Technology to generate and utilize customized microorganism-growth assay keys

Abstract

A system for generating and using an assay key comprising growth conditions for a set of microorganisms for a set of diverse QAC-based culture media under a variety of incubation conditions known to modulate the effect of QAC on growth of some microorganisms in the set. Each culture medium is characterized by a pH and includes one or more QACs and one or more growth supplements. The set of culture media includes media comprising various combinations of pH, QAC type, QAC concentration, growth supplement type, and growth supplement concentration. The assay key can be used to identify a microorganism by inoculating a variety of growth media within the key and incubating the inoculated media under conditions within the key and comparing the resulting pattern of microorganism growth across the media and conditions with growth patterns for various known microorganisms across the media and conditions that are within the key.

Claims

1. A method for identifying a microorganism, the method comprising: (a) selecting a first set of one or more culture-medium panels, each panel of the first set of one or more culture-medium panels comprising a plurality of diverse culture media, each culture medium of the plurality of diverse culture media characterized by a pH and comprising a QAC having a QAC type and a QAC concentration and a growth supplement having a growth-supplement type and a growth-supplement concentration, wherein no two culture media of the plurality of diverse culture media have the same pH, QAC type, QAC concentration, growth-supplement type, and growth-supplement concentration; (b) inoculating each of the one or more culture-medium panels of the first set of culture-medium panels with a sample of a microorganism; (c) incubating the inoculated one or more culture-medium panels of the first set of culture-medium panels at a first incubation condition comprising a first time and a first temperature; (d) determining a first growth-pattern comprising microorganism-colony-growth state for each culture medium of the plurality of diverse culture media of each culture-medium panel of the first set of culture-medium panels for the first incubation condition; and (e) comparing the first growth-pattern with an assay key comprising growth patterns for various microorganisms for the diverse culture media for the first incubation condition.

2. The method of claim 1 further comprising: (a) selecting a second set of one or more culture-medium panels that is a duplicate of the first set of one or more culture-medium panels; (b) inoculating each of the one or more culture-medium panels of the second set of culture-medium panels with the sample of the microorganism; (c) incubating the inoculated one or more culture-medium panels of the second set of culture-medium panels at a second incubation condition comprising a second time and a second temperature, wherein the second incubation condition differs from the first incubation condition in at least one of the following ways: the first time differs from the second time, the first temperature differs from the second temperature; (d) determining a second growth-pattern comprising microorganism-colony-growth state for each culture medium of the plurality of diverse culture media of each culture-medium panel of the second set of culture-medium panels for the second incubation condition; and (e) comparing the second growth-pattern with an assay key comprising growth patterns for various microorganisms for the diverse culture media for the second incubation condition.

3. The method of claim 1 wherein each culture medium includes at least one type of growth supplement from the group consisting of peptone, potato dextrose, and yeast nitrogen base.

4. The method of claim 1 wherein each QA culture medium includes at least one type of QAC from the group consisting of cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, and tetradecyltrimethylammonium bromide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings where:

(2) FIG. 1 depicts the plating scheme for the suspension-based plate assays depicted in FIGS. 2 and 13A.

(3) FIG. 2 depicts plate assays for various yeasts using culture media containing different amounts of a Quaternary Ammonium Compound (QAC, cetylpyridinium chloride in this example) and using different incubation temperatures.

(4) FIG. 3 depicts a comparison between a plate assay using the nonselective standard Sabouraud-dextrose culture medium (left) and using an exemplary QAC-based positive-selection culture medium (right).

(5) FIG. 4 depicts the plating scheme for the assays depicted in FIGS. 5A and 5B.

(6) FIGS. 5A-5B depict plate assays for 25-day-old C. auris cells using a standard Sabouraud-dextrose culture medium (FIG. 5A) and a QAC-based positive-selection medium configured to support old C. auris cells (FIG. 5B).

(7) FIG. 6 depicts the plating scheme for the assays depicted in FIGS. 7A-7C.

(8) FIGS. 7A-7C depict plate assays for a variety of environmental samples using nonselective standard media (a yeast-extract-peptone-dextrose-based medium (YPD) and a brain-heart-infusion-based medium (BHI)) and a QAC-based positive-selection medium configured to support old C. auris cells.

(9) FIG. 8 depicts the plating scheme for the assays depicted in FIGS. 9A-13B.

(10) FIGS. 9A-10B depict plate assays for a variety of organisms using a QAC-free control medium and a QAC-based positive-selection medium configured to support old C. auris cells.

(11) FIGS. 11A-12B depict plate assays for a variety of organisms using a QAC-free control medium and a QAC-based positive-selection medium.

(12) FIGS. 13A-13B depict plate assays for a variety of organisms using a standard, nonselective, Sabouraud-dextrose culture medium (FIG. 13A) and a negative-selection culture medium containing tert-butyl-hydroperoxide (tBHP) (FIG. 13B).

(13) FIGS. 14A-14B depict plate assays for a variety of organisms using a tBHP-free, tryptone-based, nonselective, control culture medium (FIG. 14A) and a negative-selection culture medium containing tert-butyl-hydroperoxide (FIG. 14B).

(14) FIGS. 15-18 depict flow charts for exemplary assays to detect C. auris.

(15) FIG. 19 depicts an exemplary assay key including a panel of culture media, incubation conditions, and microorganism-colony growth states for a variety of microorganisms for each culture medium of the panel and each incubation condition.

(16) FIG. 20 depicts an exemplary assay comprising a first test consisting of identifying candidate microorganisms using two different culture media incubated at different conditions and a second test consisting of identifying candidate microorganisms using a third culture medium.

(17) FIG. 21 depicts an exemplary assay for identifying a microorganism using panels of diverse culture media.

DETAILED DESCRIPTION

(18) In the summary above, and in the description below, reference is made to particular features of the invention in the context of exemplary embodiments of the invention. The features are described in the context of the exemplary embodiments to facilitate understanding. But the invention is not limited to the exemplary embodiments. And the features are not limited to the embodiments by which they are described. The invention provides a number of inventive features which can be combined in many ways, and the invention can be embodied in a wide variety of contexts. Unless expressly set forth as an essential feature of the invention, a feature of a particular embodiment should not be read into the claims unless expressly recited in a claim.

(19) Except as explicitly defined otherwise, the words and phrases used herein, including terms used in the claims, carry the same meaning they carry to one of ordinary skill in the art as ordinarily used in the art.

(20) Because one of ordinary skill in the art may best understand the structure of the invention by the function of various structural features of the invention, certain structural features may be explained or claimed with reference to the function of a feature. Unless used in the context of describing or claiming a particular inventive function (e.g., a process), reference to the function of a structural feature refers to the capability of the structural feature, not to an instance of use of the invention.

(21) Except for claims that include language introducing a function with “means for” or “step for,” the claims are not recited in so-called means-plus-function or step-plus-function format governed by 35 U.S.C. § 112(f). Claims that include the “means for [function]” language but also recite the structure for performing the function are not means-plus-function claims governed by § 112(f). Claims that include the “step for [function]” language but also recite an act for performing the function are not step-plus-function claims governed by § 112(f).

(22) Except as otherwise stated herein or as is otherwise clear from context, the inventive methods comprising or consisting of more than one step may be carried out without concern for the order of the steps.

(23) The terms “comprising,” “comprises,” “including,” “includes,” “having,” “haves,” and their grammatical equivalents are used herein to mean that other components or steps are optionally present. For example, an article comprising A, B, and C includes an article having only A, B, and C as well as articles having A, B, C, and other components. And a method comprising the steps A, B, and C includes methods having only the steps A, B, and C as well as methods having the steps A, B, C, and other steps.

(24) Terms of degree, such as “substantially,” “about,” and “approximately,” are used herein to denote features that satisfy their technological purpose equivalently to a feature that is “exact.” For example, a component A is “substantially” perpendicular to a second component B if A and B are at an angle such as to equivalently satisfy the technological purpose of A being perpendicular to B.

(25) Except as otherwise stated herein, or as is otherwise clear from context, the term “or” is used herein in its inclusive sense. For example, “A or B” means “A or B, or both A and B.”

(26) In the culture assays depicted herein, (e.g., FIGS. 2, 3, 5A, 5B, 7A, 7B, 7C, 9A, 9B, 10A, 10B, 11A, 11B, 12A, 12B, 13A, 13B,) a dark spot denotes the presence of a colony. Larger spots depict larger colonies. More spots depict more colonies. The density or overlap of colonies often results in a single spot.

(27) The invention is premised on the discovery that C. auris has two distinctive phenotypes: First, C. auris is relatively more resistant to quaternary ammonium compounds (“QACs” or “quats”) when compared to other yeasts. This relative resistance increases at higher temperatures (other yeasts show a decreased resistance at higher temperature while C. auris remains approximately the same). And a difference in relative sensitivity to a particular QAC between C. auris and another yeast can be enhanced by choosing a particular kind of growth-factor supplement (for example, Yeast Nitrogen Base vs. peptone). Second, C. auris is relatively less resistant to tert-butyl-hydroperoxide (“tBHP”) when compared to other yeasts. Utilizing these relative sensitivities, identification of C. auris is improved through use of a culture medium that allows C. auris growth while suppressing other yeasts (“positive selection”) and a culture medium that suppresses C. auris growth while allowing the growth of other yeasts (“negative selection”).

(28) Positive-Selection System: C. auris has a high degree of relative resistance towards QACs, such as cetylpyridinium chloride. The same is true for C. haemulonii which (together with C. lusitaniae and C. krusei) is the closest relative to C. auris. The closely-related Candida species can be distinguished, however, by their differential QAC resistance at higher-than-standard yeast incubation temperatures (preferably in the range of 37° C.-38.5° C. vs the standard 30° C.). Besides cetylpyridinium chloride, other QACs, including benzalkonium chloride, benzethonium chloride, and tetradecyltrimethylammonium bromide were successfully used to differentiate C. auris from other Candida species. As an added benefit, growth of many bacteria and molds is suppressed at the applied QAC concentrations. This makes this system an appealing choice for positive selection of C. auris within a sample containing mixed microbial species (e.g. a sample wiped from human skin).

(29) Discriminating Candida species based on their relative sensitivity to QACs is a novel approach to identifying C. auris. QACs are known to be relatively ineffective in suppressing or killing Candida species. Using short-term exposure, activity of QACs has been described as generally weak in various Candida species. See, e.g., J. L. Cadnum et al., Effectiveness of Disinfectants Against Candida auris and Other Candida Species, 38 Infection Control & Hospital Epidemiology 1240-1243 (October 2017), available at doi.org/10.1017/ice.2017.162. But differential sensitivity of colony formation ability among different Candida species to QACs added to growth media is largely unexplored. Using this discriminating phenotype is a novel and effective way to identify C. auris. Further, using the temperature and growth factor supplement dependence of the relative sensitivity to QACs is a novel and effective way to identify C. auris. This relative sensitivity is the basis for positive selection media for C. auris.

(30) FIG. 2 illustrates C. auris's relative resistance to cetylpyridinium chloride. The figure depicts eight plates containing four different culture mediums that differ with respect to the concentration of cetylpyridinium chloride in the medium. Each plate includes seven different yeasts (Candida albicans, Candida glabrata, Candida krusei, Candida auris, Candida haemulonii, Candida famata, and Saccharomyces cerevisiae) at four different calculated cell counts (62, 625, 6,250, 62,500, with the uncertainty inherent in the calculation). Four of the yeast-containing plates (one of each culture medium) were incubated at 30° C. and the other four (one of each culture medium) were incubated at 38.5° C.

(31) The plating scheme for the FIG. 2 assay is illustrated in FIG. 1. Drops of various yeast suspensions are placed on a culture medium 12 in a plate 10. The drops contain yeasts (Candida albicans, Candida glabrata, Candida krusei, Candida auris, Candida haemulonii, Candida famata, and Saccharomyces cerevisiae) as listed on the vertical axis 14. The drops contain different calculated cell counts (62, 625, 6,250, 62,500) as listed on the horizontal axis 16. The dashed lines are there to show the intersection of yeast and cell count for a particular drop. For example, the drop 18 at the intersection of C. auris and 62,500 indicates that a drop with 62,500 cells of C. auris was placed on the medium 12 at that spot.

(32) Yeasts were first streaked on Sabouraud-dextrose agar plates and pre-grown at 30° C. for 72 hours. Suspensions were made and adjusted to 2.5×10.sup.7 cells per ml and subjected to serial 1:10 dilutions. Drops were placed on solid agar plates of Sabouraud dextrose (4% dextrose and 10 g/l peptone) which contained various amounts of cetylpyridinium chloride that were added after autoclaving from a 2% stock solution in isopropanol.

(33) As shown, after 48 hours of incubation at 30° C., C. glabrata, C. krusei, and S. cerevisiae are most sensitive whereas C. auris and C. haemulonii are the most resistant species. As the amount of cetylpyridinium chloride in the medium increases (top to bottom in the figure), all but C. auris and C. haemulonii are suppressed. These two species cannot be distinguished one from the other on the data presented. Notably, C. auris is frequently misidentified as C. haemulonii in the VITEK identification system. S. Kathuria et al., Multidrug-Resistant Candida auris Misidentified as Candida haemulonii: Characterization by Matrix-Assisted Laser Desorption Ionization—Time of Flight Mass Spectrometry and DNA Sequencing and Its Antifungal Susceptibility Profile Variability by Vitek 2, CLSI Broth Microdilution, and Etest Method, 53 Journal of Microbiology 1823-1830 (June 2015), available at jcm.asm.org/content/53/6/1823.full.pdf+html. Thus, the QAC-containing medium alone may not entirely resolve the misidentification problems of the prior art, though research is ongoing.

(34) After 48 hours of incubation at 38.5° C., however, C. haemulonii shows an increased sensitivity to cetylpyridinium chloride relative to C. auris (the opposite is found for C. albicans). Thus, C. auris can be distinguished from C. haemulonii. It should be noted that C. auris, unlike many other yeasts, is known to be able to grow at high temperatures (42° C. and above) but 38.5° C. is a temperature that still fully supports the growth of all tested yeast strains if untreated with cetylpyridinium chloride. Thus, the positive selection medium is based on the relative cetylpyridinium chloride resistance of C. auris at elevated temperatures.

(35) FIG. 3 illustrates the difference in response between cell-suspension samples cultured on a standard medium (left plate) and samples cultured on an exemplary QAC-containing positive-selection medium according to the invention (right plate). C. auris colonies (top culture on both plates) are the only visibly present culture on the QAC-containing medium. The middle sample (a wipe taken with a sterile cotton swab from the surface of an unwashed grape) and the bottom sample (human-skin wipe) present growing colonies on the standard medium but not on the QAC-containing medium.

(36) A recipe for an exemplary positive-selection medium is as follows: Mix per one liter of deionized water (all % in weight/volume, subject to normal laboratory uncertainty): (1) a solidifying agent if procedure done on plates: 2% agar; (2) a sugar: 4% dextrose (autoclave separately, add after autoclaving); (3) a supplement mix: 0.7% Yeast Nitrogen Base with ammonium sulfate (commercial mix, autoclave separately, add after autoclaving); and (4) a Quaternary Ammonium Compound (all stocks dissolved at 2% in 91% isopropanol, add after autoclaving): cetylpyridinium chloride=8.5 ml or benzalkonium chloride=13 ml or tetradecyltrimethylammonium bromide=13 ml. It is possible to use other supplements such as peptone but there are differences, e.g. yeast extract is not recommended. As shown below, the nature of the supplement may critically modulate QAC sensitivity in a species-specific manner to improve positive selection of C. auris. Variations in ingredients may also improve the initial selection of C. auris within samples that may contain a multitude of other microorganisms which need to be suppressed. For example, the following adaptations (alone or in combinations) may be implemented: (1) Add extra isopropanol (or dissolve the QAC at a lower concentration in isopropanol). Could go up to 3%. C. auris of approximately 1-12 days of culture age is very resistant to isopropanol but many other organisms are not. This approach may not be appropriate if it is possible that older C. auris is present because C. auris of about 12-days of culture age or older is less resistant to isopropanol than is younger C. auris. (2) Lower the amount of Yeast Nitrogen Base. Can go down to 0.14% which makes the medium less rich. This is no problem for C. auris but fewer organisms will grow. (3) Add 25 μg/ml chloramphenicol (stock: 50 mg/ml in ethanol, a standard method to suppress bacteria). Samples are placed on solid media by, for example, streaking or dropping suspension. The inoculated medium is incubated at approximately 38.5° C. and evaluated after as early as 36 hours, preferably after about 48 to 60 hours.

(37) Two-Step Positive-Selection System: In circumstances potentially involving old C. auris cells (e.g., 12 days old or older) or certain other yeasts such as Candida lusitaniae and certain strains of Saccharomyces cerevisiae, it is preferable to use a two-step process at 37° C. The first step involves a first positive-selection medium (the “Step-1 Medium”) that has been modified to the allow uninhibited growth of colonies from old C. auris cells which may be of concern in the surveillance of surface contamination (e. g. in hospitals). Type and concentration of QAC are selected to minimize impact on old C. auris cells, isopropanol is not used as a solvent, and the incubation temperature is 37° C. This Step-1 Medium may not, however, uniquely select C. auris. For example, C. lusitaniae and certain strains of S. cerevisiae may grow on or in such a medium, even when incubated at an elevated temperature (e.g., 37° C.). The second step involves a second positive-selection medium (the “Step-2 Medium”) and cells cultured with the first medium. Because the cells for the second step come from the colonies formed in the first step, the risk of failing to detect C. auris because of the age of the cells is not present. The Step-2 Medium is formulated to suppress growth of C. lusitaniae and S. cerevisiae when incubated at an elevated temperature (e.g., 37° C.). Thus, the Step-2 Medium is designed to positively uniquely select C. auris without concern for the age of the cells. Together, the Step-1 Medium and the Step-2 Medium positively select for C. auris without significant loss of old C. auris cells and without significant risk of misidentification of another yeast as C. auris. Other organisms are also efficiently suppressed (as outlined below).

(38) A Step-1 Medium includes: (1) a higher dextrose content (relative to standard media) to inhibit bacterial growth, (2) an antibiotic to inhibit bacterial growth, (3) a nutritionally poor (with no amino acids) and defined supplement mixture to inhibit growth of bacteria, yeast, and molds with complex growth requirements, and (4) a QAC or a combination of more than one QAC to generally suppress the growth of organisms other than C. auris.

(39) An exemplary recipe for a solid Step-1 Medium is as follows: Mix per one liter of deionized water: (1) 20 g agar; (2) 60 g dextrose (add from 200 g/l stock solution after autoclaving); (3) 25 mg chloramphenicol (add from a 50 mg/ml stock in ethanol after autoclaving); (4) 6.7 g Yeast Nitrogen Base without amino acids and with ammonium sulfate (YNB) (add from 67 g/l stock in water after autoclaving); (5) 70 mg benzalkonium chloride (add from a 10 mg/ml stock in water after autoclaving); and (6) 80 mg tetradecyltrimethylammonium bromide (add from a 20 mg/ml stock in water after autoclaving).

(40) As shown in FIGS. 4, 5A, and 5B, an exemplary Step-1 Medium supports the growth of even old C. auris cells. The plating scheme for this test is shown in FIG. 4. Suspensions of ten different C. auris strains, CDC-AR Bank #0381-#390, were made in sterile deionized water, kept for 25 days at room temperature, and diluted. Drops (10 μl) of the diluted solutions were streaked on a control medium and a Step-1 Medium plate. The inoculated control medium, a standard Sabouraud-dextrose medium, was incubated at 30° C. for 48 hours. (The control conditions are known to be favorable to the growth of C. auris.) The inoculated Step-1 Medium was incubated at 37° C. for 72 hours. The number of C. auris colonies formed on the Step-1 Medium (FIG. 5B) is approximately the same as the number formed on the control medium (FIG. 5A). While the size of the colonies may differ between the control and the Step-1 Medium, having the same number of colonies indicates that the Step-1 Medium supports old C. auris cells to the same extent as the control.

(41) As shown in FIGS. 6, 7A, 7B, and 7C, an exemplary Step-1 Medium inhibits the growth of commonly occurring organisms while supporting the growth of C. auris. Samples of C. auris (pure), human skin wipes, and grape surface wipes were streaked on three different media—two control media and Step-1 Medium. One of the grape wipes (grape B) was from a grape purposefully contaminated with C. auris. The media were incubated at either 30° C. or 37° C. The results are depicted in FIGS. 7A-7C. The plating scheme for this test is shown in FIG. 6. FIG. 7A depicts an assay using yeast extract, peptone, and dextrose (YPD) medium. The YPD medium supports the growth of a wide variety of organisms while favoring yeasts and molds due to dextrose content. The inoculated YPD medium was incubated at 30° C. for 48 hours. Colonies grew for each sample, but the C. auris colonies from the grape-B wipe (plate row F in the figure) are not distinguishable from other organisms due to the intense growth of other organisms (mold). FIG. 7B depicts an assay using brain heart infusion (BHI) medium. The BHI medium supports the growth of a wide variety of organisms while favoring bacteria. The inoculated BHI medium was incubated at 37° C. for 72 hours. Colonies grew for each sample. Some of the C. auris from the grape-B wipe (plate row F in the figure) are seen to grow among other organisms (bacteria and molds, the latter growing slower as in 7A due to less favorable conditions). FIG. 7C depicts an assay using Step-1 Medium. Here, only C. auris colonies grew (plate rows A, B, and F in the figure) and can thus be clearly identified as the only growing species in a mixed sample of various organisms (compare row F in FIGS. 7A-7C).

(42) Two other tests of Step-1 Medium are depicted in FIGS. 8-10B. Suspensions of various organisms were made from samples provided by the CDC, ATCC (American Type Culture Collection), and DSMZ (German Collection of Microorganisms and Cell Cultures). Drops (10 μl) of the solutions containing approximately 2×10.sup.5 cells (calculated) were placed on two media: a control medium plate (a Step-1 Medium without a QAC) and a Step-1 Medium plate. The plating scheme is shown in FIG. 8. One inoculated control medium plate and one inoculated Step-1 Medium plate were incubated at 30° C. for 48 hours (FIGS. 9A-9B). A second inoculated control medium plate and a second inoculated Step-1 Medium plate were incubated at 37° C. for 48 hours (FIGS. 10A-10B). The difference between FIGS. 9A and 9B shows that the Step-1 Medium suppressed the growth of four organisms at 30° C.: K. ohmeri, C. krusei (CDC AR #0397), C. krusei (ATCC14243), and C. glabrata. See plate cells (A,4), (B,4), (B,5), and (B,6), respectively (denoted as (row, column)). The difference between FIGS. 10A and 10B shows that the Step-1 Medium suppressed the growth of four additional organisms at 37° C.: C. albicans, C. famata, C. parapsilosis, C. tropicalis. See plate cells (A,6), (C,6), (D,6), and (E,6) respectively. (The higher incubation temperature suppressed the growth of C. duobushaemulonii (CDC AR #0391), C. duobushaemulonii (CDC AR #0392), C. haemulonii (CDC AR #0393), C. duobushaemulonii (CDC AR #0394), and C. haemulonii (CDC AR #0395). See plate cells (A,3), (B,3), (C,3), (D,3), and (E,3) respectively. The surviving organisms were all ten strains of C. auris (plate columns 1 and 2), C. lusitaniae (plate cell (C,4)), and two strains of S. cerevisiae (plate cells (D,4) and (E,4)).

(43) Step-2 Medium includes: (1) a higher dextrose content (relative to standard media) to inhibit bacterial growth, (2) an antibiotic to inhibit bacterial growth, (3) a nutritionally rich (with amino acids) and complex supplement mixture to modulate the C. auris QAC sensitivity vis-à-vis YNB, and (4) a QAC or a combination of more the one QAC to suppress the growth of organisms other than C. auris (including by suppressing C. lusitaniae and S. cerevisiae).

(44) An exemplary recipe for solid Step-2 Medium is as follows: Mix per one liter of deionized water: (1) 20 g agar; (2) 60 g dextrose (add from 200 g/l stock solution after autoclaving); (3) 25 mg chloramphenicol (add from a 50 mg/ml stock in ethanol after autoclaving); (4) 10 g peptone (meat); and (5) 130 mg benzalkonium chloride (add from a 10 mg/ml stock in water after autoclaving).

(45) Tests of Step-2 Medium are depicted in FIGS. 8, 11A-12B. Suspensions of various organisms were made from samples provided by the CDC, ATCC, and DSMZ. Drops (10 μl) of the suspensions containing approximately 2×10.sup.5 cells (calculated) were placed on plates of two media: control medium (Step-2 Medium without a QAC) and Step-2 Medium. The plating scheme is shown in FIG. 8. One inoculated control medium plate and one inoculated Step-2 Medium plate were incubated at 30° C. for 48 hours (FIGS. 11A-11B). A second inoculated control medium and a second inoculated Step-2 Medium were incubated at 37° C. for 48 hours (FIGS. 12A-12B). The difference between FIGS. 11A and 11B shows that at 30° C. the Step-2 Medium suppressed the growth of organisms other than C. auris (plate columns 1 and 2) and C. duobushaemulonii (CDC AR #0391), C. duobushaemulonii (CDC AR #0392), C. haemulonii (CDC AR #0393), and C. duobushaemulonii (CDC AR #0394) (plate cells (A,3), (B,3), (C,3), and (D,3)). The difference between FIGS. 12A and 12B shows that at 37° C. the Step-2 Medium suppressed the growth of organisms other than C. auris (plate columns 1 and 2) and C. duobushaemulonii (CDC AR #0392) (plate cell (B,3)).

(46) There are two points of note from the tests depicted in FIGS. 8-12B: (1) The Step-2 Medium suppressed the growth of the non-C. auris survivors of the Step-1 Medium at 37° C. incubation: C. lusitaniae (plate cell (C,4)) and S. cerevisiae (plate cells (D,4) and (E,4)). (2) The non-C. auris survivor of the Step-2 Medium at 37 ° C. incubation, C. duobushaemulonii (CDC AR #0392) (plate cell (B,3)), did not survive the Step-1 Medium at 37° C. incubation. This indicates that the Step-1 Medium and Step-2 Medium can be used in concert to uniquely identify C. auris. Step-1 Medium with incubation at 37° C. would select a candidate set that includes C. auris, C. lusitaniae (plate cell (C,4)), and S. cerevisiae, but not the other organisms. A sample taken from this candidate set will survive a 37° C. incubation on a Step-2 Medium only if the sample is C. auris.

(47) Negative Selection System: C. auris has a relatively high sensitivity to the oxidizing agent tert-butyl-hydroperoxide (“tBHP”). Hence, tBHP can be used as the basis of a negative-selection medium.

(48) FIGS. 13A-13B illustrate C. auris's relative sensitivity to tBHP. FIG. 13A depicts a plate with a tBHP-containing culture medium that includes seven different yeasts (Candida albicans, Candida glabrata, Candida krusei, Candida auris, Candida haemulonii, Candida famata, and Saccharomyces cerevisiae) at five different calculated cell counts (62, 625, 6,250, 62,500, with the uncertainty inherent in the calculation). The yeast-containing plate was incubated at 30° C. The plating scheme is illustrated in FIG. 1. The culture medium comprised a Sabouraud dextrose medium with tBHP (added after autoclaving the other media ingredients from a 70% solution in water). Whereas a resuspension of cells picked from a colony will always enhance the sensitivity of this (and related) assays, it is possible to streak cells without any dilution directly from a colony if the plate is evaluated early (after approximately 24 hours, some regrowth will occur later), as depicted in FIG. 13B.

(49) FIGS. 14A-14B further illustrate C. auris's relative sensitivity to tBHP. The plating scheme for this test is shown in FIG. 8. Suspensions of various organisms were made from samples provided by the CDC, ATCC (American Type Culture Collection), and DSMZ (German Collection of Microorganisms and Cell Cultures). Drops (10 μl) of the solutions containing approximately 2×10.sup.5 cells (calculated) were placed on two media: a tBHP-free control medium plate and a tBHP-based negative-selection medium plate. The plating scheme for this test is shown in FIG. 8. The inoculated media were incubated at 30° C. for 24 hours. The standard medium (FIG. 14A) supported the growth of all the organisms. The negative-selection medium (FIG. 14B) suppressed the growth of all ten C. auris strains (plate columns 1 and 2), as well as C. tropicalis (plate cell (E,6)).

(50) In use of a negative-selection medium, samples are added to the medium by, for example, streaking or dropping suspension. The inoculated medium is incubated at approximately 30° C. and evaluated after 22-26 hours. The accuracy and robustness of such an assay may be improved by first culturing the samples on a non-selective medium, such as a standard Sabouraud-dextrose medium, and then taking samples from the cultures and applying to the negative-selection medium. For example, samples may be placed on a standard medium, incubated at 30° C. for 24-48 hours, and then samples of the cultures placed on the negative-selection medium and incubated at 30° C. for about 24 hours. Lack of growth on the negative-selection medium indicates the samples are C. auris (or C. tropicalis, if that yeast is not first eliminated by, e.g., use of a positive-selection medium).

(51) A recipe for an exemplary negative-selection medium is as follows: Mix per one liter of deionized water (all % in weight/volume, subject to normal laboratory uncertainty): (1) a solidifying agent if procedure done on plates: 2% agar; (2) a sugar: 4% dextrose (autoclave separately, add after autoclaving); (3) a supplement mix: 1% peptone; and (4) the oxidizing agent tert-butyl-hydroperoxide (tBHP): 350 μl (from a 70% solution in water, add after autoclaving).

(52) Another recipe for an exemplary solid negative-selection medium is as follows: Mix per one liter of deionized water: (1) 20 g agar; (2) 60 g dextrose (add from 200 g/l stock solution after autoclaving); (3) 25 mg chloramphenicol (add from a 50 mg/ml stock in ethanol after autoclaving); (4) 20 g tryptone; and (5) 300 μl tert-butyl-hydroperoxide (add from 70% solution in water after autoclaving).

(53) As depicted in the exemplary flow charts of FIGS. 15-17, use of the C. auris positive-selection and negative-selection media can be combined in various ways to increase the accuracy of C. auris identification (i.e., to lower the number of false positives and false negatives). FIG. 15 illustrates an assay using a defined-supplement QAC-based positive-selection medium and a tBHP-based negative-selection medium. A sample is collected from the environment of interest 1501 and is used to inoculate the defined-supplement QAC-based positive-selection medium 1502 (e.g., the Step-1 Medium described above). The inoculated medium is incubated at an elevated temperature (˜37° C.) for about 72 hours 1503. The medium is then examined 1504: if no growing cultures are present, then one can conclude (within a margin of uncertainty) that the sample does not include C. auris 1505, if cultures have grown, then a candidate sample from the culture is collected 1506 for further analysis using a tBHP-based negative-selection medium. The candidate sample is used to inoculate a standard non-selective medium (e.g., a Sabouraud-dextrose medium) 1507, which is then incubated at standard temperature (˜30° C.) for about 36-48 hours 1508. A candidate sample is then collected from the resulting cultures 1509 and used to inoculate a tBHP-based negative-selection medium 1510. The inoculated negative-selection medium is incubated at a standard yeast-incubation temperature (˜30° C.) for 22-26 hours 1511. The medium is then examined 1512: if no cultures have grown, then one can conclude (within a margin of uncertainty) that the sample does include C. auris 1514, if cultures have grown, then one can conclude (within a margin of uncertainty) that the sample does not include C. auris 1513.

(54) An assay using a defined-supplement QAC-based positive-selection medium and a complex-supplement QAC-based positive-selection medium is depicted in FIG. 16. As with the previous flows: a sample is collected from the environment 1601 and is used to inoculate a defined-supplement QAC-based positive-selection medium 1602 (e.g., the Step-1 Medium described above), the inoculated medium is incubated at an elevated temperature 1603, and then examined for the presence of growing cultures 1604. If there are no cultures present, one can conclude the sample does not include C. auris 1605. Otherwise, a candidate sample is collected from the culture 1606 and used to inoculate a complex-supplement QAC-based positive-selection medium 1607 (e.g., the Step-2 Medium described above), which is then incubated at an elevated temperature (˜37° C.) for about 36-48 hours 1608. The medium is then examined 1609: if no cultures have grown, then one can conclude (within a margin of uncertainty) that the sample does not include C. auris 1610, if cultures have grown, then one can conclude (within a margin of uncertainty) that the sample does include C. auris 1611.

(55) The assay illustrated in FIG. 17 adds a negative-selection component to the assay of FIG. 16. Steps 1701 through 1709 of FIG. 17 are identical to steps 1601 through 1609 of FIG. 16 respectively, except that if incubation of the complex-supplement QAC-based positive-selection medium 1708 yields growing cultures 1709, then a candidate sample is collected from the complex-supplement QAC-based positive-selection medium 1711 and subject to a negative-selection assay. The candidate sample is used to inoculate a standard non-selective medium (e.g., a Sabouraud-dextrose medium) 1712, which is then incubated at standard temperature (˜30° C.) for about 24-48 hours 1713. A candidate sample is then collected from the resulting cultures on the non-selective medium 1714 and used to inoculate a tBHP-based negative-selection medium 1715. The inoculated negative-selection medium is incubated at a standard yeast-incubation temperature (˜30° C.) for about 22-26 hours 1716. The medium is then examined 1717: if no growing cultures are present, then one can conclude (within a margin of uncertainty) that the sample does include C. auris 1719, if growing cultures are present, then one can conclude (within a margin of uncertainty) that the sample does not include C. auris 1718.

(56) While the steps of the exemplary assays depicted in FIGS. 15-17 are illustrated as proceeding sequentially, certain steps may also proceed in parallel. For example, the flow depicted in FIG. 18 includes three assays performed in parallel. In this exemplary assay, a common sample is used to inoculate three different media: a defined-supplement QAC-based positive-selection medium 1803, a complex-supplement QAC-based positive-selection medium 1813, and a tBHP-based negative-selection medium 1823. Each inoculated medium is incubated: the defined-supplement QAC-based positive-selection medium at an elevated temperature (˜37° C.) for about 72 hours 1805, the complex-supplement QAC-based positive-selection medium at an elevated temperature (˜37° C.) for about 36-48 hours 1815, and the tBHP-based negative-selection medium at a standard yeast-incubation temperature (˜30° C.) for 22-26 hours 1825. The incubated media are examined for cultures 1807, 1817, 1827. If cultures grew on the defined-supplement QAC-based positive-selection medium, then the assay is a positive indication of C. auris (C. auris present in the sample) 1811. Otherwise, it is a negative indication of C. auris (C. auris not present in the sample) 1809. If cultures grew on the complex-supplement QAC-based positive-selection medium, then the assay is a positive indication of C. auris (present in the sample) 1821. Otherwise, it is a negative indication of C. auris (not present in the sample) 1819. If cultures grew on the tBHP-based negative-selection medium, then the assay is a negative indication of C. auris (not present in the sample) 1831. Otherwise, it is a positive indication of C. auris (present in the sample) 1829. Three positive results suggest the presence of C. auris in the sample. Three negative results suggest the absence of C. auris in the sample. Mixed results call for further investigation. It should be noted that direct evaluation of an environmental sample may not be possible in this manner since only the defined-supplement QAC medium is specifically designed to prevent the growth of unwanted microorganisms, such as bacteria or molds. Thus, it may not be easy to identify yeast colonies among the background growth of other organisms on the other media. However, this approach is feasible if single candidate yeast colonies are to be tested that have been isolated previously by other methods.

(57) Another exemplary parallel assay would include a first step of inoculating and incubating a defined-supplement QAC-based positive-selection medium, as described above with reference to items 1801 to 1811 in FIG. 18. If the first step yields cultures (a positive indication of C. auris), those cultures are used to: (1) inoculate and incubate a complex-supplement positive-selection medium, as described above with reference to items 1813-1821 of FIG. 18, and, in parallel, (2) inoculate and incubate a tBHP-based negative-selection medium, as described above with reference to items 1823-1831 of FIG. 18 (perhaps with intervening culturing on a nonselective culture medium).

(58) Exemplary Applications. The positive/negative selection systems may be most valuable in monitoring of patient/visitor/staff colonization or environmental contamination in a hospital environment and lends itself to large-scale testing. Such monitoring and, if needed, epidemiological investigation and source identification, is of particular importance for C. auris which shows a high propensity for patient-to-patient transmission. The positive/negative selection systems may be especially important in a healthcare setting in a less-developed country where sophisticated diagnostics are not readily available. Initial clues to the nature of the pathogen can thus be obtained, leading to more targeted testing. The positive/negative selection systems may be incorporated in automated diagnostic systems, such as VITEK, that use similar metabolic and growth criteria. This would aid accurate identification of C. auris.

(59) The strategy that resulted in the development of the described method to identify C. auris relies on the comparatively high resistance of the organism to QACs and the relative enhancement of such resistance by other factors, namely temperature and kind of growth supplement. Below, the pH of the growth medium is identified as another important factor.

(60) The applications of this strategy are quite broad. The following example serves to illustrate how a well-planned use of QACs and the described factors may result in definition of a pattern of growth/non-growth responses in various derived media that can identify or assist in identifying a species among otherwise practically indistinguishable microorganisms.

(61) As an example, a group of 36 yeast strains with Candida-like morphology representing 29 different species was used to define single species or subgroups of species depending on their QAC sensitivity at different temperatures, pH values and in the presence of different supplements (e.g. peptone, potato dextrose, and YNB with amino acids).

(62) Preliminary tests were designed with several representative yeast strains with a range of benzalkonium chloride (“BAC”) concentrations, a mix of supplements, neutral pH, and intermediate temperature (30° C.). The observed growth characteristics made it likely that two BAC concentrations (“low”=1.5 mg/l and “high”=10 mg/l) could be used in conjunction with the three different growth supplements (used separately). Low and high pH and temperature values were defined to elicit species-specific growth responses: two different pH conditions (pH 3.3 and pH 7.5, adjusted with concentrations of HCl or 100 mg/ml NaOH) and incubation temperatures (23° C. and 37° C.). Commonly used concentrations for peptone (20 g/l), potato dextrose (24 g/l), and yeast nitrogen base with amino acids (6.7 g/l) were applied. As shown in Table A 1900 depicted in FIG. 19, this results in a panel of 12 different media which can be used at 23° C. or 37° C. This results in 24 different conditions (listed left-to-right in Table A 1900). For example: “pH7.5/low BAC/Pep-23° C.” refers to a plate with pH 7.5, 1.5 g/l BAC (low BAC) containing peptone as a growth supplement and incubated at 23° C.

(63) All 36 strains were streaked and precultured for 48 h on Sabauroud-dextrose medium at 30° C. before cell samples were suspended in water (as described before). 10-15 μl drops of these suspensions were applied to the different media and plates were incubated in parallel at 37° C. and 23° C. These drops were classified as growing (+) or non-growing (−) after 3 days or 5 days of incubation, respectively.

(64) A selected subset of the analyzed 36 strains is shown in Table A 1900. As expected, Candida auris shows growth and thus resistance to BAC under virtually all conditions. The other end of the spectrum is represented by Candida valida which shows BAC sensitivity under all conditions. Candida guilliermondii is a notable example where BAC sensitivity is extremely temperature-dependent and growth is observed under all conditions at 23° C. but not 37° C. Candida rugosa shows a pronounced pH dependency of BAC resistance since growth at the high concentration appears to be restricted to the low pH. Most strains represent a more complicated and often unique pattern that can serve as an identifying “fingerprint.” Thus, Table A 1900 can serve as a key for subsequently identifying microorganisms. Similar keys may be produced for other groups of microorganisms.

(65) The species Candida famata is represented in the table by two different isolates, one from the German Collection of Microorganisms (DSZM, DSM3428=A), the other has been isolated for this study from a natural source (apricot, New Zealand=B). While overall very similar, some differences were found between these two different isolates of the same species and such strain-dependent differences need to be taken into account if a unique pattern is to be associated with one (or more) species. Such a high degree of resolution may be considered a disadvantage for assignment of a species but there are applications where distinction of strains within the same species is highly desirable, e.g. within the species of Saccharomyces cerevisiae for the winemaking/brewery industry.

(66) Several ways of applying these data sets to the preliminary identification of an unknown yeast whose morphology suggest relationship to Candida are envisioned. For instance, the yeast can be tested under all 24 conditions and compared to the best matching reference pattern. It should be noted that this is not very labor intensive since it just means to place drops out of the same suspension on 2×12 plates. All or a selected subset of such media could be provided in the form of wells of (micro)array plates, possibly also in liquid which would make the inoculation step and analysis of the result amenable to automation. In another implementation, the analysis could proceed by progression through a key and successive use of a subset of media/incubation conditions, as shown in FIG. 20. Here, the initial grouping is achieved after applying pH7.5/low BAC/peptone medium and pH7.5/high BAC/peptone at 23° C. and 37° C., as shown in Table B 2000. In this example, it is assumed that clinically important Candida albicans is then narrowed down to the species level by only one additional step utilizing a pH7.5/high BAC/potato dextrose medium, as shown in Table C 2002. Similar steps can be applied to the other subgroups as well (not shown here).

(67) A general approach to a key-based assay in depicted in FIG. 21. A sample is collected 2100 and used to inoculate 2102 a variety of media of one or more panels. Each medium of a panel reflects a set of QAC content, growth medium, and pH values and is unique relative to the others (though this does not preclude using multiples of some media as, e.g., a redundancy measure). The panels are incubated 2104 under predetermined conditions. For example, two identical inoculated panels may be incubated at different conditions, one at a higher temperature than the other or for longer than the other. After incubation, the growth pattern is examined 2106 and compared with growth-pattern key to identify a set of potential microorganisms 2108. One then determines if the identification is satisfactory 2112. If it is, the assay is complete 2110. If it is not, an additional panel(s) are inoculated and incubated, and the growth pattern is compared with the key, knowing the growth pattern produced from the previous panel(s). The system is applicable for identification of many other microorganisms, including those whose molecular characterization (e.g. through PCR) does not yet exist.

(68) While the foregoing description is directed to the preferred embodiments of the invention, other and further embodiments of the invention will be apparent to those skilled in the art and may be made without departing from the basic scope of the invention. And features described with reference to one embodiment may be combined with other embodiments, even if not explicitly stated above, without departing from the scope of the invention. The scope of the invention is defined by the claims which follow.