupling fonate (CMC), diisopropylcarbodiimide (DIC), (1-bis(dim include EDC, HATU, HBTU, NHS, DCC, HOBT, or ethylamino)methylene)-1H-1,2,3-triazolo 4,5-b]pyridinium PyBOP or any combination thereof. 3-oxid hexafluorophosphate) (HATU), Woodwards 0273. In embodiments, agents that promote coupling Reagent, N,N'-carbonyl diimidazole, N-hydroysuccinimide include EDC, DCC, CMC, DIC, or HATU, or any combi (NHS), or N-hydroxysulfosuccinimide (sulfo-NHS), or any nation thereof. combination thereof. 0274. In embodiments, agents that promote coupling 0269. In embodiments, agents that promote coupling include glutaraldehyde, formaldehyde, or paraformalde include aldehydes, acrylates, amides, imides, anhydrides, hyde, or any combination thereof. US 2017/0211121 A1 Jul. 27, 2017

0275. In embodiments, a chemical moiety 101 is capable 0285. In embodiments, an electrophilic functional group of forming a non-covalent interaction with the Surface of a comprises —CHO, —C(O)CHI, microorganism (e.g., any non-covalent interaction described herein). In embodiments, a non-covalent interaction com : prises: ionic, ion-ion, dipole-dipole, ion-dipole, electro : static, London dispersion, Vander Waals, hydrogen bonding, No or JC-TU, or any combination thereof. O N O, O N O, O O 0276. In embodiments, a chemical moiety 101 is capable of forming a non-covalent interaction with the Surface of a Nr. Nyrulu microorganism, wherein said non-covalent interaction com ne C. : prises ionic interactions, van der Waals interactions, hydro \7 N phobic interactions, JC-T interactions, or hydrogen bonding, 2 Or or any combination thereof. s' O O 0277. In embodiments, a chemical moiety 101 comprises a nucleophilic functional group. In embodiments, a chemical moiety 101 comprises a group formed from a nucleophilic functional group. 0278. In embodiments, a nucleophilic functional group is: amino, amido, hydrazino, hydroxyamino, hydroxy, or 0286. In embodiments, an electrophilic functional group thio. In embodiments, a nucleophilic functional group is: comprises —CHO. —C(O)CHI, amino, hydrazino, hydroxyamino, or thio. In embodiments, a nucleophilic functional group comprises: amino, : hydrazino, hydroxyamino, hydroxy, or thio. In embodi : ments, a nucleophilic functional group is carboxamide, No N-hydroxycarboxamide, carboxyl hydrazide, or guanidino. 0279. In embodiments, a nucleophilic functional group is NH, NHNH CONHOH, CONHNH - ONH, —OH, or —SH. In embodiments, a nucleophilic functional group is NH, -NHNH – CONHNH, or - ONH. 0287. In embodiments, chemical moiety 101 comprises a 0280. In embodiments, a chemical moiety 101 comprises group that is alkyl, alkenyl, alkynyl, phenyl, heteroaryl, an electrophilic functional group. haloalkyl, hydroxy, carbonyl, acyl halide, alkoxycarbonyl) 0281. In embodiments, a chemical moiety 101 comprises oxy, carboxy, haloketone, alkoxy, alkoxyol (hemiacetal or) a group formed from an electrophilic functional group. hemiketal, dialkoxy (e.g., ketal or acetal), trialkoxy(orthoe 0282. In embodiments, an electrophilic functional group ther), carbamoyl, amino, ammonio, imino, imido, Succina comprises an aldehyde, a ketone, a carboxylic acid, a mido, maleidido, hydroxySuccinamido, biotin, D-Biotin, carboxylic ester, a carboxylic acid halide (e.g., acetyl chlo azido, azo, cyanate, isocyanato, nitroxy, cyano, isocyano, ride), or a carboxylic acid anhydride (e.g., acetic anhydride). nitroSooxy, nitro, nitroso, oxime, Sulfanyl, Sulfinyl, Sulfonyl, Sulfino, Sulfo, thiocyanato, isothiocyanato, thioyl, phos 0283. In embodiments, an electrophilic functional group phate, or boronate. comprises an aldehyde, an a-halo ketone, a maleimide, a Succinimide, a hydroxysuccinimide, an isothiocyanate, an 0288. In embodiments, spacer moiety 102 is hydropho isocyanate, an acyl azide, a Sulfonyl chloride, a tosylate bic. In embodiments, spacer moiety 102 is hydrophilic. ester, a glyoxal, an epoxide, an oxirane, a carbonate, an 0289. In embodiments, spacer moiety 102 is peptidic imidoester, an anhydride, a fluorophenyl ester, a hydroxym (e.g., derived from peptide linkages). ethyl phosphine derivative, a carbonate, a haloacetyl, a 0290. In embodiments, spacer moiety 102 comprises chlorotriazine, a haloacetyl, an alkyl halide, an aziridine, or inorganic linkages. In embodiments, spacer moiety 102 an acryloyl derivative. In embodiments, an electrophilic comprises organic linkages. In embodiments, spacer moiety functional group is an aldehyde, an a-halo ketone, a maleim 102 comprises only organic linkages. ide, a Succinimide, a hydroxysuccinimide, an isothiocya 0291. In embodiments, spacer moiety 102 is oligomeric. nate, an isocyanate, an acyl azide, a Sulfonyl chloride, a In embodiments, spacer moiety 102 is polymeric. In tosylate ester, a glyoxal, an epoxide, an oxirane, a carbonate, embodiments, spacer moiety 102 comprises segments (e.g., an imidoester, an anhydride, a fluorophenyl ester, a 1 to about 300, 1 to about 200, 1 to about 100, 1 to about 50, hydroxymethyl phosphine derivative, a carbonate, a halo 1 to about 25, or 1 to about 10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or acetyl, a chlorotriazine, a haloacetyl, an alkyl halide, an 10 segments) of methylene (—CH2—), ethylene glycol aziridine, or an acryloyl derivative. (—CHCHO ), iminoethylene ( CHCH-NH ), vinyl alcohol ( CHCHOH ), lactic acid (-CH(CH)—C 0284. In embodiments, an electrophilic functional group (O)—O—), acrylic acid ( CH-CH(COH)—), meth comprises an aldehyde, an a-halo ketone, a maleimide, a acrylic acid ( CHC(CH)(COH)—), or methyl meth Succinimide, or a hydroxysuccinimide group. acrylate (—CHC(CH)(COCH)—). US 2017/0211121 A1 Jul. 27, 2017 20

0292. In embodiments, spacer moiety 102 comprises a 0297. In embodiments, spacer moiety 102 comprises segment that is

H H V A N : in N-1- N-n M V H.J. : N B : : V / B-N A V H H HO In embodiments, t is an integer of 1 to about 300 (e.g., 1 to 0293. In embodiments, n, m, p, and q independently is an about 200, 1 to about 100, 1 to about 50, 1 to about 25, or integer of 1 to about 300 (e.g., 1 to about 200, 1 to about 100, 1 to about 10). In embodiments, t is independently 1, 2, 3, 1 to about 50, 1 to about 25, or 1 to about 10). In 4, 5, 6, 7, 8, 9, or 10. embodiments, each of n, m, p, and q is independently 1, 2, 0298. In embodiments, spacer moiety 102 comprises: 3, 4, 5, 6, 7, 8, 9, or 10. 0294. In embodiments, spacer moiety 102 comprises O

Y NNH : O

0299. In embodiments, spacer moiety 102 is a polymer R comprising repeating groups, comprising alkyl, alkoxy, ester, acrylic, amino, hydroxyl, or acyl hydrazine functional groups, or any combination thereof In embodiments, R is independently hydrogen or a group 0300. In embodiments, the spacer moiety 102 is: that is C-C alkyl, C-C alkenyl, or C-C alkynyl. In embodiments, o is an integer of 1 to about 300 (e.g., 1 to about 200, 1 to about 100, 1 to about 50, 1 to about 25, or 1 to about 10). In embodiments, o is independently 1, 2, 3, .-his. N-1so s N-n} , or 4, 5, 6, 7, 8, 9, or 10. 0295) In embodiments, spacer moiety 102 comprises -n. HO

R O H wherein n, m, p, and q are as defined herein : N 0301 In embodiments, each of n, m, o, p, q, r, S, or t N : independently is an integer of 1 to 100, of 10 to 90, of 10 to H 80, of 10 to 70, of 10 to 60, of 10 to 50, of 10 to 40, of 10 O R * to 30, of 10 to 20, or of 1 to 10. 0302) In embodiments, a chemical moiety 103 comprises a group that is a nucleophilic functional group. In embodiments, R is independently hydrogen or a group 0303. In embodiments, a chemical moiety 103 comprises that is C-C alkyl, C-C alkenyl, or C-C alkynyl. In a group formed from a nucleophilic functional group. embodiments, r is an integer of 1 to about 300 (e.g., 1 to 0304. In embodiments, a nucleophilic functional group about 200, 1 to about 100, 1 to about 50, 1 to about 25, or is: amino, amido, hydrazino, hydroxyamino, hydroxy, or 1 to about 10). In embodiments, r is independently 1, 2, 3, thio. In embodiments, a nucleophilic functional group is: 4, 5, 6, 7, 8, 9, or 10. amino, hydrazino, hydroxyamino, or thio. 0305. In embodiments, a nucleophilic functional group 0296 In embodiments, spacer moiety 102 comprises comprises: amino, hydrazino, hydroxyamino, hydroxy, or thio. In embodiments, a nucleophilic functional group is carboxamide, N-hydroxycarboxamide, carboxyl hydrazide, or guanidino. Sc's. 0306 In embodiments, a nucleophilic functional group is R. R. NH, -NHNH, CONHOH, -CONHNH, ONH, —OH, or —SH. In embodiments, a nucleophilic functional group is NH, -NHNH, -CONHNH, or - ONH. In embodiments, R is independently hydrogen or a group 0307. In embodiments, a chemical moiety 103 comprises that is C-C alkyl, C-C alkenyl, or C-C alkynyl. In a group that is a electrophilic functional group. embodiments, s is an integer of 1 to about 300 (e.g., 1 to 0308. In embodiments, a chemical moiety 103 comprises about 200, 1 to about 100, 1 to about 50, 1 to about 25, or a group formed from a electrophilic functional group. 1 to about 10). In embodiments, s is independently 1, 2, 3, 0309. In embodiments, an electrophilic functional group 4, 5, 6, 7, 8, 9, or 10. comprises an aldehyde, a ketone, a carboxylic acid, a US 2017/0211121 A1 Jul. 27, 2017 carboxylic ester, a carboxylic acid halide (e.g., acetyl chlo thiol, maleimide, Succinimide, hydroxySuccinimide, bioti ride), or a carboxylic acid anhydride (e.g., acetic anhydride). nyl, anhydride, chlorotriazine, epoxide, isocyanate, or iso 0310. In embodiments, an electrophilic functional group thiocyanate is capable of forming non-covalent interaction comprises an aldehyde, an O-halo ketone, a maleimide, a with an amplifier group (e.g., an amplifier group 104). Succinimide, a hydroxysuccinimide, an isothiocyanate, an 0315. In embodiments, a chemical moiety 103 is formed isocyanate, an acyl azide, a Sulfonyl chloride, a tosylate from a chemical structure comprising a group that is carbo nyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, ester, a glyoxal, an epoxide, an oxirane, a carbonate, an Succinimide, hydroxySuccinimide, biotinyl, anhydride, chlo imidoester, an anhydride, a fluorophenyl ester, a hydroxym rotriazine, epoxide, isocyanate, or isothiocyanate. In ethyl phosphine derivative, a carbonate, a haloacetyl, a embodiments, said group that is carbonyl, alkenyl, alkynyl, chlorotriazine, a haloacetyl, an alkyl halide, an aziridine, or hydroxyl, amino, thiol, maleimide, Succinimide, hydroxy an acryloyl derivative. In embodiments, an electrophilic Succinimide, biotinyl, anhydride, chlorotriazine, epoxide, functional group is an aldehyde, an a-halo ketone, a maleim isocyanate, or isothiocyanate has formed a covalent bond to ide, a Succinimide, a hydroxysuccinimide, an isothiocya an amplifier group (e.g., an amplifier group 104). In embodi nate, an isocyanate, an acyl azide, a Sulfonyl chloride, a ments, said group that is carbonyl, alkenyl, alkynyl, tosylate ester, a glyoxal, an epoxide, an oxirane, a carbonate, hydroxyl, amino, thiol, maleimide, Succinimide, hydroxy an imidoester, an anhydride, a fluorophenyl ester, a Succinimide, biotinyl, anhydride, chlorotriazine, epoxide, hydroxymethyl phosphine derivative, a carbonate, a halo isocyanate, or isothiocyanate has formed a non-covalent acetyl, a chlorotriazine, a haloacetyl, an alkyl halide, an interaction with an amplifier group (e.g., an amplifier group aziridine, or an acryloyl derivative. 104). 0311. In embodiments, an electrophilic functional group 0316. In embodiments, a chemical moiety 103 comprises comprises an aldehyde, an O-halo ketone, a maleimide, a a group that is carbonyl, alkenyl, alkynyl, hydroxyl, amino, Succinimide, or a hydroxysuccinimide group. thiol, maleimide, Succinimide, hydroxySuccinimide, or 0312. In embodiments, an electrophilic functional group biotinyl. comprises —CHO. —C(O)CHI, 0317. In embodiments, a chemical moiety 103 comprises a carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, Succinimide, hydroxysuccinimide, or biotinyl : functional group. : No 0318. In embodiments, a chemical moiety 103 comprises:

= --~~.us ls CNes N C N

0313. In embodiments, an electrophilic functional group comprises —CHO, —C(O)CHI,

: : No

0319. In embodiments, a chemical moiety 103 comprises a group formed from a chemical structure comprising a 0314. In embodiments, a chemical moiety 103 comprises group that is carbonyl, alkenyl, alkynyl, hydroxyl, amino, a chemical structure that is carbonyl, alkenyl, alkynyl, thiol, maleimide, Succinimide, hydroxySuccinimide, or hydroxyl, amino, thiol, maleimide, Succinimide, hydroxy biotinyl functional group. Succinimide, biotinyl, anhydride, chlorotriazine, epoxide, 0320 In embodiments, a linker group L has the structure isocyanate, or isothiocyanate. In embodiments, said group of substructure (II): that is carbonyl, alkenyl, alkynyl, hydroxyl, amino, thiol, maleimide, Succinimide, hydroxysuccinimide, biotinyl, anhydride, chlorotriazine, epoxide, isocyanate, or isothio (II) cyanate is capable of forming a covalent bond to an amplifier x-his-hy group (e.g., an amplifier group 104). In embodiments, said group that is carbonyl, alkenyl, alkynyl, hydroxyl, amino, US 2017/0211121 A1 Jul. 27, 2017 22

0321 wherein -continued 0322 X represents a chemical moiety 101 (e.g., any : chemical moiety 101 as described herein; No 0323 R represents a spacer moiety 102 (e.g., any spacer moiety 102 as described herein); 0324 Y represents a chemical moiety 103 (e.g., any chemical moiety 103 as described herein); and O 0325 each of and k independently is an integer of 0 to O NH H. 1OO. 1". 0326 In embodiments, X is O

: : : V YNH SNH, o-NH. O 22 22 2 nea O n N : r I, O N O, O N O, NH2, SNH, o-NH, O C N C an. O 4N. O 2 s S 2

N N 0329. In embodiments, Xis capable of forming a covalent c.1N, or 2n, bond to a microorganisms surface. In embodiments, X O 2 s S 2 forms a covalent bond to a microorganism’s Surface. 0330. In embodiments, X is capable of forming one or 0327. In embodiments, R is more non-covalent interactions with a microorganism’s Sur face. In embodiments, X forms one or more non-covalent

: : : interactions with a microorganism’s Surface. -hs. N-1s 1n 0331. In embodiments, Y is capable of forming a covalent bond to an amplifier group 104 (e.g., a chemical or bio O O chemical amplifier). In embodiments, Y forms a covalent bond to an amplifier group Such as an amplifier group 104 R (e.g., a chemical or biochemical amplifier.) N-n Nlp ". 1. *, 0332. In embodiments, Y is capable of forming one or HO more non-covalent interactions to an amplifier group 104 O R O (e.g., a chemical or biochemical amplifier). In embodiments, Y forms one or more non-covalent interactions to an ampli - - nN.1- N. - N : fier group Such as an amplifier group 104 (e.g., a chemical H H s or biochemical amplifier.) O O R * H H 0333. In embodiments, a linker group L is: V A : N M =N P1 . or *-N B-*, (13) / \ V / R. R. B-N M V HN -r-ol. H H wherein each of n, m, o, p, q, r, S, or t is as described herein S O (e.g., an integer of 1 to about 300). 0328. In embodiments, Y is HNy US 2017/0211121 A1 Jul. 27, 2017

-continued -continued (14) (18) O Oy-o-y HN-PEG / cott HN-PEG s S O O S O HNy HN O y-st (19) O (15) O HN-PEG-NH N. O.

O HN-PEG / O HN NH - S O y (20) O O HN 'V \/ y Yo-Na', O-N O

O O

(16) HN -PEG-NH2, HNy O S

WGA-Biotin, PolymixinB-Biotin, monoclonal antibody, polyclonal antibody, biotinylated monoclonal antibody, biotinylated polyclonal antibody, europium chelate-anti y body, horseradish peroxidase-conjugated antibody, and anti O body variants (e.g., Fab: fragment, antigen-binding (one arm), F(ab')2: fragment, antigen-binding, including hinge region (both arms); Fab: fragment, antigen-binding, includ ing hinge region (one arm); ScFV: single-chain variable fragment, di-scFv: dimeric single-chain variable fragment; (17) sdAb: single-domain antibody; Bispecific monoclonal anti bodies; trifunctional antibody; and BiTE: bi-specific T-cell engager), HN - PEG / s. 0334 Exemplary amplifier groups include those described in, e.g., International Publication No. WO 2016/ S O 015027 and in International Application No. PCT/US16/ 42589, each of which is incorporated by reference in its entirety.

HN 0335. In embodiments, an amplifier group (e.g., an ampli fier group 104) comprises a catalyst, a fluorophore, or a y colormetric dye. In embodiments, an amplifier group (e.g., O an amplifier group 104) is a catalyst, a fluorophore, or a colormetric dye. US 2017/0211121 A1 Jul. 27, 2017 24

0336. In embodiments, an amplifier group (e.g., an ampli coordination complex, a ruthenium coordination complex, a fier group 104) comprises an enzyme, a catalyst, or a rhenium coordination complex, a palladium coordination nanoparticle. In embodiments, an amplifier group (e.g., an complex, a platinum coordination complex, a Samarium amplifier group 104) is an enzyme, a catalyst, or a nano coordination complex, a terbium coordination complex, or a particle. dysprosium coordination complex. 0349. In embodiments, an amplifier group (e.g., an ampli 0337. In embodiments, a chemical amplifier group com fier group 104) comprises a chemiluminophore. In embodi prises a catalyst, a fluorophore, a nanoparticle, or a color ments, an amplifier group (e.g., an amplifier group 104) metric dye. In embodiments, a chemical amplifier group is comprises a quantum dot. In embodiments, an amplifier a catalyst, a fluorophore, a nanoparticle, or a colormetric group (e.g., an amplifier group 104) comprises an enzyme. dye. In embodiments, an amplifier group (e.g., an amplifier group 0338. In embodiments, an amplifier group (e.g., an ampli 104) comprises an iron coordination catalyst. In embodi fier group 104) comprises a catalyst. In embodiments, an ments, an amplifier group (e.g., an amplifier group 104) amplifier group (e.g., an amplifier group 104) is a catalyst. comprises a europium coordination complex. In embodi 0339. In embodiments, an amplifier group (e.g., an ampli ments, an amplifier group (e.g., an amplifier group 104) fier group 104) comprises a fluorophore. In embodiments, an comprises a ruthenium coordination complex. In embodi amplifier group (e.g., an amplifier group 104) is a fluoro ments, an amplifier group (e.g., an amplifier group 104) phore. Exemplary fluorophores include those described in comprises a rhenium coordination complex. In embodi Table 1 of International Application No. PCT/US16/42589, ments, an amplifier group (e.g., an amplifier group 104) which is incorporated by reference in its entirety. comprises a palladium coordination complex. In embodi 0340. In embodiments, an amplifier group (e.g., an ampli ments, an amplifier group (e.g., an amplifier group 104) fier group 104) comprises a colormetric dye. In embodi comprises a platinum coordination complex. In embodi ments, an amplifier group (e.g., an amplifier group 104) is a ments, an amplifier group (e.g., an amplifier group 104) colormetric dye. comprises a Samarium coordination complex. In embodi 0341 In embodiments, an amplifier group (e.g., an ampli ments, an amplifier group (e.g., an amplifier group 104) fier group 104) comprises an enzyme. In embodiments, an comprises a terbium coordination complex. In embodi amplifier group (e.g., an amplifier group 104) is an enzyme. ments, an amplifier group (e.g., an amplifier group 104) 0342. In embodiments, an amplifier group (e.g., an ampli comprises a dysprosium coordination complex. fier group 104) comprises a nanoparticle. In embodiments, 0350. In embodiments, an amplifier group 104 comprises an amplifier group (e.g., an amplifier group 104) is a a moiety that is: nanoparticle. 0343. In embodiments, an amplifier group (e.g., an ampli fier group 104) comprises a lanthanide. (III) 0344. In embodiments, an amplifier group (e.g., an ampli fier group 104) comprises a lanthanide that is europium, strontium, terbium, Samarium, or dysprosium. In embodi ments, an amplifier group (e.g., an amplifier group 104) comprises a lanthanide selected from the group consisting of europium, strontium, terbium, Samarium, and dyspro sium. 0345. In embodiments, an amplifier group (e.g., an ampli fier group 104) comprises an organic fluorophore. 0346. In embodiments, an amplifier group (e.g., an ampli fier group 104) comprises a fluorophore that is a coordina tion complex. 0347 In embodiments, an amplifier group (e.g., an ampli fier group 104) comprises a europium coordination complex. In embodiments, a coordination complex is a europium

coordination complex. In embodiments, an amplifier group (IV) (e.g., an amplifier group 104) comprises a ruthenium coor dination complex. In embodiments, a coordination complex is a ruthenium coordination complex. In embodiments, an amplifier group (e.g., an amplifier group 104) comprises a rhenium coordination complex. In embodiments, a coordi nation complex is a rhenium coordination complex. In embodiments, an amplifier group (e.g., an amplifier group 104) comprises a palladium coordination complex. In embodiments, a coordination complex is a palladium coor dination complex. In embodiments, an amplifier group (e.g., an amplifier group 104) comprises a platinum coordination complex. In embodiments, a coordination complex is a platinum coordination complex. 0348. In embodiments, an amplifier group (e.g., an ampli fier group 104) comprises a chemiluminophore, a quantum dot, an enzyme, an iron coordination catalyst, a europium US 2017/0211121 A1 Jul. 27, 2017

-continued -continued (V) (VIII)

COO

0351. In embodiments, an amplifier group 104 comprises a moiety that is:

(VI)

(IX)

a R all lo Osi-O N F3. CNN s 1SN-(N)V tu y lo sul NTSN 2 V / H (X) OH

(VII) '',

Osilo rsa eO:

ap1 N-/YN OS 0352. In embodiments, an amplifier group 104 is a cata N N/S3 \N lyst or enzyme. In embodiments, an amplifier group is a (N horseradish peroxidase, alkaline phosphatase, acetylcholin Na esterase, glucose oxidase, beta-D-galactosidase, or beta lactamase. O P-Q) 0353. In embodiments, amplifier group 104 is horserad A. ish peroxidase. I 0354. In embodiments, amplifier group 104 is a fluoro phore or colormetric dye. 0355 Suitable fluorophores and colormetric dyes are well known to those skilled in the art and are described in The Molecular Probes(R Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11" Ed. (2010) and Gomes, Fernandes, and Lima J. Biochem. Biophys. Methods 65 (2005) pp. 45-80, which are herein incorporated by reference in their entirety. Exemplary fluorophores also include those described in, e.g., International Publication US 2017/0211121 A1 Jul. 27, 2017 26

No. WO 2016/015027 and in International Application No. U.S. Pat. No. 9,056,138, U.S. Pat. No. 9,118,028, U.S. Pat. PCT/US16/42589, each of which is incorporated by refer No. 9,133,205, U.S. Pat. No. 9,187,690, U.S. Pat. No. ence in its entirety. 9,193,746, U.S. Pat. No. 9,312,496, U.S. Pat. No. 9,337,432, 0356. Examples of suitable fluorophore or colormetric U.S. Pat. No. 9,343,685, U.S. Pat. No. 9,391,288, and U.S. dyes include, but are not limited to, ethidium bromide, Pat. No. 9,537,107, which are incorporated by reference in propidium iodide, SYTOX green, phenanthridines, acri their entirety. Exemplary organometallic compounds, tran dines, indoles, imidazoles, cyanine, TOTO, TO-PRO, sition metal complexes, or coordination complexes also SYTO, 5-carboxy-2,7-dichlorofluorescein, 5-Carboxyfluo include those described in, e.g., International Publication rescein (5-FAM), 5-Carboxynapthofluorescein, 5-Car No. WO 2016/015027 and in International Application No. boxytetramethylrhodamine (5-TAMRA), 5-FAM (5-Car PCT/US16/42589, each of which is incorporated by refer boxyfluorescein), 5-HAT (Hydroxy Tryptamine), 5-ROX ence in its entirety. (carboxy-X-rhodamine), 6-Carboxyrhodamine 6G, 0358. In embodiments, amplifier group 104 is a lan 7-Amino-4-methylcoumarin, 7-Aminoactinomycin D thanide coordination complex. (7-AAD), 7-Hydroxy-4-methylcoumarin, 9-Amino-6- 0359. In embodiments, a lanthanide coordination com chloro-2-methoxyacridine, ACMA (9-Amino-6-chloro-2- plex is a complex between a lanthanide (e.g., Eu or Tb) and methoxyacridine), Acridines, Alexa Fluors, Alizarin, Allo a tetradentate ligand. phycocyanin (APC), AMCA (Aminomethylcoumarin), 0360. In embodiments, a lanthanide coordination com Bodipy, Carboxy-X-rhodamine, Catecholamine, Fluorescein plex is a complex between a lanthanide (e.g., Eu or Tb) and (FITC), Hydroxycoumarin, Lissamine Rhodamine, Mono a cryptate ligand. bromobimane, Oregon Green, Phycoerythrin, SYTO. Thi 0361. In embodiments, amplifier group 104 is a coordi adicarbocyanine (DiSC3). Thioflavin, X-Rhodamine, C or nation complex of Lanthanum (La), Cerium (Ce), Praseo TetramethylRodaminelsoThioCyanate. dymium (Pr), Neodymium (Pm), Samarium (Sm), Europium 0357. In embodiments, amplifier group 104 is an orga (Eu), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), nometallic compound, transition metal complex, or coordi Holmium (Ho), Erbium (Er), Thulium (Tm). Ytterbium nation complex. Exemplary examples are described in but (Yb), Lutetium (Lu), Ruthenium (Ru), Rhodium (Rh), Pal not limited to EP 0180 492, EPO 321 353, EPO 539 435, ladium (Pd), Osmium (Os), Iridium (Ir), or Platinum (Pt). EP0539 477, EPO 569 496, EP139675, EP64484, U.S. Pat. 0362. In embodiments, amplifier group 104 is a coordi No. 4,283,382, U.S. . No. 4,565,790, U.S. Pat. No. nation complex of a rare earth metal collectively refers to 17 4,719, 182, U.S. Pat. No. 4,735,907, U.S. Pat. No. 4,808,541, elements consisting of a group of 15 elements from lantha U.S. Pat. No. 4,927,923, U.S. Pat. No. 5,162,508, U.S. Pat. num having an atomic number of 57 to lutetium having an No. 5,220,012, U.S. Pat. No. 5,324,825, U.S. Pat. No. atomic number of 71 (lanthanides), and two additional 5 346,996, U.S. Pat. No. 5,373,093, U.S. Pat. No. 5,432,101, elements consisting of Scandium having an atomic number J.S. Pat. No. 5,457,185, U.S. Pat. No. 5,512,493, U.S. Pat. of 21 and yttrium having an atomic number of 39. Specific o. 5,527,684, U.S. Pat. No. 5,534,622, U.S. Pat. No. examples of rare earth metals include europium, terbium, N 627,074, U.S. Pat. No. 5,696,240, U.S. Pat. No. 6,6,100,394, lanthanum, cerium, praseodymium, neodymium, prome .S. Pat. No. 6,340,744, U.S. Pat. No. 6,524,727, U.S. Pat. thium, Samarium, gadolinium, dysprosium, holmium, o. 6,717,354, U.S. Pat. No. 7,067,320, U.S. Pat. No. erbium, thulium, ytterbium, lutetium, Scandium and yttrium, 364,597, U.S. Pat. No. 7,393,599, U.S. Pat. N 56,023, with europium and terbium being preferable, and europium .S. Pat. No. 7,465,747, U.S. Pat. No. 7,625, 9 o S. Pat. being more preferable. o. 7,854,919, U.S. Pat. No. 7,910,088, U.S.s Pat. No. 0363. In embodiments, amplifier group 104 is a coordi 955,859, U.S. Pat. No. 7,968,904, U.S. Pat. N 07,926, nation complex of a lanthanide (e.g., Europium or Terbium) .S. Pat. No. 8,012,609, U.S. Pat. No. 8,017, S. Pat. with diethylenetriaminetetraacetic acid or a cryptate ligand. o. 8,018,145, U.S. Pat. No. 8,048,659, U. at. No. 0364. In embodiments, amplifier group 104 is a coordi 067,100, U.S. Pat. No. 8,129,897, U.S. Pat. N 8, 74,001, nation complex of a lanthanide (e.g., Europium or Terbium) .S. Pat. No. 8,183,586, U.S. Pat. No. 8, 193, 1 S. Pat. with diethylenetriaminetetraacetic acid. o. 8,221,719, U.S. Pat. No. 8,288,763, U.S. Pat. No. 0365. In embodiments, amplifier group 104 is a coordi 362,691, U.S. Pat. No. 8,383.249, U.S. Pat. N 8, 92,783, nation complex of a lanthanide (e.g., Europium or Terbium) .S. Pat. No. 8,632,753, U.S. Pat. No. 8,663,603, U.S. Pat. with a cryptate ligand. o. 8,722,881, U.S. Pat. No. 8,754,206, U.S. Pat. No. 0366. In embodiments, a signaling agent (e.g., a chemical 8,890,402, U.S. Pat. No. 8,969,862, U.S. Pat. No. 9,012,034, signaling agent) comprises or is formed from:

(1) (2)

Eu-cryptate-maleimide Eu-cryptate-NHS US 2017/0211121 A1 Jul. 27, 2017 27

-continued (3) (4)

- C COO- COO>> COO COO Eu-N1-ITC (Delfia)

(5) (6) C W y H H2 N-( )- N1 N N

COO COO

COO Eu-N1-amino Eu-N1-DTA

(7) (8)

Eu-N1-iodoacetamido

(9) (10) US 2017/0211121 A1 Jul. 27, 2017 28

-continued (11)

(12)

0367. In embodiments, a signaling agent may comprise may consist, for example, of ligands from the group of one or more paramagnetic metal chelates in order to form a porphyrins, bipyridyls, phenanthrolines or other heterocy contrast agent. Preferred paramagnetic metal ions have clical compounds. atomic numbers 21-29, 42, 44, or 57-83. This includes ions 0370. In embodiments, a signaling agent capable of bind of the transition metal or lanthanide series which have one, ing a microorganism Surface comprises an antibody (e.g., and more preferably five or more, unpaired electrons and a monoclonal or polyclonal), modified antibodies (e.g., bioti nylated monoclonal antibody, biotinylated polyclonal anti magnetic moment of at least 1.7 Bohr magneton. Preferred body, europium chelate-antibody, horseradish peroxidase paramagnetic metals include chromium (III), manganese conjugated antibody), antibody variants (e.g., Fab: fragment, (II), manganese (III), iron (II), iron (III), cobalt (II), nickel antigen-binding (one arm), F(ab')2 fragment, antigen-bind (II), copper (II), praseodymium (III), neodymium (III), ing, including hinge region (both arms); Fab': fragment, Samarium (III), gadolinium (III), terbium (III), dysprosium antigen-binding, including hinge region (one arm); scEv: (III), holmium (III), erbium (III), europium (III) and ytter single-chain variable fragment; di-ScFV: dimeric single bium (III). Additionally, a signaling agent of the present chain variable fragment, SdAb: Single-domain antibody; invention may also comprise one or more Superparamag Bispecific monoclonal antibodies; trifunctional antibody; netic particles: and BiTE: bi-specific T-cell engager), WGA-Biotin, Poly 0368. In embodiments, a signaling agent may comprise mixinB-Biotin, lectin, natural peptide, synthetic peptides, synthetic and/or natural ligands, synthetic and/or natural one or more metals that are included in a metal complex polymers, synthetic and/or natural glycopolymers, carbohy along with or as a part of a fluorescent compound: The metal drate-binding proteins and/or polymers, glycoprotein-bind complex includes metal complexes having Al, Zn, Be, or the ing proteins and/or polymers, charged Small molecules, like; a rare-earth metal such as Tb, Eu, or Dy; or a transition other proteins, bacteriophages, and/or aptamers. metal Such as Pt or Ir as a central metal, and having an 0371. As used herein, the term “antibody” referrers to any oxadiazole, thiadiazole, phenylpyridine, phenylbenzimida herein-described type of antibody, modified antibody, or Zole, or quinoline structure as a ligand. Such as aluminum antibody fragment or type of antibody, modified antibody, or quinolinol complexes, benzoquinolinol beryllium com antibody fragment as known in the art. Thus, “a signaling plexes, benzoxazole Zinc complexes, benzothiazole Zinc agent comprising an antibody' includes, as examples, a complexes, azomethyl Zinc complexes, porphyrin Zinc com signaling agent comprising an unmodified monoclonal anti plexes, and europium complexes. body, a Fab fragment, and a trifunctional antibody. 0369. In embodiments, a signaling agent may comprise a 0372. In embodiments, a signaling agent capable of bind luminophore (donor) which features high luminescence ing a microorganism surface comprises a lanthanide coor quantum efficiency and long luminescence decay time (>100 dination complex, biotin, antibody, and/or an enzyme. ins). Preferred luminophores are cationic, metalorganic com 0373) In embodiments, a signaling agent capable of bind plexes of palladium, rhodium, platinum, ruthenium, ing a microorganism Surface comprises or is formed from a osmium, rare earths (in particular, europium and lantha structure comprising an antibody, lectin, natural peptide, num). The organic portion of these metalorganic complexes synthetic peptides, synthetic and/or natural ligands, Syn US 2017/0211121 A1 Jul. 27, 2017 29 thetic and/or natural polymers, synthetic and/or natural 0385. In embodiments, a signaling agent capable of bind glycopolymers, carbohydrate-binding proteins and/or poly ing to a microorganism Surface comprises: mers, glycoprotein-binding proteins and/or polymers, charged Small molecules, other proteins, bacteriophages, 0386 an antibody; and and/or aptamers. 0387 a europium coordination complex. 0374. In embodiments, a signaling agent capable of bind ing a microorganism Surface comprises an amplifier group 0388. In embodiments, a signaling agent capable of bind 104 that comprises a lanthanide coordination complex, ing to a microorganism Surface comprises a linker group L and/or an enzyme and streptavidin and/or an antibody and/or that comprises NH-PEG-Biotin (2K), NH-PEG-Biotin aptamer. (4K), sulfo-NHS-Biotin, WGA-Biotin, or polymixinB-Bio tin. 0375. In embodiments, a signaling agent capable of bind ing a microorganism Surface comprises a binding moiety 0389. In embodiments, a signaling agent capable of bind comprising a polyclonal and/or monoclonal antibody. ing to a microorganism Surface comprises a Europium 0376. In embodiments, a signaling agent capable of bind complex comprises: ing a microorganism Surface comprises a binding moiety comprising a modified antibody. Exemplary modified anti bodies include a biotinylated monoclonal antibody, bioti (III) nylated polyclonal antibody, a europium chelate-antibody, and a horseradish peroxidase-conjugated antibody. 0377. In embodiments, a signaling agent capable of bind ing a microorganism Surface comprises a binding moiety comprising an antibody variant. Exemplary antibody Vari ants include Fab: fragment, antigen-binding (one arm): F(ab')2 fragment, antigen-binding, including hinge region (both arms); Fab': fragment, antigen-binding, including hinge region (one arm); scFv: single-chain variable frag ment; di-scFV: dimeric single-chain variable fragment; sdAb: single-domain antibody; Bispecific monoclonal anti bodies; trifunctional antibody; and BiTE: bi-specific T-cell engager), 0378. In embodiments, a signaling agent capable of bind ing a microorganism Surface comprises WGA-Biotin or (IV) PolymixinB-Biotin. 0379. In embodiments, a signaling agent capable of bind ing a microorganism Surface comprises a binding moiety comprising a synthetic and/or natural ligand and/or peptide. 0380. In embodiments, a ligand and/or peptide is selected from bis(zinc-dipicolylamine), TAT peptide, serine pro teases, cathelicidins, cationic dextrins, cationic cyclodex trins, salicylic acid, lysine, and combinations thereof. 0381. In embodiments, a signaling agent capable of bind ing a microorganism Surface comprises a binding moiety comprising a synthetic and/or natural polymer and/or gly copolymer. 0382. In embodiments, a natural and/or synthetic poly mer is linear or branched and selected from amylopectin, Poly(N3-(dimethylamino)propyl)methacrylamide), poly (ethyleneimine), poly-L-lysine, polyI2-(N,N-dimethyl amino)ethyl methacrylate, and combinations thereof. 0383. In embodiments, a natural and/or synthetic poly mer and/or glycopolymer comprises moieties including, but (V)

not limited to, chitosan, gelatin, dextran, trehalose, cellulose, mannose, cationic dextrans and cyclodextrans, quaternary amines, pyridinium tribromides, histidine, lysine, cysteine, arginine, Sulfoniums, phosphoniums, or combinations thereof including, but not limited to, co-block, graft, and alternating polymers. COO 0384. In embodiments, a signaling agent capable of bind ing a microorganism Surface comprises a binding moiety 0390. In embodiments, a signaling agent capable of bind comprising a glycoprotein selected from mannose-binding ing to a microorganism Surface comprises a Europium lectin, other lectins, annexins, and combinations thereof. complex comprises: US 2017/0211121 A1 Jul. 27, 2017 30

EP139675; EP64484; US 2013/0217063; US 2014/ 0278136; US 2014/0323340; US 2014/0363817; US 2015/ 0064703; US 2015/0337351; US 2016/0010138; U.S. Pat. No. 3,798,320; U.S. Pat. No. 4,565,790; U.S. Pat. No. 4,647,536; U.S. Pat. No. 4,808,541; U.S. Pat. No. 4,927,923; U.S. Pat. No. 5,457,185; U.S. Pat. No. 5,489,401; U.S. Pat. No. 5,512,493; U.S. Pat. No. 5,527,684; U.S. Pat. No. 5,627,074; U.S. Pat. No. 5,665,554; U.S. Pat. No. 5,695,946; U.S. Pat. No. 6,284.470; U.S. Pat. No. 6,385,272; U.S. Pat. a No. 6,844,028: U.S. Pat. No. 7,341,841; U.S. Pat. No. 7,629,029; U.S. Pat. No. 7,868,144;U.S. Pat. No. 8,178,602; O R O Sa 2O Sa a U.S. Pat. No. 8,895,255; PCT/US2016/042589; and WO/2016015027 each of which is incorporated herein by reference in their entireties. N 0399. Any of the above aspects and embodiments can be N1-S- N SN combined with any other aspect or embodiment as disclosed 2 V / H in the Drawings, in the Summary of the Invention, and/or in OH the Detailed Description, including the below Examples. 0400 Although methods and materials similar or equiva lent to those described herein can be used in the practice or testing of the present invention, Suitable methods and mate rials are described below. All publications, patent applica '', tions, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In addition, the materials, methods, and examples | are illustrative only and are not intended to be limiting. EXAMPLES Os O rSa aO. p1 N- S Example 1 a -S- The Present Invention Provides Rapid and Accurate s N7 \N Determination of an Antimicrobials Minimum a X Inhibitory Concentration (MIC) 04.01. In this example, the present invention for rapidly O ". N h determining an antimicrobials minimum inhibitory concen tration (MIC) against Staphylococcus aureus or Pseudomo I nas aeruginosa was compared to a standard method requir ing an overnight incubation of a S. aureus or P. aeruginosa. 0402. A culture of Staphylococcus aureus (ATCC strain 29213) was grown using Mueller-Hinton (MH) broth over night at 37° C. with vigorous shaking. Concurrently, two sterile 96-well microplates were prepared with serial dilu tions of clindamycin from 32 ug/ml to 0.125 ug/ml and a 0391 As disclosed in the below working Examples and no-clindamycin control, all in MH broth. The S. aureus throughout the Specification and Drawings, the present concentration from the overnight culture was then set to invention provides, at least: 5x10 CFU/ml using the McFarland standard technique for 0392 >89.9% MIC agreement (+1 dilution) between optical density readings at 600 nm. The microplates, each presently-disclosed methods and CLSI standard with well containing 200 uL were inoculated with prepared no major/very major errors for seventy-five Strains of dilutions of antimicrobial and incubated at 37° C. for 3.5 twelve bacterial species (including B-lactams with hours for determining antimicrobial Susceptibly using a gram-negative rods); herein-disclosed invention (e.g., the “fast-AST' technique) 0393 Equivalent MICs between the presently-dis or incubated at 37° C. overnight (>12 hours) for the ODoo closed method for direct-from-positive-blood culture control. The “fast-AST microplate was removed from the and CLSI standard blood culture sample processing shaking incubator after 4 hours and a horseradish peroxidase 0394) Detection of gram-positive and negative species (HRP) conjugate of a polyclonal rabbit-anti-S. aureus anti down to 2x10 CFU/ml: body (Fitzgerald Industries International, Inc.) was added to 0395. Non-specific binding of a microorganism by a each well. The plate was then shaken at room temperature signaling agent; for 20 minutes to allow binding, and afterwards, the 0396 Use of Europium formulations; microplate was centrifuged at 4,000xg in order to pellet the 0397) Semi-automated device use with data output. remaining intact bacteria. The MH broth was then aspirated 0398. Additional teaching relevant to the present inven and sterile broth added for a total of 3 washes. After the final tion are described in one or more of the following: aspiration, a stabilized development Solution consisting of US 2017/0211121 A1 Jul. 27, 2017

3.3',5,5'-tetramethylbenzidine (TMB) and hydrogen perox strains that are antimicrobial resistant to, respectively, the ide (ThermoFisher) was added, and the optical density at MICs for Paeruginosa, S. aureus, and E. coli Strains that are 650 nm and 450 nm were monitored for 10 minutes with a antimicrobial-sensitive. microplate reader (Vmax, Molecular Devices). After an 0407. The MIC for the susceptible P. aeruginosa strain, overnight incubation, the ODoo control microplate was ATCC 27853, was determined as described in Example 1, removed from the incubator, and the optical density at 600 with the key difference that imipenem was used as the nm was read directly (Vmax, Molecular Devices). Finally, antimicrobial (a serial dilution from 32 g/ml to 0.125ug/ml the MIC was determined per CLSI standards from the data, was used). The MIC for the resistant P aeruginosa strain, as shown in FIG. 5. The MIC determined by both techniques ATCC BAA-2108, was determined similarly. The same is the same: 0.125 ug/ml 96-well microplate was used for both strains, with 48 wells 0403. Similarly, a culture of Pseudomonas aeruginosa dedicated to each strain. The experiment was repeated 3 (ATCC strain 27853) was grown using MH broth overnight times with similar results, and the resulting data are shown at 37° C. with vigorous shaking. Concurrently, two sterile in FIG. 7. The MIC for the susceptible strain is 2 ug/ml; the 96-well microplates were prepared with serial dilutions of MIC for the resistant strain is 32 ug/ml. ceftazidime from 32 g/ml to 0.125 ug/ml and a no-ceftazi 0408 For S. aureus, the same procedure as described dime control, all in MH broth. The P. aeruginosa concen above in Example 1 was used, with the exceptions that tration from the overnight culture was then set to 5x10 methicillin was used as the antimicrobial and a resistant CFU/ml using the McFarland standard technique for optical strain, ATCC 43300, was used. For E. coli, the same density readings at 600 nm. These microplates, each well procedure as described above for P. aeruginosa was used, containing 200 uL, were inoculated with the prepared anti except E. coli susceptible (25922) and resistant (35218) microbial dilutions and incubated at 37° C. for 3.5 hours for strains were used and ampicillin was used as the antimicro determining antimicrobial Susceptibly using a herein-dis bial, and a HRP-conjugate of a polyclonal rabbit-anti-E. coli closed invention or incubated at 37° C. overnight (>12 antibody (Abcam) was used as the chemical moiety that hours) for the OD control. The “fast-AST microplate allows the signaling agent to bind the bacterium. The was removed from the shaking incubator after 4 hours and “fast-AST values after a five-minute incubation with detec a solution of a horseradish peroxidase (HRP) conjugate of a tion solution were compared with ODoo overnight controls, polyclonal rabbit-anti-P aeruginosa antibody (Abcam) was and the data is compiled in FIG. 8. added to each well. The plate was then shaken at room 04.09. These data show that the present invention is temperature for 20 minutes to allow binding, and afterwards, accurately able to differentiate an antimicrobials MIC for a the microplate was centrifuged at 4,000xg in order to pellet strain of bacteria that is resistant to the antimicrobial and a the remaining intact bacteria. The MH broth was then strain of the same bacteria that is sensitive to the antimi aspirated and sterile broth added for a total of 3 washes. crobial. After the final aspiration, a stabilized development solution consisting of 3,3',5,5'-TMB and hydrogen peroxide (Ther Example 3 moFisher) was added, and the optical density at 650 nm and 450 nm were monitored for 10 minutes with a microplate The Present Invention Provides a Detectible Signal reader (Vmax, Molecular Devices). The data shown in FIG. at Microbial Concentration That is Two-Hundred 6 depicts from the 5 minute point after the start of incubation Fold Less Concentrated than is Required for a with detection solution. After an overnight incubation, the Standard Method ODoo control microplate was removed from the incubator, 0410. In this example, the microbial concentration and the optical density at 600 nm was read directly (Vmax, required to provide a detectable signal in present invention Molecular Devices). Finally, the MIC was determined per for was compared to a standard method requiring an over CLSI standards from the data, as shown in FIG. 6. The MIC night incubation of a S. aureus. determined by both techniques is the same: 4 g/ml. 0411 S. aureus was cultured overnight as described in 04.04 The accuracy of the present invention in determin Example 1. A serial dilution of the overnight colony was ing MIC is clearly demonstrated in that the slope of the made in a 96-well microplate and the absorbance was read downward region of the data (in FIG. 5 and in FIG. 6) is at 600 nm. These values were compared with McFarland nearly identical between the present invention and the standards to obtain the bacteria concentration in CFU/ml. overnight cultures. The quantifiable region of the curve is shown, in FIG. 9 (ODoo); the experiment was repeated three times with 04.05 These data show that the present invention is similar results. A similar dilution series of S. aureus was accurately able to determine an antimicrobials MIC which treated with S. aureus-specific signaling agents for 20 min, is at least as accurate as a standard method requiring an as described in Example 2. Following Example 2s proce overnight incubation of a bacterial culture. dure, the microorganisms were centrifuged and washed three times, and a detection solution was added. Example 2 0412. In FIG. 9, the resulting absorbance is shown vs. the McFarland standard-determined S. aureus concentration. The Present Invention Provides Rapid and Accurate The “fast-AST signal is visible from the starting bacteria Determination of an Antimicrobials Minimum concentration of a Clinical Laboratory Standards Institute Inhibitory Concentration (MIC) for (CLSI)-standard AST experiment (i.e., 5x10 CFU/ml), as Antimicrobial-Resistant Bacteria shown by the arrow. In contrast, the optical signal does not enable accurate quantification until ~10 CFU/ml. 0406. In this example, the present invention was used to 0413. These data show that the present invention is able compare the MICs for P aeruginosa, S. aureus, and E. coli to provide a detectible and usable signal at microbial culture US 2017/0211121 A1 Jul. 27, 2017 32 concentration that is two-hundred fold less concentration S. aureus and K. pneumoniae results are shown in FIG. 12A than that required for a standard method. to FIG. 12C. There was excellent agreement between MIC values obtained from the present invention “fast-AST' and Example 4 the CLSI-obtained results for all experiments except for the erythromycin experiment with S. aureus and the tetracycline The Present Invention Provides MICs Values and imipenem experiments with K. pneumonia; however, Similar to Those Obtained from the CLSI according to the FDA, the discrepancies between the present Reference Method Across Multiple Species and invention and the CLSI results are “minor errors', see, FIG. Strains of Pathogenic Bacteria, Yet in Significantly 12C Less Time than Required for the CLSI Method 0419. These data show that the present invention is 0414. In this example, the present invention for rapidly accurately able to determine, for two dissimilar bacterial determining an antimicrobials MIC for a plurality of patho species, a plurality of antimicrobials MIC which are at least genic bacteria was compared to the Clinical Laboratory as accurate as the CLSI method, yet takes significantly less Standards Institute (CLSI) method. time to determine the MIC; thus, the present invention 0415. As shown in FIG. 10, MIC determinations for six greatly reduces time before a patient is provided an appro bacteria were obtained after 3.5-hour incubations, whereas priate treatment regimen, i.e., a specific antimicrobial and at the CLSI AST reference method determinations were a particular dosage. obtained after sixteen hour incubations (for amplicillin Example 6 treated cultures) or twenty-four hour incubations (for oxa cillin-treated cultures). The drug, signaling agent/chemical Using Multiple S. aureus and E. coli Clinical moiety (“antibody-HRP conjugate'), and bacteria strains are Strains, across a Wide Variety of Antimicrobials, listed in FIG. 11. A for the signaling agent/chemical moiety, the Present Invention Provides MICs Values wheat germ agglutinin (WGA) HRP conjugate was used for Similar to Those Obtained from the CLSI S. epidermidis testing, and the “fast-AST' assay follows the Reference Method, Yet in Significantly Less Time procedure of Example 2 above. All clinical isolates were de-identified samples and were sub-cultured a minimum of 0420. In this example, the present invention was used to two times before use. A total of eighty-seven individual rapidly determine a plurality of antimicrobials’ MICs when samples were tested, including, but not limited to, the treating S. aureus (a gram-positive bacterium) or E. coli (a following bacterial species: E. coli, S. aureus, P. aeruginosa, gram-negative bacterium) and compared to MIC values K. pneumoniae, E. faecalis, Coagulase-Negative Staphyllo obtained by the CLSI method. cocci, P. mirabilis, E. faecium, E. clocae, and A. baumannii. 0421. As in Example 5, SensiTitre(R) plate (Thermo It is noteworthy that the bacterial species tested in this Fisher) was used to perform these experiments. The same example (except P. mirabilis) are together responsible for procedure was used as described in Example 2, except 50 L >90% of positive blood cultures in many clinical laborato of inoculum was added to each well, according to Thermo ries. Thus, the present invention has clear clinical relevance Fisher's instructions. The CLSI reference method was per to human infectious diseases. The MIC values between the formed for twenty-four hours (oxacillin and Vancomycin) present invention and the CLSI method are highly similar, and eighteen hours (levofloxacin) and for all experiments yet the present invention requires a three and half hour using the present invention (“fast-AST method) was per incubation whereas the CLSI method requires sixteen hour formed in four hours (including a three and half hour or twenty-four hour incubations. incubation). Results are shown in FIG. 13A to FIG. 13C and 0416) These data show that the present invention is FIG. 14A to FIG. 14D. The dark lines in FIG. 13 A to FIG. accurately able to determine an antimicrobials MIC which 13C and FIG. 14A to FIG. 14D show the CLSI breakpoints is at least as accurate as the CLSI method, yet takes for each antimicrobial. Essential Agreement “EA' and Cat significantly less time to determine the MIC; thus, the egorical Agreement “CA' are defined as by the FDA in their present invention greatly reduces time before a patient is Class II Guidance Document for Automated AST Systems. provided an appropriate treatment regimen, i.e., a specific Additionally, for one clinical species of S. aureus, multiple “fast-AST' assays and CLSI standard reference assays, as antimicrobial and at a particular dosage. described above, were run over the course of one month to Example 5 determine consistency of results; see FIG. 15. 0422 These data show that the present invention (“fast With S. aureus and K. pneumoniae Samples, across AST’ procedure) provides consistent results with the CLSI a Wide Variety of Antimicrobials, the Present reference method when tested with multiple antimicrobials Invention Provides MICs Values Similar to Those on S. aureus and E. coli clinical Strains, yet takes signifi Obtained from the CLSI Reference Method, Yet in cantly less time to determine the MIC; thus, the present Significantly Less Time invention greatly reduces time before a patient is provided an appropriate treatment regimen, i.e., a specific antimicro 0417. In this example, the present invention was used to bial and at a particular dosage. rapidly determine a plurality of antimicrobials’ MICs when treating S. aureus (a gram-positive bacterium) or K. pneu Example 7 moniae (a gram-negative bacterium) and compared to MIC values obtained by the CLSI method. The Present Invention Provides Rapid and Accurate 0418 Commercial, full-panel dried antimicrobial plates, Determination of a Plurality of Antimicrobials SensiTitre(R) (ThermoFisher) were used in the method of the MICs for an Antimicrobial-Resistant Bacterium. present invention as described above in Example 2, where 0423. In this example, the present invention was used to bacterial viability was assessed at four hours. Representative determine the MICs for a plurality of antimicrobials for E. US 2017/0211121 A1 Jul. 27, 2017

coli strains that are antimicrobial resistant to the MICs for E. time relative to standard methods, can provide a patient with coli strains that are antimicrobial-sensitive. an appropriate treatment regimen, i.e., a specific antimicro 0424 Both Escherichia coli (QC strain, ATCC 25922) bial and at a particular dosage. and clinical resistant E. coli (“Clinical) were cultured under Example 8 standard sterile conditions in Mueller-Hinton (MH) broth overnight at 37°C. with shaking. The E. coli concentration The Present Invention Provides Rapid and Accurate from the overnight culture was then set to 5x10 CFU/ml Determination of a Plurality of Antimicrobials using the McFarland standard technique for optical density MICs for an Antimicrobial-Sensitive Bacterium readings at 600 nm. Concurrently, two sterile 96-well 0428. In this example, the present invention was used to determine the MICs for a plurality of antimicrobials for an microplates were prepared with serial dilutions of a specified S. aureus strain that is antimicrobial sensitive. antimicrobial (see below) and a no-antimicrobial (Saline) 0429 S. aureus (QC strain 29213) was cultured under control, all in MH broth. The microplates, each well con standard sterile conditions in Mueller-Hinton (MH) broth taining 200 uL, were inoculated with the prepared antimi overnight at 37° C. with shaking. The S. aureus concentra crobial dilutions and incubated at 37° C. for 3 hours, 45 tion from the overnight culture was then set to 5x10 minutes for determination using the present invention (the CFU/ml using the McFarland standard technique for optical “fast-AST' technique). The “fast-AST microplates were density readings at 600 nm. Concurrently, two sterile removed from the shaking incubator after 3 hours, 45 96-well microplates were prepared with serial dilutions of a minutes and centrifuged for 2.5 minutes at 2500 g in order specified antimicrobial (see below) and a no-antimicrobial to pellet. The MH broth was then aspirated and 100 uL of (saline) control, all in MH broth. water was added to each well of both microplates. Then, 10 0430. The microplates, each well containing 100 uL. uL of the chemical moiety (here, Europium-Cryptate for were inoculated with the prepared antimicrobial dilutions mulation) was added to each well (to 20 ng/well) and 10 LIL and incubated at 37° C. for 3 hours, 45 minutes for deter of 5% Glutaraldehyde (as the signaling agent) was added to mination using the present invention (the “fast-AST' tech each well. The two microplates were then shaken at 300 rpm nique). The “fast-AST microplates were removed from the for 30 minutes. After, both plates were centrifuged for 2.5 shaking incubator after 3 hours, 45 minutes and centrifuged minutes at 2500 g to pellet. The solution was aspirated and for 2.5 minutes at 2500xg in order to pellet. The MH broth a wash of 200 uL PBS-tween was added to each well, was then aspirated and 100 uL of 25 mM PBS was added to followed by a centrifugation to pellet. After aspiration of each well of both microplates. Then, 104 of the chemical moiety (here, Europium-Cryptate formulation) was added to solution, a second identical wash of 200 uL PBS-tween each well (to 20 ng?well) and 10 uL of 0.005% Glutaralde occurred, followed by a final centrifugation to pellet. The hyde (as the signaling agent) was added to each well. The plate was then read using time resolved fluorescence on a two microplates were then shaken at 300 rpm for 30 min BioTek H1 plate reader. This process was carried out with utes. After, both plates were centrifuged for 2.5 minutes at the following antimicrobial preparations: Imipenem at 2500xg to pellet. The solution was aspirated and a wash of diluted concentrations from 8 ug/ml to 0.12 g/ml (FIG. 16); 200 uL PBS-tween was added to each well, followed by a Ampicillin at diluted concentrations from 32 g/ml to 0.25 centrifugation to pellet. After aspiration of solution, a second ug/ml (FIG. 17); Ceftazidime at diluted concentrations from identical wash of 200 uL PBS-tween occurred, followed by 32 ug/ml to 0.03 ug/ml (FIG. 18); Gentamicin at diluted a final centrifugation to pellet. 200 uL PBS-tween was added concentrations from 16 g/ml to 0.06 ug/ml (FIG. 19); to each well. The plate was then read using time resolved Levofloxacin at diluted concentrations from 8 ug/ml to 0.06 fluorescence on a BioTek H1 plate reader. This process was ug/ml (FIG. 20); Trimethethoprim/Sulfamethoxazole (SXT) carried out with the following antimicrobial preparations: at diluted concentrations from 32 ug/ml to 0.5 g/ml (FIG. Vancomycin at diluted concentrations from 32 g/ml to 0.25 21); Ciprofloxacin at diluted concentrations from 4 g/ml to ug/ml (FIG. 24); Penicillin at diluted concentrations from 8 0.015 ug/ml (FIG. 22); and Cetriaxone at diluted concen ug/ml to 0.0625 ug/ml (FIG. 25); and Teicoplanin at diluted trations from 64 g/ml to 0.12 ug/ml (FIG. 23). concentrations from 16 g/ml to 0.0125 ug/ml (FIG. 26). 0431. As seen in FIG. 24 to FIG. 26, S. aureus (QC strain 0425. As seen in FIG. 16 to FIG. 23, Escherichia coli 29213) the present invention determined MICs that were (QC strain, ATCC 25922) and the clinical resistant E. coli similar to those obtained from a standard CLSI reference (“Clinical) had similar MICs for Imipenem, Ceftazidime, method: Vancomycin: 0.5-2 g/ml; Penicillin: 0.25-2 ug/ml; 0426 Gentamicin, Levofloxacin, Ciprofloxacin, and and Teicoplanin 0.25-1 lug/ml. Cetriaxone whereas the two strains had dissimilar MICs for 0432. These data show that the present invention is Ampicillin and Trimethethoprim/Sulfamethoxazole (SXT). accurately able to determine a plurality of antimicrobials Accordingly, the data shows that clinical resistant E. coli MICs; thus, the present invention, in a greatly reduced strain is resistant to Ampicillin and Trimethethoprim/Sul amount of time relative to standard methods, can provide a famethoxazole (SXT). Thus, if a patient presents with an patient with an appropriate treatment regimen, i.e., a specific infection with this (or a similar strain), Ampicillin and antimicrobial and at a particular dosage. Trimethethoprim/Sulfamethoxazole (SXT) should not be Example 9 administered; instead, Imipenem, Ceftazidime, Gentamicin, Levofloxacin, Ciprofloxacin, and Cetriaxone should be The Present Invention Provides Rapid and Accurate administered. Determination of an Antimicrobials MIC Directly 0427. These data show that the present invention is from Blood Culture Samples and without the Need accurately able to differentiate an antimicrobials MIC for a for Sub-Culturing and an Overnight Growth clinically-relevant strain of bacteria that is resistant to one or Incubation more antimicrobials and the antimicrobials MIC for a strain 0433. In this example, the present invention was used for of the same bacteria that is sensitive to the antimicrobial; rapidly determining an antimicrobials MIC directly from a thus, the present invention, in a greatly reduced amount of blood culture sample. US 2017/0211121 A1 Jul. 27, 2017 34

0434 One clinical sample for each of E. coli, S. aureus, Example 10 and K. pneumoniae were obtained. The isolates were shipped on agar slants, sub-cultured, and stored at -80° C. Streptavidin Conjugated to Europium Binds to The samples were removed from the freezer, allowed to Biotinylated Wheat Germ. Agglutinin, which warm to room temperature, and streaked on a 5% sheep Specifically Binds to Gram Positive Bacteria blood-trypic soyagar (TSA) petri dish (ThermoEisher). The 0440. In this example, Europium was used as a chemical petri dish was placed in an incubator at 35° C. overnight. A moiety in signaling agents that comprise wheat germ agglu single colony was picked and the streaking process was tinin, which specifically binds to gram-positive bacteria. repeated on a new plate, followed by a second, 35° C. 0441 Bacteria (S. aureus) were inoculated across a overnight incubation. A total of three to five colonies were 96-well plate in concentrations ranging from 1x10 to 1x10 picked and dispersed into 1 mL of sterile saline (Hardy in MES buffer at pH 6. To each well containing the bacteria, Diagnostics) and the concentration was determined by opti and the corresponding control wells, 2 ug of biotinylated cal density measurement at 600 nm (Molecular Devices wheat germ agglutinin (Sigma) was added and the reaction M2). The sample was diluted in two steps to 2 CFU/ml in 40 solution was allowed to incubate for 15 minutes in order to mL of sterile cation adjusted Mueller Hinton Broth (MHB, facilitate the labeling of the exterior of the bacteria within Hardy Diagnostics) in a covered flask. the well with the chosen reporter. Then, a commercially available streptavidin-Europium (e.g., from Perkin-Elmer) 0435 The flask was loaded into a shaker incubator over was added to a final concentration per well of 0.4 pg/ml. night at 35° C. to mimic the performance of a BD After incubation for a further 15 min, the test plate was BACTEC(R) blood culture system. The flask was put at 4°C. centrifuged, using a Thermo Scientific Heraeus Multifuge after 10 hours, at which point the E. coli concentration was X3, at a speed of 2500xg for 2.5 minutes in order to pellet determined to be ~1x10 CFU/ml. This is the approximate the bacteria in the bottom of the plate while leaving any concentration at which commercial blood culture systems, unassociated reporter in the Supernatant. The plate was then such as the BD BACTEC and bioMerieux BacT/Alert, aspirated, using a BioTek Multiflo X plate washer, to remove register positive blood cultures. The 10-hour incubation time the Supernatant and unreacted reporter, before the addition was determined by streaking blood culture samples on 5% of wash buffer. This wash procedure was repeated two sheep blood TSA-petri dishes, incubating these at 35° C. additional times in order to thoroughly remove any unre overnight, and determining the colony count. acted reporter. Finally, to the aspirated wells, was added the reading buffer before the addition of Delfia Enhancement 0436 The sub-culture “control sample was taken by Solution. The plate was then incubated for 15 minutes to streaking this “positive' blood culture onto a TSA plate and allow for the europium enhancement before measurement of incubating overnight at 35° C. A standard CLSI broth the europium using time resolved fluorescence on a BioTek microdilution reference method was then performed, as H1 plate reader, as shown in FIG. 29. described previously. 0442. These data show that the use of Europium as a 0437 Centrifugation-based separation was then per chemical moiety in a signaling agent is accurately able to formed by following the SepsiTyper (Bruker Daltonics) quantify bacterial concentrations in a solution. protocol. Briefly, 1 mL of lysis buffer (Bruker Daltonics) was added to 5 mL of the MHB broth with 1x10 CFU/ml Example 11 E. coli. The mixture was aliquoted into six microfuge tubes, vortexed for 10 seconds, and then spun at 13,000 rpm for 2 Streptavidin Conjugated to Europium Binds to min. The Supernatant was removed and discarded, 1 mL of Biotinylated Polymixin B, Which Specifically washing buffer (Bruker) was added to each tube, and the Binds to Gram Negative Bacteria tubes were centrifuged at 13,000 rpm for 1 min. The 0443) In this example, Europium was used as a chemical Supernatant was again removed and discarded. Each pellet moiety in signaling agents that comprise Polymixin B, was resuspended in 500 uL of sterile saline by pipetting which specifically binds to gram-negative bacteria. Bacteria up-and-down. The solutions were mixed and the bacteria (E. coli) were inoculated across a 96-well plate in concen concentration was determined using a Promega Bactitre trations ranging from 1x10 to 1x10 in MES buffer at pH 6. GloTM bacteria cell viability kit. To each well containing the bacteria, and the corresponding control wells, biotinylated polymixin (Hycult Biosciences) 0438. The samples were diluted into MHB at a concen was added for a final dilution of 1:200 and the reaction tration of -5x10 CFU/ml. A “fast-AST' assay (as described solution was allowed to incubate for 15 minutes in order to in Example 2) was then performed, and the MIC determi facilitate the labeling of the exterior of the bacteria within nations were compared. The “fast-AST method on clinical the well with the chosen reporter. Then, a commercially samples provided similar MIC values as a standard method available streptavidin-Europium (e.g., from Perkin-Elmer) which requires sub-culturing, see, FIG. 27 and FIG. 28. was added to a final concentration per well of 0.4 ug/ml. 0439. These data show that the present invention (“fast After incubation for a further 15 min, the test plate was AST’ procedure), when used directly on clinical samples, centrifuged, using a Thermo Scientific Heraeus Multifuge provides consistent results with a standard MIC-determining X3, at a speed of 2500xg for 2.5 minutes in order to pellet method which requires a Sub-culturing step prior to an the bacteria in the bottom of the plate while leaving any overnight growth; thus, the present invention greatly reduces unassociated reporter in the Supernatant. The plate was then time before a patient is provided an appropriate treatment aspirated, using a BioTek Multiflo X plate washer, to remove regimen, i.e., a specific antimicrobial and at a particular the Supernatant and unreacted reporter, before the addition dosage. of wash buffer. This wash procedure was repeated two US 2017/0211121 A1 Jul. 27, 2017

additional times in order to thoroughly remove any unre acted reporter. Finally, to the aspirated wells, was added the (3) reading buffer before the addition of Delfia Enhancement Solution. The plate was then incubated for 15 minutes to allow for the europium enhancement before measurement of the europium using time resolved fluorescence on a BioTek HN NH2: H1 plate reader, as shown in FIG. 30. 0444 These data show that the use of Europium as a chemical moiety in a signaling agent is accurately able to quantify bacterial concentrations in a solution. Example 12 Europium Detector Provides Larger Signal Range and, Therefore, More Accurate MIC Data 0445. This example compared the ability of Europium and HRP as chemical moieties in signaling agents (com prising an antibody that specifically binds to bacteria) to accurately determining MICs. (6) 0446. Using 96-well plates containing cation-adjusted Mueller Hinton broth and appropriate antimicrobial dilu HN tions, bacteria were prepared by diluting colonies into Saline to reach a McFarland value of 0.5, which was verified using a spectrophotometer. This was diluted 1:20 into saline and 10 ul of inoculum was added to each well. Bacterial anti COO COO' COO microbial testing plates were incubated at 35°C., shaking at Europium-N1-amino (Eu-N1-amino) 150 rpm for 3 hours and 45 minutes. After this incubation, cationic magnetic beads and anti-S. aureus antibodies (con jugated to either horseradish peroxidase or Europium; cus 0451. The reaction solution was allowed to incubate for tom conjugation performed by Cisbio Assays) were added to 30 minutes in order to facilitate the labeling of the exterior each well and incubated for 20 minutes. Using an automated of the bacteria within the well with the chosen reporter. plate washer, magnetic beads were captured and the contents Then, the test plate was centrifuged, using a Thermo Sci of each well were washed three times with PBS-Tween 20 entific Heraeus Multifuge X3, at a speed of 2500xg for 2.5 minutes in order to pellet the bacteria in the bottom of the (0.1%). Then, wells were imaged directly using time plate while leaving any unassociated reporter in the Super resolved fluorescence (Europium) or TMB was added and natant. The plate was then aspirated, using a BioTek Multiflo allowed to incubate for 15 minutes, after which the reaction X plate washer, to remove the Supernatant and unreacted was stopped by addition of 1 M sulfuric acid and absorbance reporter, before the addition of wash buffer. This wash at 450 nm was measured for each well. procedure was repeated one (Eu-Cryptate-diamine) or two 0447. As shown in FIG. 31, using Europium as chemical (EuropiumN1-amino) additional times in order to thor moiety determined SXT's MIC more accurately than the oughly remove any unreacted reporter. Wells containing MIC determined using HRP as chemical moiety. Europium Cryptate-diamine were reconstituted in reading 0448. These data show that the use of Europium as a buffer and read using time resolved fluorescence on a chemical moiety in a signaling agent is accurately able to BioTek H1 plate reader, as shown in FIG. 32. Finally, to the determine an antimicrobials MIC. aspirated wells treated with Europium N1-amino, was added the reading buffer before the addition of Delfia Enhancement Example 13 Solution. The plate was then incubated for 15 minutes to allow for the europium enhancement before measurement of Embodiments of Europium Formulations, Which the europium using time resolved fluorescence on a BioTek Non-Specifically Label Bacteria, are Effective at H1 plate reader, as shown in FIG. 32. Detecting Bacteria and Quantifying Bacteria 0452. These data show that Europium formulations are Concentrations accurately able to quantify bacterial concentrations in a 0449 In this example, Europium formulations are non Solution when the Europium formulations are non-specifi specifically bound to bacteria. cally bound to bacteria. 0450 Bacteria (Ecoli) were inoculated across a 96-well Example 14 plate in concentrations ranging from 1 e5 to le9 in MES buffer at pH 6 (Europium Cryptate-diamine) or HEPES pH Europium Can Be Attached to Amines via 7.5 (EuropiumN1-amino). To each well containing the bac Isothiocyanate or to Carboxylic Acids via NH2 for teria, and the corresponding control wells, Europium Non-Specifically Labeling Bacteria When Cryptate-diamine (Compound (3); Cisbio) or Europium Quantifying Bacteria Concentrations N1-amino (Compound (6); PerkinElmer) was added at 66 0453. In this example, Europium formulations are non ng?well, then EDC/NHS (at 0.1 and 0.3 mg/ml) or glutar specifically bound to bacteria. aldehyde (0.5% final concentration) were added as indi 0454 Klebsiella pneumoniae or E. coli were inoculated cated. across a 96-well plate in concentrations ranging from 1x10 US 2017/0211121 A1 Jul. 27, 2017 36 to 1x10 in MES buffer at pH 6 (Europium Cryptate Example 15 diamine: Compound (3)) or HEPES pH 7.5 (Europium ITC: Compound (4)). To each well containing the bacteria, and Glutaraldehyde Can Be Used to Non-Specifically the corresponding control wells, Europium Cryptate-di Link Europium-Cryptate to the Bacterial Surface amine (Cisbio) or Europium ITC (PerkinElmer) was added 0457. In this example, Europium formulations are non at 66 ng?well, then EDC/NHS (at 0.1 and 0.3 mg/ml) to wells specifically bound to bacteria with glutaraldehyde. containing Europium Cryptate. 0458 Klebsiella pneumoniae, E coli, or Staph aureus were inoculated across a 96-well plate in concentrations ranging from 1x10 to 1x10 in MES buffer at pH 6. To each (3) well containing the bacteria, and the corresponding control wells, Europium Cryptate-diamine (Compound (3); Cisbio) was added at 66 ng/well, then a 5% solution of glutaralde hyde to wells containing Europium Cryptate.

(3)

Europium-cryptate-diamine (Eu-Cryptate-diamine) (4)

SCN

r Europium-cryptate-diamine (Eu-Cryptate-diamine) COO Eu-N1-ITC (Delfia) 0459. The reaction solution was allowed to incubate for 30 minutes in order to facilitate the labeling of the exterior 0455 The reaction solution was allowed to incubate for of the bacteria within the well with the chosen reporter. 30 minutes in order to facilitate the labeling of the exterior Then, the test plate was centrifuged, using a Thermo Sci of the bacteria within the well with the chosen reporter. entific Heraeus Multifuge X3, at a speed of 2500xg for 2.5 minutes in order to pellet the bacteria in the bottom of the Then, the test plate was centrifuged, using a Thermo Sci plate while leaving any unassociated reporter in the Super entific Heraeus Multifuge X3, at a speed of 2500xg for 2.5 natant. The plate was then aspirated, using a BioTek Multiflo minutes in order to pellet the bacteria in the bottom of the X plate washer, to remove the Supernatant and unreacted plate while leaving any unassociated reporter in the Super reporter, before the addition of wash buffer. This wash natant. The plate was then aspirated, using a BioTek Multiflo procedure was repeated once to thoroughly remove any X plate washer, to remove the Supernatant and unreacted unreacted reporter. Wells containing EuropiumCryptate-di reporter, before the addition of wash buffer. This wash amine were reconstituted in reading buffer and read using procedure was repeated one (EuropiumCryptate-diamine) or time resolved fluorescence on a BioTek H1 plate reader, as two (PerkinElmer) additional times in order to thoroughly shown in FIG. 34. remove any unreacted reporter. Wells containing Europium 0460. These data show that Europium formulations are Cryptate-diamine were reconstituted in reading buffer and accurately able to quantify bacterial concentrations in a read using time resolved fluorescence on a BioTek H1 plate Solution when the Europium formulations are non-specifi reader, as shown in FIG. 33. Finally, to the aspirated wells cally bound to bacteria. treated with Eu-N1, was added the reading buffer before the addition of Delfia Enhancement Solution. The plate was then Example 16 incubated for 15 minutes to allow for the europium enhance ment before measurement of the europium using time EDC/NHS Can Be Used to Non-Specifically resolved fluorescence on a BioTek H1 plate reader, as shown Couple Europium-Cryptate to the Bacterial Surface in FIG. 33. 0461. In this example, Europium formulations are non 0456. These data show that Europium formulations are specifically bound to bacteria with EDC/NHS. accurately able to quantify bacterial concentrations in a 0462 Klebsiella pneumoniae or E coli were inoculated Solution when the Europium formulations are non-specifi across a 96-well plate in concentrations ranging from 1x10 cally bound to bacteria. to 1x10 in MES buffer at pH 6. To each well containing the US 2017/0211121 A1 Jul. 27, 2017 37 bacteria, and the corresponding control wells, Europium Cryptate-diamine (Compound (3); Cisbio) was added at 66 (3) ng?well, then EDC/NHS (at 0.1 and 0.3 mg/ml) to wells containing Europium Cryptate.

(3)

Europium-cryptate-diamine (Eu-Cryptate-diamine)

0467. The reaction solution was allowed to incubate for 30 minutes in order to facilitate the labeling of the exterior of the bacteria within the well with the chosen reporter. Europium-cryptate-diamine (Eu-Cryptate-diamine) Then, the test plate was centrifuged, using a Thermo Sci entific Heraeus Multifuge X3, at a speed of 2500xg for 2.5 minutes in order to pellet the bacteria in the bottom of the 0463. The reaction solution was allowed to incubate for plate while leaving any unassociated reporter in the Super 30 minutes in order to facilitate the labeling of the exterior natant. The plate was then aspirated, using a BioTek Multiflo of the bacteria within the well with the chosen reporter. X plate washer, to remove the Supernatant and unreacted Then, the test plate was centrifuged, using a Thermo Sci reporter, before the addition of wash buffer. This wash entific Heraeus Multifuge X3, at a speed of 2500xg for 2.5 procedure was repeated twice to investigate the effect of minutes in order to pellet the bacteria in the bottom of the multiple washes on overall data quality. Wells containing plate while leaving any unassociated reporter in the Super EuropiumCryptate-diamine were reconstituted in reading natant. The plate was then aspirated, using a BioTek Multiflo buffer and read using time resolved fluorescence on a X plate washer, to remove the Supernatant and unreacted BioTek H1 plate reader, as shown in FIG. 36A to FIG. 36C. reporter, before the addition of wash buffer. This wash 0468. These data show that Europium formulations can procedure was repeated once to thoroughly remove any be non-specifically bound to bacteria using various washes unreacted reporter. Wells containing EuropiumCryptate-di comprising glutaraldehyde. amine were reconstituted in reading buffer and read using Example 18 time resolved fluorescence on a BioTek H1 plate reader, as shown in FIG. 35. A Two-Step Tagging Process. Using 0464. These data show that Europium formulations are NH-PEG-Biotin Followed by accurately able to quantify bacterial concentrations in a Streptavidin-Europium (Eu-SAV) Can Solution when the Europium formulations are non-specifi Non-Specifically Label Bacteria cally bound to bacteria. 0469. In this example, Europium formulations are non specifically bound to bacteria using a two-step process. 0470 E coli was inoculated across a 96-well plate in Example 17 concentrations ranging from 1x10 to 1x10 in MES buffer at pH 6. To each well containing the bacteria, and the Effect of Glutaraldehyde Wash Cycles on corresponding control wells, Amine-PEG-Biotin (Laysan Non-Specifically, Cryptate Labeled Bacteria Bio) was added at 1 mg/well, then EDC/NHS (at 0.1 and 0.3 mg/ml) to wells containing Amine-PEG-Biotin. The reaction 0465. In this example, Europium formulations are non solution was allowed to incubate for 15 minutes in order to specifically bound to bacteria with various washes compris facilitate the functionalization of the exterior of the bacteria ing glutaraldehyde. within the well with the biotin species. To each reaction well, streptavidin-europium (Eu-SAV) (PerkinElmer) was 0466 E coli or Staph aureus were inoculated across a added at 400 ng/well. The reaction solution was allowed to 96-well plate in concentrations ranging from 1x10 to 1x10 incubate for 15 minutes in order to facilitate the coupling in MES buffer at pH 6. To each well containing the bacteria, between the biotin and streptavidin. Then, the test plate was and the corresponding control wells, EuropiumCryptate centrifuged, using a Thermo Scientific Heraeus Multifuge diamine (Compound (3); Cisbio) was added at 66 ng/well, X3, at a speed of 2500xg for 2.5 minutes in order to pellet then a 5% solution of glutaraldehyde to wells containing the bacteria in the bottom of the plate while leaving any Europium Cryptate. unassociated reporter in the Supernatant. The plate was then US 2017/0211121 A1 Jul. 27, 2017

aspirated, using a BioTek Multiflo X plate washer, to remove distinguishing antimicrobial resistant bacteria from bacteria the Supernatant and unreacted reporter, before the addition that have undergone filamentous growth. When not consid of wash buffer. This wash procedure was repeated twice to ered, Such filamentous growth improperly identifies a bac investigate the effect of multiple washes on overall data terium as more resistant than it really is. Difficulty in quality. Wells containing Eu-SAV were reconstituted in determining antimicrobial resistance using a “volume’ reading buffer and read using time resolved fluorescence on approach is seen in FIG. 39. a BioTek H1 plate reader, as shown in FIG. 37. 0477. In order to avoid this, in one embodiment, at the 0471. These data show that Europium formulations can end of an incubation period, each broth microdilution is be non-specifically bound to bacteria using a two-step loaded into a filter comprising one or more pre-determined process comprising NH2-PEG-Biotin followed by Eu-SAV. pore sizes (see FIG. 40). The pore sizes are chosen such that a plurality of “normal bacteria is able to pass through the Example 19 filter, but filamentous bacteria greater than a certain length are trapped. The pore size may be >0.2 microns and <10 A Two-Step Bacteria Tagging Process with microns. NHS-LC-LC-Biotin Followed by Eu-SAv Can 0478. This filter may be designed for parallel sample Non-Specifically Label Bacteria processing, such as a 96-, 384- or 1586-well plate. A filter 0472. In this example, Europium formulations are non may be applied during the AST process, as shown in FIG. specifically bound to bacteria using another two-step pro 40. CCSS, 0479. This example further illustrates the key advantage 0473 E coli was inoculated across a 96-well plate in of designing a rapid AST platform that determines intact concentrations ranging from 1x10 to 1x10 in MES buffer bacteria presence by Surface area as opposed to the conven at pH 6. To each well containing the bacteria, and the tional metabolic approach, which is essentially a volumetric corresponding control wells, Amine-LC-LC-Biotin measurement. (Thermo-Fisher) was added at 1 mg/well, then EDC/NHS (at 0.1 and 0.3 mg/ml) to wells containing Amine-PEG Example 21 Biotin. The reaction solution was allowed to incubate for 15 minutes in order to facilitate the functionalization of the Methods for Preparing and Using A Signaling exterior of the bacteria within the well with the biotin Agent Comprising a Fluorescent Nanoparticle species. To each reaction well, streptavidin-europium (Per 0480. In this example, methods for preparing and using a kin-Elmer) was added at 400 ng/well. The reaction solution signaling agent comprising a fluorescent nanoparticle are was allowed to incubate for 15 minutes in order to facilitate described. the coupling between the biotin and streptavidin. Then, the 0481 First, 20 mg of fluorescein dilaurate (FL-DL) were test plate was centrifuged, using a Thermo Scientific Her weighed into a clear glass scintillation vial and 1000 mg of aeus Multifuge X3, at a speed of 2500xg for 2.5 minutes in Ethanol were added. The FL-DL was then dissolved in the order to pellet the bacteria in the bottom of the plate while vial via vortexing. Afterwards, 10 mg of DSPE-PEG-2k leaving any unassociated reporter in the Supernatant. The amine (Laysan Bio) were added to this mixture and dis plate was then aspirated, using a BioTek Multiflo X plate solved by vortexing. Separately, 40 g of DI water was washer, to remove the Supernatant and unreacted reporter, weighed in a beaker, and a stir-bar was added. The beaker before the addition of wash buffer. This wash procedure was was then placed on a magnetic stirrer and stirred at 200 repeated twice to investigate the effect of multiple washes on RPM. Next, the Ethanol solution was added to the beaker in overall data quality. Wells containing EuropiumCryptate a drop-wise fashion, and the Subsequent solution was then diamine were reconstituted in reading buffer and read using introduced into a Microfluidics homogenizer and processed time resolved fluorescence on a BioTek H1 plate reader, as one time at 6000 psi. 200gm of DI water was then added to shown in FIG. 38. the resulting mixture and Tangential Flow Filtration (TFF) 0474 These data show that Europium formulations can was used to purify and concentrate the nanoparticles about be non-specifically bound to bacteria using a two-step 12-fold to 20 mL and were then collected in a glass scin process comprising NHS-LC-LC-Biotin followed by Eu tillation vial. This collected nanoparticle formulation was SAV. filtered through a 0.2 Lum filter, and nanoparticle size and concentration were determined by NanoSight (Malvern), Example 20 with an average size reading of 102 nm. 0482. The fluorophore-comprising nanoparticles were Filamentous Bacteria Can Be Isolated from a functionalized with positively charged Small molecules to Solution Using a Filter System Having Pores form a signaling agent useful in the present invention. Ranging in Size from >0.2 Microns to <10 0483 Both Escherichia coli (ATCC 11303) and ampicil Microns: Thereby Providing More Accurate lin-resistant E. coli (ATCC 39936) were cultured under Chemical Sensitivity Data standard sterile conditions in LB broth at 37° C. Concen 0475. This Example illustrates an embodiment using fil trations were determined by measuring absorbance at 600 tering to exclude bacteria that have undergone filamentous nm (McFarland), and a concentration of 5x10 CFU/ml was growth in response to antimicrobial treatment. set by dilution. Ampicillin was then weighed into sterile 0476 Gram negative rods in particular first undergo water and added in appropriate concentrations to sterile 3 filamentous growth in response to Sub-inhibitory concentra mL microfuge tubes. Bacteria and nanoparticles at a con tions of cell wall-acting antimicrobials (such as beta centration of 8x10 nanoparticles/ml were both added to lactams). Although these will eventually be inhibited, meta these amplicillin-coated sterile microfuge tubes. The tubes bolic “volume” approaches have significant difficultly in were then capped and placed at 37° C. for 1.5 hours with US 2017/0211121 A1 Jul. 27, 2017 39 continuous shaking, after which they were opened and each beads. However, here, the S. aureus strain was ATCC 12600 was passed through a 0.2 um filter. Next, 100 uL of each and three antimicrobials were used: certazidime, oxacillin, filtrate was added to a well of a 96-well plate and 150 uL of and Vancomycin. a development solution (5% tetramethylammonium hydrox 0491. After the incubation period, magnetic beads with ide in ethanol) was added to each well. After 5 minutes the fixed cationic charges of 0.5 p.m size (ChemiGell Fluidmag.) plate was read at 490 nm excitation/530 nm emission in a were added concurrently with the signaling agents. The pH SpectraMax M2 microplate reader (Molecular Devices). was adjusted to -8.4 by the addition of 50 uL of borate 0484 FIG. 41 shows the result of an assay using the buffer. The microplate was agitated on an orbital shaker for above-prepared signaling agent comprising a nanoparticle. 20 minutes. The microplate was then placed on a magnetic Here, Escherichia coli and ampicillin-resistant E. coli capture plate comprising 24 neodymium N52 magnetics. treated with and without 100 ug/ml amplicillin, a concentra The MH broth was then aspirated and PBS with 0.1% tion well above the MIC. An assay for free signaling agents, Tween-20 added for a total of three washes. After the final which are not associated with intact bacteria, was performed aspiration, a stabilized development Solution consisting of after intact bacteria were removed by filtration through a 0.2 3.3',5,5'-tetramethylbenzidine (TMB) and hydrogen perox um filter. Control groups containing no amplicillin show low ide (ThermoFisher) was added, and the optical density at fluorescent signals as does the amplicillin-resistant E. coli 650 nm and 450 nm were monitored for ten minutes with a group treated with amplicillin. The E. coli treated with microplate reader (Vmax, Molecular Devices). The data ampicillin above the MIC show a significant increase in shown in FIG. 44 depicts from the five-minute point after the fluorescence, indicating efficacy of this antimicrobial. start of incubation with detection solution. As expected increasing amounts of the antimicrobials, which cause bac 0485 FIG. 42 shows the result of an assay for E. coli for terial cell lysis, reduces the number of intact bacteria. varying amplicillin concentrations. The fluorescent signal is 0492. These data show that functionalized magnetic par low when the amplicillin concentration is below the MIC, ticles can capture intact bacteria and enable quantification of ~15 ug/ml, and it rises at this value, indicating the efficacy the intact bacteria (when bound to a signaling agent) fol of the antimicrobial in this range lowing an antimicrobial treatment. Such magnetic capture may be used together with or in place of other separation Example 22 techniques in order to collect intact bacteria for use in the present invention. The Present Invention Can Be Performed Using Magnetic Beads to Isolate Intact Bacteria Example 23 Centrifugation of Bacterial Solutions Provides 0486 In this example, magnetic beads, which are asso More Accurate Counts of Intact Bacteria When ciated with an agent capable of binging intact bacteria, are Compared to Isolation of Intact Bacteria by used to isolate intact bacteria from a solution. Functionalized Magnetic Beads 0487. Magnetic beads reactive to E. coli were prepared 0493. In this example, the method for isolating intact from N-hydroxySuccinimidyl ester-activated 1 micron mag bacteria using functionalized magnetic beads (as described netic beads according to the manufacturers instructions in Example 21+) is compared to a method for isolating intact (ThermoEisher). Briefly, the supplied beads were magneti bacteria using centrifugation. cally captured and the storage solution was aspirated. The 0494 Capture of intact bacteria using functionalized beads were then washed with ice-cold 0.1 M hydrochloric magnetic beads was performed as described above. For the acid, followed by the addition of a polyclonal goal-anti centrifugation data, bacteria were washed three times by a lipopolysaccharide (LPS) antibody (Antibodies-Online process of centrifugation at 2500xg for 2.5 minutes, manu Inc.). The reaction was shaken for 2 hours, with Vortexing ally aspiration, and addition of PBS-Tween. Magnetic beads every 5 minutes for the first 30 minutes, per the manufac were not used for centrifugation washes. Bacteria were turer's instructions. The beads were then washed thoroughly treated with varying concentrations of Vancomycin and stored in phosphate buffered saline, pH 7.4, at 4°C. until (VAN). SC. 0495 FIG. 45 shows that centrifugation provides much 0488 Signaling agents comprise a moiety capable of higher and more accurate bacterial numbers and MICs than binding to a microorganism (e.g., an antibody that binds to isolation by functionalized magnetic beads. E. coli) and a chemical moiety capable of providing a signal 0496 These data show that centrifugation of bacterial or contributing to production of a signal (e.g., horseradish cultures for isolating intact bacteria is Superior to methods peroxidase (HRP)). using functionalized magnetic particles. Isolation using cen 0489. The anti-LPS magnetic beads and anti-E. coli sig trifugation may be used together with (e.g. magnetic isola naling agents were added simultaneously to a McFarland tion) or in place of other separation techniques in order to standard-determined dilution series of E. coli in MH broth. collect intact bacteria for use in the present invention. The reaction was allowed to proceed for 20 min, followed by magnetic bead capture with a 96-well microplate magnetic Example 24 stand (V&P Scientific). The wells were washed three times, Chemical Amplification via TAML Nanoparticle followed by the addition of the detection solution described Amplifier Allows for Signaling with Optimal in Example 1. The optical densities at 450 nm and 650 nm Sensitivity in the 1x10 to 1x10 CFU/ml Range were read for 10 min. The value at 5 min is plotted in FIG. Using Standard Optical Detection Equipment 43. 0497. In this example, methods for preparing and using a 0490 The procedure of Example 1 was then used through signaling agent comprising tetra-amino metalorganic ligand the addition of signaling agents and functionalized magnetic (TAMLR) catalysts are described. US 2017/0211121 A1 Jul. 27, 2017 40

0498 Chemical amplification in enabled with a propri 6. The method of claim 1, wherein the signaling agent can etary nanoparticle amplifier, which adapts cutting-edge form a covalent bond with the Surface of a microorganism in nanoparticle formulation techniques from drug delivery and the presence of one or more agents that promote coupling, a small-molecule catalyst from green chemistry. Each the agents selected from the group consisting of glutaralde “nanolabel” comprises >6x10" densely packed iron-contain hyde, formaldehyde, paraformaldehyde, EDC, DCC, CMC, ing, tetra-amino metalorganic ligand (TAMLR) catalysts DIC, HATU, Woodward's Reagent, N,N'-carbonyl diimida shielded by a polymer shell functionalized with specific Zole, acrylates, amides, imides, anhydrides, chlorotriazines, ligands (FIG. 46). Each TAML molecule has molar activities epoxides, isocyanates, isothiocyanates, organic acids, mono within 5-fold those of horseradish peroxidase, a gold-stan mers, polymers, silanes, silcates, NHS, and sulfo-NHS, or dard immunoassay enzyme label (FIG. 46B). After specific any combination thereof. binding, nanolabels are chemically triggered to release their 7. The method of claim 6, wherein the agent that promotes contents into Solution, enabling homogeneous catalysis of coupling is glutaraldehyde. the optical signal. The high number of catalysts per binding 8. The method of claim 1, wherein multiple antimicrobials event enables quantification of as few as 200 intact bacteria are tested in parallel. (FIG. 46A) and 100-fold sensitivity enhancements over 9. The method of claim 1, wherein the determining signal standard enzyme immunoassays (FIG. 46C). In addition to levels comprises measuring the signal levels associated with obviating the need for the development of new detection intact microorganisms. technologies, standard optical detection enables compatibil 10. The method of claim 9, wherein the method further ity with standard dried antimicrobial panel microplates. Such comprises a step of determining whether a microorganism is as SensiTitre plates. resistant, intermediately resistant, or Susceptible to one or What is claimed is: more antimicrobials and/or determining one or more anti 1. A method for determining antimicrobial susceptibility microbial minimum inhibitory concentrations (MIC) based of microorganisms comprising: upon the signal levels associated with intact microorgan incubating a liquid Suspension of microorganisms in the isms. presence of an antimicrobial under conditions that 11. The method of claim 1, wherein the microorganisms promote growth of the microorganisms; are bacteria, fungi, protozoa, and/or archaea. adding a signaling agent capable of binding to a surface 12. The method of claim 1, wherein the method does not of the microorganisms; involve a step of capturing microorganisms on a solid surface prior to or during incubation and/or does not include separating the microorganisms bound by the signaling a step of growing microorganisms on a solid Surface prior to agent from unbound signaling agent; and or Subsequent to the incubating step. determining signal levels associated with the microorgan 13. The method of claim 1, wherein the separating the isms as compared to one or more controls, thereby microorganisms is performed by centrifugation, magnetic determining the antimicrobial susceptibility of the separation, filtration, electrophoresis, dielectrophoresis, pre microorganisms; cipitation, or agglutination, or a combination thereof. wherein the signaling agent comprises a structure that is: 14. The method of claim 1, wherein the one or more controls comprise a positive control measured from micro organisms under otherwise identical conditions but without antimicrobials or with one or more antimicrobials for which the microorganisms are not susceptible. 15. The method of claim 1, wherein the microorganisms are obtained from a biological sample from a subject having an infection of the microorganisms and/or are obtained from a culture derived from the biological sample. 16. The method of claim 15, wherein the biological sample is selected from the group consisting of blood or blood components, bronchoalveolar lavage, cerebrospinal fluid, nasal Swabs, sputum, stool, throat Swabs, vaginal Swabs, urine, and wound Swabs, or a combination thereof. 17. The method of claim 1, wherein the steps of incubat ing the liquid Suspension of microorganisms and adding the signaling agent occur in a cartridge comprising a plurality of chambers and the step of determining signal levels associ ated with the microorganisms comprises determining sig 2. The method of claim 1, wherein the antimicrobial naling levels in the plurality of chambers. Susceptibility of the microorganisms is determined in less 18. The method of claim 17, wherein the cartridge further than 5 hours. comprises one or more control chambers that do not contain 3. The method of claim 1, wherein adding the signaling antimicrobials or one or more antimicrobials for which the agent occurs during the incubating step. microorganisms are not susceptible. 4. The method of claim 1, wherein adding the signaling 19. A method for determining antimicrobial susceptibility agent occurs after the incubating step. of microorganisms comprising: 5. The method of claim 1, wherein the signaling agent can incubating a liquid Suspension of microorganisms in the form a non-covalent bond with the Surface of a microorgan presence of an antimicrobial and a signaling agent ism. under conditions that promote growth of the microor US 2017/0211121 A1 Jul. 27, 2017

ganisms, wherein the signaling agent is capable of 26. A method for determining antimicrobial susceptibility binding to a surface of the microorganisms; of microorganisms comprising: separating the microorganisms bound by the signaling incubating a liquid Suspension of microorganisms in a agent from the unbound signaling agent; and cartridge comprising a plurality of chambers, each determining signal levels associated with the microorgan chamber containing one or more antimicrobials, under conditions that promote growth of the microorganisms; isms as compared to one or more controls, thereby adding a signaling agent to the plurality of chambers, determining the antimicrobial susceptibility of the wherein the signaling agent is capable of binding to a microorganisms; Surface of the microorganisms; wherein the signaling agent comprises a structure that is: removing unbound signaling agent, and determining signaling levels in the plurality of chambers as compared to one or more controls, thereby deter mining the Susceptibility of microorganisms to the one or more antimicrobials; wherein the signaling agent comprises a structure that is:

20. The method of claim 19, wherein the antimicrobial Susceptibility of the microorganisms is determined in less than 5 hours. 21. The method of claim 19, wherein the one or more controls comprise a positive control measured from micro organisms under otherwise identical conditions but without 27. The method of claim 26, wherein the antimicrobial antimicrobials or with one or more antimicrobials for which Susceptibility of the microorganisms is determined in less the microorganisms are not susceptible. than 5 hours. 22. The method of claim 19, wherein the signaling agent 28. The method of claim 26, wherein the cartridge further can form a non-covalent bond with the Surface of a micro comprises one or more control chambers that do not contain organism. antimicrobials or one or more antimicrobials for which the 23. The method of claim 19, wherein the signaling agent microorganisms are not susceptible. can form a covalent bond with the Surface of a microorgan 29. The method of claim 26, wherein the signaling agent ism in the presence of one or more agents that promote can form a covalent bond with the Surface of a microorgan coupling, the agents selected from the group consisting of ism in the presence of one or more agents that promote glutaraldehyde, formaldehyde, paraformaldehyde, EDC, coupling, the agents selected from the group consisting of DCC, CMC, DIC, HATU, Woodward's Reagent, N,N'- glutaraldehyde, formaldehyde, paraformaldehyde, EDC, carbonyl diimidazole, acrylates, amides, imides, anhydrides, DCC, CMC, DIC, HATU, Woodward's Reagent, N,N'- chlorotriazines, epoxides, isocyanates, isothiocyanates, carbonyl diimidazole, acrylates, amides, imides, anhydrides, organic acids, monomers, polymers, silanes, silcates, NHS, chlorotriazines, epoxides, isocyanates, isothiocyanates, and sulfo-NHS, or any combination thereof. organic acids, monomers, polymers, silanes, silcates, NHS, 24. The method of claim 23, wherein the agent that and sulfo-NHS, or any combination thereof promotes coupling is glutaraldehyde. 30. The method of claim 26, wherein the microorganisms 25. The method of claim 19, wherein the microorganisms are obtained from a biological sample from a subject having are obtained from a biological sample from a subject having an infection of the microorganisms and/or are obtained from an infection of the microorganisms and/or are obtained from a culture derived from the biological sample; and a culture derived from the biological sample; and wherein the biological sample is selected from the group wherein the biological sample is selected from the group consisting of blood or blood components, bronchoal consisting of blood or blood components, bronchoal Veolar lavage, cerebrospinal fluid, nasal Swabs, sputum, veolar lavage, cerebrospinal fluid, nasal Swabs, sputum, stool, throat Swabs, vaginal Swabs, urine, and wound stool, throat Swabs, vaginal Swabs, urine, and wound Swabs, or a combination thereof. Swabs, or a combination thereof. k k k k k