ASSAYS FOR IMPROVING AUTOMATED ANTIMICROBIAL SUSCEPTIBILITY TESTING ACCURACY

Abstract

Phenotypic antimicrobial susceptibility testing (AST), the gold-standard diagnostic that indicates whether an antimicrobial will be clinically effective, often suffer the slowest times-to-result for the most resistant pathogens. Here we introduce novel assays to be performed in parallel with standard AST assays that enable rapid, same-shift reporting of AST results for a plurality of pathogens. The assays developed here are further capable of detecting resistance to carbapenems, the most powerful class of beta-lactams commonly used as last-resort antimicrobials.

Claims

1. A method for performing automated antimicrobial susceptibility testing of a carbapenem antimicrobial comprising: performing a dilution assay, comprising: inoculating a microbial solution to achieve a final concentration C.sub.0 into a plurality of fluid wells defining a dilution series of a carbapenem antimicrobial; and measuring in each of the plurality of fluid wells a signal associated with microbial growth; and in parallel with the dilution assay, performing a carbapenemase assay on the same microbial sample comprising: measuring a signal associated with carbapenem degradation in a first well comprising a carbapenem antimicrobial, a microbial concentration of C.sub.R, a buffer system at <0.05 M, a pH indicator, and optionally ionic zinc referenced to a second well comprising a similar microbial concentration of C.sub.R, a buffer system at <0.05 M, a pH indicator, and optionally ionic zinc; and combining the data derived from the dilution assay with that derived from the carbapenemase assay to define and label the microorganism as carbapenem susceptible or carbapenem resistant.

2. The method of claim 1, further comprising comparing the signal associated with the first well to a at least one well comprising a carbapenem antimicrobial, a microbial concentration of C.sub.R, a buffer system at <0.05 M, a pH indicator, and a metal ion chelating agent.

3. The method of claim 1, wherein the carbapenemase assay comprises the first well comprises a microbe sample, zinc sulfate, sodium phosphate buffer, fluorescein, and imipenem; the second well comprises a microbe sample, zinc sulfate, sodium phosphate buffer, and fluorescein; and a third well comprises a microbe sample, fluorescein, imipenem, and EDTA; and wherein the carbapenemase activity is proportional to the difference in fluorescence between the first and second sets of assay wells, and the metallo-carbapenemase activity is proportional to the difference in fluorescence between the first and third sets of assay wells.

4-36. (canceled)

37. A method for performing multi-assay rapid antimicrobial susceptibility testing sequences, the method comprising: inoculating two or more different concentrations of a sample comprising a microorganism derived from a clinical sample into a plurality of dilution wells of a test panel, at least a portion of the plurality of wells containing one or more dilutions of a plurality of antimicrobials for inoculation of the sample at concentration C.sub.0, and at least two wells comprising a carbapenemase assay comprising: i. one or more assay wells comprising a carbapenem, ionic zinc, a pH indictor, a buffer system at <0.05 M, and a microbe sample at concentration C.sub.R comprising <110.sup.8 CFU intact microbes; and ii. one or more assay wells comprising saline, ionic zinc, a pH indictor, a buffer system at <0.05 M, and a microbe sample at concentration C.sub.R comprising <110.sup.8 CFU intact microbes; loading the test panel into an automated rapid antimicrobial susceptibility testing system for performing a multi-assay testing sequence; and operating the testing system to: move the loaded test panel to an incubation assembly comprising a nest assembly adapted to: i) house at least one test panel, and ii) facilitate incubation of one or more test panels in order to undergo the multi-assay testing sequence the incubation assembly comprising: an agitation system configured to generate a repeated motion of the nest assembly; incubate and optionally agitate the inoculated sample in the incubation assembly; at least once, periodically measure an amount of sample growth in a plurality of control wells of the plurality of wells; responsive to determining that a level of growth in the control wells meets or exceeds a threshold level of growth, stop incubation; detecting signal from the carbapenemase and/or beta-lactamase assay for one or more carbapenems and/or beta-lactam/beta-lactamase inhibitors on incubated samples in the test panel; perform one or more end point assays on incubated samples in the test panel; measure an optical output from the sample in the plurality of wells of the test panel, the optical output corresponding to an amount of the microorganism remaining in each of the plurality of wells; and report at least one of: a minimum inhibitory concentration of and/or a qualitative susceptibility interpretation for the microorganism remaining in each of the plurality of wells and the plurality of antimicrobials, such that the results of the carbapenemase and/or beta-lactamase assays influence algorithmic MIC determinations of one or more carbapenems and/or beta lactams.

38. The method of claim 37, wherein the buffer system limits changes in pH due to the differences in the components in assay wells (i) and (ii) not caused by the microbes.

39. A method for performing automated antimicrobial susceptibility testing of a microorganism to an antimicrobial comprising: performing a dilution assay, comprising: inoculating the microorganism into a plurality of fluid wells defining a dilution series of an antimicrobial to achieve a concentration C.sub.0 in each well; measuring in each of the plurality of fluid wells a signal associated with microbial growth; and in parallel with the dilution assay, performing an assay for inducible resistance to the antimicrobial, the assay comprising: measuring a signal associated with resistance to the antimicrobial in a first well comprising an antimicrobial and the microorganism at concentration C.sub.R; measuring a signal associated with induced resistance in a second well, comprising different contents than the first well and thereby acting as a control for the first well; and combining the data derived from the dilution assay with that derived from the inducible resistance assay to define a minimum inhibitory concentration (MIC) of the carbapenem antimicrobial and label the microorganism as carbapenem susceptible or carbapenem resistant.

40. The method of claim 39, further comprising comparing the signal measured in each of the plurality of fluid wells and based on said comparison, defining a minimum inhibitory concentration (MIC) of the antimicrobial.

41. The method of claim 39, wherein the signal associated with induced resistance to the antimicrobial is a signal associated with enzyme catalyzed degradation of the antimicrobial.

42. The method of claim 39, wherein the second well of the induced resistance assay either a) comprises an inhibitor of a resistance factor or b) does not comprise the microbial inoculum.

43. The method of claim 39, wherein the induced resistance assay comprises a third well comprising EDTA.

44. The method of claim 39, wherein C.sub.R>C.sub.0.

45. The method of claim 39, wherein C.sub.R10C.sub.0.

46. The method of claim 39, wherein C.sub.0 is approximately 110.sup.5 to 110.sup.7 CFU/mL intact microorganisms.

47. The method of claim 39, wherein C.sub.R is less than approximately 110.sup.8 CFU/mL intact microorganisms.

48. The method of claim 39, wherein two or more different induced resistance assays are performed in parallel for a single sample with inoculation at concentrations C.sub.R1 and C.sub.R2.

49. The method of claim 48, wherein C.sub.R1=C.sub.R2.

50. The method of claim 48, wherein C.sub.R1C.sub.R2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIG. 1 depicts exemplary data from beta-lactamase inhibitor assay tests of clavulanic acid and tazobactam with three microbial strains.

DETAILED DESCRIPTION

Overview

[0109] Generally, this disclosure focuses on beta lactamase and/or carbapenemase assays that are performed in parallel with standard phenotypic AST assays on an automated analyzer, e.g., as described in U.S. Pat. No. 10,161,948 to Vacic et al, which is incorporated by reference herein for all purposes. In several exemplary embodiments described herein, both types of assays are run on the same cartridge such that only a single cartridge is required for all tests for the automated analyzer.

[0110] Those of skill in the art will appreciate that the integration of these two assays into a single automated process involves several departures from existing industry standard processes. First, commercially available AST systems generally require all wells of the multi-well AST testing vessel (e.g., microtiter plate, multi-well cassette, etc.) be loaded with substantially equivalent quantities of microbial sample, so as to ensure that differences in growth observed across antimicrobial dilution series are not due to differences in initial loading of the wells. By contrast, in some of the embodiments of this disclosure, the time-to-result for carbapenemase and beta-lactamase assays is reduced by increasing the quantity of microorganisms in the reservoirs for these tests relative to the lower microorganism concentration present in the plurality of wells in the panel that are used for MIC determinations, as is discussed in greater detail below.

[0111] Second, existing automated AST systems generally take one of two approaches to carbapenemase or beta-lactamase testing: first, in an automated sample in which beta lactam or carbapenem resistance is suspected based upon algorithmic interpretations of growth curve results, the incubation period will be continued until the algorithmic interpretation of the growth curves is confident the resistance either is or is not present. This may add significant time before AST results are available due to the inducible nature of some resistance mechanisms, as known to those skilled in the art. This growth curve-based approach has the advantage of automation, but it can extend the time-to-result by hours, and in laboratories staffed in discontinuous shifts (e.g., without an overnight shift), extended assay times may shift the result from the same day to the following morning. In a second approach, a carbapenemase or beta lactamase assay is performed offline, independently of the automated AST assay. This approach may allow a result to be obtained and reported sooner, but it may also be more resource intensive than a fully automated process, and the separation of the AST and resistance assays raises a risk that data from both assays will not be associated with the same patient record during the period where the AST results are interpreted and prescribing decisions are made. The systems and methods presented herein leverage the advantages of both approaches yet can reduce or eliminate delays in delivery of results and integrate data from both assays into a single output. In the preferred embodiment, the results from the carbapenemase and/or beta-lactamase assays are used to influence the algorithmic MIC determinations of results from the appropriate carbapenem and beta lactam dilution series. In additional embodiments, the results from the carbapenemase and/or beta-lactamase assays may be provided to the user.

[0112] In some embodiments of this disclosure, beta-lactamase or carbapenemase activity is measured indirectly, for example by detecting a change in pH caused by the liberation of free acids by degradation of the antimicrobial. Those of skill in the art will appreciate that pH in particular may be affected by factors other than enzyme-catalyzed degradation due to microbial resistance. The potential for these other factors to confound the results of AST and carbapenemase/beta-lactamase probe-based assays can be reduced by the inclusion of one or more carbapenemase or beta-lactamase inhibitors. By comparing the signal from an inhibited well to one containing the probe and microbes alone, the efficacy of the inhibitor may be tested. For example, tazobactam's inhibitory effects on a clinical Escherichia coli sample may be determined by setting up two wells containing E. coli and nitrocefin and adding tazobactam to the second well. By extending the dilution series, minimum inhibition concentrations may be obtained.

[0113] In some embodiments, such tests would be performed by an automated AST analyzer, in parallel with AST tests, and the combined results would add increased accuracy to the tests. Since some beta-lactamases may be induced by the presence of beta-lactams, it may be advantageous to perform beta-lactamase inhibitory testing after microbial growth in the presence a beta-lactam. In the case where the presence of the beta-lactam inhibits microbial growth, no useful data from beta-lactamase inhibitory testing may be obtained.

[0114] Any beta-lactamase probe may be utilized for these assays. Engineered probes with less-broad activity may also be used. However, the key to the assay concept is that the probe is sufficiently broad to ensure a plurality of potential beta-lactamase variants that can be inhibited by the inhibitor will degrade the probe for which the assay results will influence the MIC-determining algorithm of the AST platform.

[0115] By combining inhibitors in a single test well, information about the presence of beta-lactamase classes may be obtained. In this case, inhibitors may include drugs and potential drugs as well as agents with unacceptable toxicities for human use, such as EDTA, which is well-known to inhibit metallo-beta-lactamases (MBLs) but is toxic. An assay for carbapenemase activity may be developed by including inhibitors of non-carbapenemase ESBLs in one well, together with the clinical sample and nitrocefin, and comparing this to a sample comprising the clinical sample and nitrocefin without the inhibitors. Such inhibitors include, but are not limited to, clavulanate, cloxacillin, and tazobactam. Additional accuracy may be obtained by adding a third well comprising known inhibitors of carbapenems, including but not limited to avibactam, vaborbactam, and EDTA.

[0116] Information on carbapenemase presence may also be obtained by utilizing a carbapenem antimicrobial as a competitor to a beta-lactamase probe. In this case, one or more inhibitors may be added to inhibit non-carbapenemase beta-lactamase activity in all assay wells, in addition to a clinical microbe sample and a broad-spectrum probe, such as nitrocefin. Note a more narrow-spectrum probe may be advantageous here.

[0117] One well would then comprise a high concentration of a carbapenem, which would not be present in the second well. By comparing the rate of nitrocefin degradation in each well, the presence of a carbapenemase may be inferred: if the carbapenem retards the generation of the signal resulting from nitrocefin degradation, a carbapenemase is likely present. In alternative embodiments, oxacillin may be used in one or more additional wells as a competitor, specifically for the detection of OXA carbapenemases.

[0118] Exemplary carbapenemase assays may also utilize the acidimetric technique. Currently disclosed acidimetric carbapenemase assays utilize >10.sup.9 CFU/mL microbes, complicating their use in automated AST testing following standard laboratory workflows, where <510.sup.7 CFU/mL microbes are commonly available. Furthermore, standard laboratory workflows for AST testing utilize intact microbes of which a plurality are viable. These standard workflow requirements challenge previously-disclosed carbapenemase assays, which rely on greater microbial quantities and lysed microbes. These requirements are due to the fact that many carbapenemases may be intracellular and may be expressed in low concentration at early timepoints due to their inducible nature.

[0119] Here we demonstrate the surprising finding that acidimetric carbapenemase assays may be performed using less than one-tenth ( 1/10.sup.th) of the number of intact microbes by utilizing four assay wells to provide an appropriately normalized result. Additionally, the assay utilizes a fluorometric, rather than a colorimetric, pH probe, increases assay incubation times to 4 hours, and increases carbapenem concentrations to >1 mg/mL.

[0120] Here we further demonstrate the counter-intuitive finding that faster AST results may be made available by utilizing different microorganism concentrations in different test reservoirs of an automated AST panel. This is particularly surprising because existing automated AST platforms, including the Vitek2 (bioMrieux), MicroScan (Danaher/Beckman-Coulter), Phoenix (Becton-Dickinson), and SensiTitire (ThermoFisher) provide dedicated hardware specifically to ensure all reservoirs on the panel inoculated with microorganisms (e.g. all reservoirs except the contamination control that is inoculated with zero microorganisms) receive a substantially equivalent quantity of microorganisms. This is the case because MICs are relative measurements, in which the growth in each reservoir of a dilution series of an antimicrobial is compared with the growth in other reservoirs of the same dilution series (and may be compared with that for other antimicrobials).

[0121] In contrast, here we demonstrate that AST result accuracy may be enhanced through the inoculation of two or more different quantities of microorganisms in different automated AST panel reservoirs. In particular, microorganisms inoculated into reservoirs comprising dilution series of antimicrobials may be of a substantially equivalent concentration, C.sub.0, and microorganisms inoculated into specific resistance mechanism-determining assays may be of a different concentration, C.sub.R. In particular, the concentrations may be such that C.sub.R=C.sub.0, where is preferably greater than 1 and most preferably greater than 10.

[0122] Here we further demonstrate that only two wells may be required for carbapenemase-mediated resistance determinations by the addition of buffer to these wells at concentrations between 0.0001 and 0.1 M, preferably 0.0005 and 0.01 M. Buffer systems are defined as chemicals that donate or accept protons in order to maintain stable solution pH and, thus, resist pH changes. Thus, the addition of a buffer system to the wells of the carbapenemase assay is surprising because the preferred mechanism for detecting resistance is carbapenemase-mediated pH changes. This advance is important because it not only decreases the number of wells required for the assay but additionally may eliminate the need for sterile saline during AST panel preparation. These advantages may lower costs and/or enable greater throughputs and/or increase the number of drugs that may be tested in parallel. In addition, this advance may confer stability to solutions during manufacturing, which may be particularly beneficial for minimizing the need to change solution pH during the manufacturing process.

[0123] In an exemplary embodiment, the assay comprises a minimum of 2 wells and utilizes a sample comprising intact microbes. The first well (Well.sub.1) comprises a buffer system, a carbapenem, ionic zinc, a pH indictor, and a microbe sample comprising <110.sup.8 CFU microbes. The second well (Well.sub.2) comprises a buffer system, ionic zinc, a pH indictor, and a microbe sample comprising <110.sup.8 CFU microbes. The following formula can then be utilized to normalize the result:

Normalized Activity=(Well.sub.2Well.sub.1) [Formula 2]

[0124] The above says that imipenem degradation is the change in pH (e.g. loss of signal) due to the presence of microbes less the change in pH (e.g. loss of signal) due to microbes in solution only.

[0125] In additional embodiments, acidimetric methods may be utilized for testing inhibitors. For example, the addition of a chelator, such as EDTA, and/or an inhibitor with known carbapenemase activity, such as avibactam or vaborbactam, to an additional well of the carbapenemase assay described above may serve as an assay for inhibitor activity. EDTA, in particular, may be advantageous in that it may enable the type of carbapenemase to be determined. For example the differential in signal between wells comprising EDTA and absent EDTA may be used to determine the presence of carbapenemases that require metal ions for functionality, termed metallo-carbapenemases, such as KPCs. Typically, high sensitivity assays that are based on amplification (e.g., catalytic) can be performed only once since chemistries necessary for those assays usually destroy the target microorganism. Thus, the systems and methods described herein typically use two types of assays to address this issue. In some cases, a preliminary (e.g., checkpoint) assay can be performed first and can be repeated periodically to interrogate growth of uninhibited microorganisms (i.e., without antimicrobial presence). These checkpoint assays can be performed in wells referred to herein as control wells. Examples of typical control wells are a growth well containing microorganisms in nutrient broth and a contamination control well containing nutrient broth only. The system interrogates growth/no growth optically (e.g., absorbance, fluorescence metabolic dye, etc.) and once a particular ratio and/or kinetic change between the control wells is achieved and detected, one or more end point assays (e.g., an amplification assay or growth assay) can be initiated on samples disposed in other portions of the test panel (e.g., the rest of, or the entire, test panel). The samples, for example, can include microorganisms originating from a clinical sample.

[0126] In some cases, the systems and methods described herein can be implemented to provide faster testing than some conventional systems. For example, though some automated systems may speed time to obtain results, the time-to-results for carbapenemase-expressing strains rarely meet the 6-hour definition of same-shift results for many clinical laboratories. Because of this slow time-to-results and because AST results are complex and may utilize expert interpretation for clinical action, such conventional systems can result in a day delay between the onset of susceptibility testing and clinical action for patients infected with these difficult-to-treat samples.

[0127] In some embodiments, the systems and methods described herein comprise a system with a stable, dried reagent, where uninoculated control wells are not a necessity and carbapenemase production can be determined by comparing only three conditions: Well 1: bacteria and pH indicator, Well 2: bacteria, imipenem (or other suitable carbapenem) and pH indicator, Well 3: bacteria, imipenem, pH indicator, and EDTA. In this embodiment, carbapenemase activity=Well 1Well 2. This indicates the bacterial-induced hydrolysis of the imipenem. A value of greater than 20,000 is indicative of an organism that produces a carbapenemase. Additionally, metallo-carbapenemase inhibition=Well 2Well 3. If this value is greater than 20,000, it indicates that the EDTA was able to substantially inhibit the carbapenemase activity in the sample, indicating a metallo-carbapenemase. KPC and NDM are both carbapenemases. NDM is a metallo-carbapenemase.

[0128] In these embodiments, the assay can be performed without control wells without significant decrease in assay performance. While not wishing to be bound by any theory, it is believed that the use of highly shelf stable reagents facilitates the consistent performance of the assay without the need for such controls. For instance, in one embodiment the reagents are dried rather than an aqueous solution and are consequently sufficiently stable to minimize or eliminate well to well variability in the amount or activity of the carbapenem.

TABLE-US-00001 TABLE 1 Carbapenemase assay results for 26 microbial strains within 4 hours. Known Other known Carbapenemase Species Strain Carbapenemase resistance Activity E. coli CDC CNP 73 KPC 3480.1845 E. coli CDC CRE 26 NDM 41326.61 E. coli CDC CRE 03 KPC-3 7149.729 E. coli CDC CRE 07 NDM 27447.359 E. coli CDC BIT 01 KPC 8803.9255 E. coli CDC CNP 38 NDM 19534.8825 E. coli CDC CRE 17 NDM 36400.9235 E. coli CDC CNP 27 None ESBL+ 2401.2335 E. coli CDC CNP 54 None Amp beta lactamase 1950.443 E. coli SML 19 None 2540.0545 E. coli SML20 None 2734.3825 E. coli AR0346 None ESBL+ 2560.861 E. coli AR0348 None 2960.1665 E. coli AR0349 None ESBL+ 2861.008 E. coli AEL18 None 1551.4885 E. coli AEL19 None 696.4825 E. coli AEL 20 None 882.3165 E. coli TRICORE 71 None 1138.2225 E. coli TRICORE 72 None 2554.797 E. coli 25922 None 2106.4115 K. pneumoniae CDC CNP 03 IMP 3662.106 K. pneumoniae CDC CNP 08 OXA-181 1323.2385 K. pneumoniae CDC CNP 09 VIM 2315.76 K. pneumoniae 700603 None 2659.8725 K. pneumoniae SML86 None 1861.1485 K. pneumoniae SML87 None 3075.4205

TABLE-US-00002 TABLE 2 Carbapenemase assay results for various concentrations of imipenem within 4 hours. Carbapenemase Activity in various concentrations of imipenem Known 50 125 250 500 1250 2500 Species Strain Carbapenemase g/mL g/mL g/mL g/mL g/mL g/mL E coli ATCC 25922 none 70.5 522 599.5 558.5 463.5 282 K pneumoniae ATCC BAA- KPC 1817 4702.5 4270.5 3708 3251 3114.5 2342 E coli AR 114 KPC3 930.5 3793.5 3342.5 2784.5 2173.5 1825.5 E coli AR 69 NDM 1111 3367 2827 2300.5 1908.5 1902.5 K pneumoniae AR 34 IMP 1034.5 2776.5 2456.5 1807.5 1148 637.5

TABLE-US-00003 TABLE 3 Carbapenemase assay performed with dried reagents. Known Carbapenemase Species Strain Carbapenemase Activity E coli ATCC 25922 none 6116 K pneumoniae ATCC BAA-1705 KPC2 19742 K pneumoniae ATCC BAA-2814 KPC3 18057 K pneumoniae ATCC 700603 none 2026

EXAMPLES

[0129] Example 1. Microbes 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 50 I of inoculum was added to wells of a 384-well plate. For each strain, 25 I of solutions containing either 0.25 mg/mL nitrocefin alone (Nitro), 0.25 mg/mL nitrocefin and 4g/mL clauvulanate (Nitro Clay), or 0.25 mg/mL nitrocefin and 4g/mL tazobactam (Nitro Tazo) were added to duplicate wells. Inoculated plates were incubated at 35 C., shaking at 150 rpm for 4 hours. Absorbance was read at 490 nm and values shown in FIG. 1 are the absorbance in the indicated condition minus the absorbance from microbial inoculum alone without nitrocefin. A greater value indicates microbial ability to degrade nitrocefin, and thus, the presence of a beta lactamase. A reduction in absorbance value in the presence of an inhibitor indicates the beta lactamase present is sensitive to the inhibitor. For the data presented here, EC 25922 does not contain a beta lactamase, but the other two organisms do. Further, the inhibitors are both active against each of the strains with known beta lactamases.

[0130] Example 2. Microbes were prepared by diluting colonies into saline to reach a McFarland value of 0.5, which was verified using a spectrophotometer. 100 L of the microbe solution was added to each of 2 wells in a 96-well plate. 100 L of saline was added to 2 separate wells on the plate. 100 L of an imipenem solution containing 5 mg/mL imipenem, 0.1 mM ZnSO.sub.4, and 10 M Fluorescein Na salt, pH 8, was added to one of the microbe-containing wells (Well.sub.2) and one of the saline wells (Well.sub.1). 100 L of a negative control solution containing 0.1 mM ZnSO.sub.4, and 10 M Fluorescein Na salt pH 7.5, was added to one of the microbe-containing wells (Well.sub.4) and one of the saline wells (Well.sub.3). Plates were incubated, shaking at 35 C. for 4 hours and fluorescence of the wells was read at Em=490 nm/Ex=510 nm. The values reported were calculated to determine the hydrolysis of imipenem by the microbes using Equation 1. These data are tabulated in Table 1.

[0131] Example 3. Microbes were prepared by diluting colonies into saline to reach a McFarland value of 0.5, which was verified using a spectrophotometer. 25 L of the microbe solution was added to each of 2 wells in a 384-well plate. 25 L of saline was added to 2 separate wells on the plate. 25 L of an imipenem solution containing imipenem, 0.1 mM ZnSO.sub.4, and 10 m Fluorescein Na Salt, pH 8, was added to one of the microbe-containing wells (Well.sub.2) and one of the saline wells (Well.sub.1). 25 L of a negative control solution containing 0.1 mM ZnSO.sub.4, and 10 M Fluorescein Na Salt, pH 7.5, was added to one of the microbe containing wells (Well.sub.4) and one of the saline wells (Well.sub.3). Plates were incubated, shaking at 35 C. for 3 hours and fluorescence of the wells was read at Em=490/Ex=510. This organization of the assay was performed multiple times, varying the concentration of imipenem in solution. The assay was run with final imipenem concentrations of 50 g/mL, 125 g/mL, 250 g/mL, 500 g/mL, 1250 g/mL, and 2500 g/mL. The values reported were calculated to determine the hydrolysis of imipenem by the microbes using Equation 1. These data are tabulated in Table 2.

[0132] Example 4. Microbes were prepared by diluting colonies into saline to reach a McFarland value of 0.5, which was verified using a spectrophotometer. 100 L of the microbe solution was added to each of 2 wells in a vacuum-dried 96-well plate (Well.sub.2, Well.sub.4). 100 L of saline was added to 2 separate wells on the plate (Well.sub.1 and Well.sub.3). Following addition of microbial or saline solutions, the final concentrations of the contents in each well were: 300 g/mL imipenem, 0.05 mM ZnSO.sub.4, and 10 M Fluorescein Na Salt, pH 7.8, (Well.sub.1, Well.sub.2); only 0.05 mM ZnSO.sub.4 and 10 M Fluorescein Na Salt, pH 7.8, were present in the third and four wells (Well.sub.3, Well.sub.4). Plates were incubated, shaking at 35 C. for 4 hours and fluorescence of the wells was read at Em=490/Ex=510. The values reported were calculated to determine the hydrolysis of imipenem by the microbes using Equation 1. These data are tabulated in Table 3.

[0133] Example 5. A starting solution comprising 0.01875 mg/mL Fluorescein Sodium Salt (Sigma PN 30181) and 0.5 mM Zinc Sulfate was made in deionized water. Solution pH was monitored using an Accumet AR-15 pH meter. Three independent preparations found that the starting pH of this solution was approximately 6.7+/0.1. Upon addition of 250 g/mL of imipenem monohydrate (Chem Impex PN 30802), the solution pH was observed to drop to pH<5 within 5 minutes, eliminating fluorescein fluorescence.

[0134] Example 6. Three batches of the same starting solution as in Example 5 were prepared with different sodium hydroxide concentrations: solution 1 had 0.01 mM NaOH (pH 6.94), solution 2 had 0.02 mM NaOH (pH 7.44), and solution 3 had 0.03 mM NaOH (pH 7.80). The solution pH was monitored. After addition of 250 g/mL imipenem, the solution pH dropped to pH<5 within 5-10 minutes for all three solutions.

[0135] Example 7. Four batches of the same starting solution as in Example 5 were prepared with different concentrations of pH 8 sodium phosphate buffer: solution 1 had 0.1 M sodium phosphate buffer, solution 2 had 0.01 M sodium phosphate buffer, solution 3 had 0.005 M sodium phosphate buffer, and solution 4 had 0.001 M sodium phosphate buffer. The solution pH was again monitored following the addition of 250 g/mL of imipenem. Solutions 1 and 2 were stable at pH 7.92 and 7.86, respectively, for over 15 minutes. Solution 3 showed a pH drop from 7.72 to 7.68 after 15 minutes but remained stable for the next 10 minutes with no drop. Solution 4 showed a pH drop from 7.00 to 6.93 over 15 minutes and to 6.82 over 60 minutes. Solutions 1-4 and the same solutions except without imipenem were loaded into different wells of a 384-well plate after approximately 60 minutes and vacuum dried. These panels were then used for carbapenemase testing following the procedure of Example 4 using two ATCC bacteria, BAA-2814, which is carbapenemase positive, and 25922, which is carbapenemase negative. The difference between the signals in the 4 wells outlined in Examples 1-4 is shown in Table 4. These data demonstrate that Solutions 2-4 accurately identify the positive carbapenemase strain. The data in Table 5 demonstrate the similar performance of 2 wells to the 4 wells of Table 4 for Solutions 2-4. Thus, these data demonstrate the saline alone control wells may be eliminated without compromising the assay performance.

[0136] Example 8. Solution 4 and the similar solution without imipenem of Example 7 were filled into multiple wells of 384-well microplates, followed by vacuum drying. Multiple different bacterial strains listed in Table 6 were inoculated into each of 2 wells. The results from the 2-well carbapenemase assay following 3 hours of incubation with 10 representative gram negative bacteria are shown in Table 6. The 2-well assay accurately identifies all carbapenemase-producing organisms without false positives.

[0137] Example 9. Ethylenediaminetetraacetic acid (EDTA) was added at 0.01 mM to Solution 4 from Example 7. This was vacuum dried in wells of a 384-well microplate in parallel with Solution 4 with and without imipenem. Multiple different bacterial strains listed in Table 7 were inoculated into each of 3 wells. The results from the 3-well carbapenemase assay following 3 hours of incubation with 7 representative gram negative bacteria are shown in Table 7. The 3-well assay accurately identifies all carbapenemase-producing organisms without false positives and the incorporation of EDTA enables the accurate identification of metallo-carbapenemases (KPCs).

[0138] Example 10. Microbes were prepared by diluting colonies into saline to reach a McFarland value of 0.5, which was verified using a spectrophotometer. 50 L of the microbe solution was added to each of 3 wells in a 384-well plate containing dried test reagents. Upon reconstitution with 50 L, the final concentrations in the test wells were: Well 1: 1.875 g/ml Fluorescein, 50 M Zinc Sulfate, 0.2 mM Sodium Phosphate Buffer pH 8; Well 2: 1.875 g/ml Fluorescein, 50 M Zinc Sulfate, 0.2 mM Sodium Phosphate Buffer pH 8, 250 g/ml Imipenem; Well 3: 1.875 g/ml Fluorescein, 10 M Ethylenediaminetetraacetic Acid (EDTA), 0.2 mM Sodium Phosphate Buffer pH 8, 250 g/ml Imipenem. Samples were incubated at 35 C. for 3 hours and fluorescence was read at Em=490 nm/Ex=510 nm. Data is shown in Table 8.

TABLE-US-00004 TABLE 4 BAA-2814 25922 Solution 1 320.8 238.6 Solution 2 1662.2 382.4 Solution 3 2420.9 533.2 Solution 4 8143.9 329.6

TABLE-US-00005 TABLE 5 BAA-2814 25922 Solution 1 320.8 238.6 Solution 1 - two wells 994.2 434.8 Solution 2 1662.2 382.4 Solution 2 - two wells 2779.1 1499.3 Solution 3 2420.9 533.2 Solution 3 - two wells 4296.6 1342.4 Solution 4 8143.9 329.6 Solution 4 - two wells 8641.7 827.4

TABLE-US-00006 TABLE 6 Strain Carbapenemase with zinc SD 963- M. morganii (MM) NDM 3643.667 CDC-CarbaNP-26 AR-0057 SD 879- M. morganii (MM) KPC 2 3589.167 CDC-CRE-22, AR-0133 SD 412- P. aeruginosa (PA) VIM 4 2239.167 CDC-CarbaNP-23, AR-0054 SD 1151- E. cloacae (ECL) NDM-1 2771.833 CDC-CarbaNP 07 AR-0038 SD 1232- P. mirabilis (PM) NDM-1 178.5 CDC-CRE 48 AR-0159 SD 2008- KP NDM-1 5739.333 SD 2009- KP NDM-1 4123.833 ATCC 700603 none 359.833 ATCC BAA-2814 KPC 2 6708.333 ATCC 25922 none 622.667

TABLE-US-00007 TABLE 7 Carba Test with Carbapenemase Carba Test Value EDTA E cloacae IMP 4375 1816.5 M morganii NDM 3551 1923.5 M morganii KPC 1848 1288.5 E cloacae NDM 2218.5 2084.5 K pneumoniae NDM 3603 2222.5 K pneumoniae KPC 3925.5 3705.5 E coli 25922 none 1149 1541.5

TABLE-US-00008 TABLE 8 Metallo- Carbapenemase carbapenemase Species Carbapenemase Activity Inhibition K pneumoniae none 18206 7941 E coli KPC 45351 7644 E cloacae KPC 36099 8594 E cloacae KPC 27792 10752 K pneumoniae KPC 29583 13917 K pneumoniae KPC 40267 2659 K pneumoniae KPC 41300 2929 K pneumoniae KPC 42933 4052 E coli NDM 34069 27006 E coli NDM 49524 34505 K pneumoniae NDM 30167 21460