Assays for improving automated antimicrobial susceptibility testing accuracy

1. A method for performing automated antimicrobial susceptibility testing of a carbapenem antimicrobial comprising

(a) performing a dilution assay comprising

inoculating a microbial inoculum of concentration C 0 into a plurality of fluid wells defining a dilution series for the carbapenem antimicrobial; and

measuring in each of the plurality of fluid wells a signal associated with microbial growth; and

(b) in parallel with the dilution assay, performing a carbapenemase assay comprising

measuring a signal associated with carbapenem degradation in a first well comprising the carbapenem antimicrobial and a microbial inoculum with concentration C R , wherein C R >10 x C 0 ;

measuring a signal associated with carbapenem degradation in one or more second wells comprising different contents than the first well and thereby acting as controls for the first well; and

(c) comparing the signals measured from the carbapenemase assay wells; and

(d) 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 measured in each of the plurality of fluid wells to define a minimum inhibitory concentration (MIC) of the carbapenem antimicrobial.

3. The method of claim 1 , wherein carbapenem degradation is determined by a signal associated with an indicator.

4. The method of claim 3 , wherein the indicator is a fluorescent or optical pH indicator.

5. The method of claim 4 , wherein the pH indicator is one or more of fluorescein, pyranine, tinopal, or a derivative thereof.

6. The method of claim 1 , wherein the carbapenem is selected from imipenem and biapenem and their derivatives.

7. The method of claim 1 , wherein the first well further comprises ionic zinc.

8. The method of claim 7 , wherein ionic zinc is one or more of zinc sulfate, zinc chloride or zinc hydroxide.

9. The method of claim 1 , wherein the assay wells are optically interrogated after less than or equal to 3, 6, 8, or 10 hours of incubation under conditions that promote microbial growth.

10. The method of claim 1 , wherein the one or more second wells comprise

(a) a first control well comprising a microbial inoculum at C R , a pH indicator and ionic zinc;

(b) a second control well comprising a pH indicator and ionic zinc; and

(c) a third control well comprising the carbapenem, a pH indicator and ionic zinc.

11. The method of claim 1 , wherein the carbapenemase assay comprises one or more more additional wells comprising a beta-lactamase inhibitor.

12. An automated antimicrobial susceptibility test assay cartridge for a beta-lactam/beta-lactamase inhibitor antimicrobial comprising:

a. two or more assay wells comprising an antimicrobial dilution series for a beta-lactam antimicrobial inoculated at a concentration C 0 with a microbial inoculum; and

b. a series of assay wells for a beta-lactamase inhibitor assay probe inoculated at a concentration C R from the same microbial inoculum comprising:

i. one or more assay wells comprising a beta-lactamase probe; and

ii. two or more assay wells comprising the same beta-lactamase probe and a beta-lactamase inhibitor at one or more concentrations.

13. The cartridge of claim 12 , wherein the probe is chromogenic or fluorometric.

14. The cartridge of claim 12 , wherein the probe is nitrocefin.

15. The cartridge of claim 12 , wherein the inhibitor is one or more of clavulanate, cloxacillin, tazobactam, avibactam, relebactam and vaborbactam.

16. An automated antimicrobial susceptibility test assay cartridge for one or more carbapenems comprising:

a. two or more assay wells comprising an antimicrobial dilution series for a carbapenem antimicrobial;

b. a series of assay wells for a carbapenemase assay comprising:

i. one or more assay wells comprising a beta-lactamase probe; and

ii. two or more assay wells comprising the same beta-lactamase probe and two or more inhibitors selected from the list of clavulanate, cloxacillin, and tazobactam;

and adapted for inoculation with microorganisms from a single microbial inoculum, wherein the results of the carbapenemase assay influence algorithmic MIC determinations of one or more carbapenems.

17. An automated antimicrobial susceptibility test assay cartridge for one or more carbapenems and/or beta-lactam/beta-lactamase inhibitors comprising:

a. two or more assay wells comprising an antimicrobial dilution series for a carbapenem antimicrobial;

b. a series of assay wells for a carbapenemase assay comprising:

i. one or more assay wells comprising saline, a carbapenem, ionic zinc, and a pH indictor;

ii. one or more assay wells comprising saline, a carbapenem, ionic zinc, a pH indictor, and a microbe sample comprising <1×10 8 CFU intact microbes;

iii. one or more assay wells comprising saline, ionic zinc, and a pH indictor;

iv. one or more assay wells comprising saline, ionic zinc, a pH indictor, and a microbe sample comprising <1×10 8 CFU intact microbes;

and adapted for inoculation with microorganisms from a single microbial inoculum and wherein the results of the carbapenemase assay influence algorithmic MIC determinations of one or more carbapenems.

18. The cartridge of claim 17 , wherein the carbapenem is imipenem or biapenem.

19. The cartridge of claim 17 , wherein the pH probe is fluorescein.

20. The cartridge of claim 17 , wherein the ionic zinc is selected from zinc sulfate or zinc chloride.

21. The cartridge of claim 17 , wherein one or more additional wells comprise beta lactamase inhibitors.

22. A method for performing multi-assay rapid antimicrobial susceptibility testing sequences, the method comprising

(a) inoculating two or more different concentrations of a sample comprising a microorganism derived from a clinical sample into a plurality of wells of a test panel, at least a portion of the plurality of wells containing one or more antimicrobials of a plurality of antimicrobials for inoculation of the sample;

(b) loading the test panel into an automated rapid antimicrobial susceptibility testing system for performing a multi-assay testing sequence; and

(c) 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 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;

perform a 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.