Patent Application
20190300927
This disclosure relates to the preparation and testing of clinical microbiological samples.
The use of resazurin-based formulations for bacterial growth determinations and AST is well-established. Resazurin, which absorbs blue light but exhibits no fluorescence, is a redox-sensitive compound that can be reduced to fluorescent resorufin (Ex=560 nm, Em=590 nm) by cellular processes. Thus, cellular viability can be monitored by increases in fluorescence intensity.
“Alamar Blue™” became a commercially-available resazurin formulation through the advances described in U.S. Pat. No. 5,501,959, which included ferricyanide and ferrocyanide salts together with methylene blue as stabilizers to prevent resazurin reduction during storage. The patent describes the ferricyanide, ferrocyanide, and methylene blue as “poising agents” that inhibit resazurin reduction and should be kept to a minimum concentration. As such, the useful concentration range (w/w) of methylene blue is defined as ⅕th to 1/10th that of resazurin.
Although this Alamar Blue formulation meets necessary stability criteria, Pseudomonas aeruginosa, an important non-fastidious gram-negative bacterial pathogen, does not effectively metabolize it,
Another frequently used molecule for the determination of bacterial viability is iodonitrotetrazolium (INT), which is converted to a highly absorbent Forazan (Abs.=490 nm) upon reduction by an actively metabolizing bacterium. However, though this molecule is readily metabolized by other species of bacteria, it is not capable of detecting P. aeruginosa growth in <5 hours,
The present disclosure provides methods for assaying microorganisms using resazurin. In one embodiment, this method may include incubating the microorganism under conditions promoting microorganism growth in a reservoir comprising nutrient broth in the presence of a metabolic probe formulation. This metabolic probe formulation may comprise resazurin at a concentration CR, one or more stabilizing salts that maintain the potential of the growth media between +0.3 and +0.45 volts in the absence of cellular growth, and one or more enhancing agents that maintain the redox potential of the growth media above −0.1 volts and are present at a concentration CE, where CE≥0.5×CR. The method may further comprise measuring the fluorescence of resorufin at one or more timepoints. The salts may be potassium ferrocyanide, ferric, and/or ferricenium. The salts may be present in both oxidized and reduced forms. The salt pair may be any of potassium ferricyanide, potassium ferrocyanide, ferrous/ferric, and ferricenium/ferrocene.
In an embodiment of this disclosure, the one or more enhancing agents that maintain the redox potential of the growth media above −0.1 volts are selected to inhibit reduction of resorufin to dihydroresorufin. The enhancing agents are redox indicators selected from the group consisting of methylene blue, toluidine blue, azure I, and gallocyanine. The resazurin concentration, CR, may be selected to be sufficient to be detectable while being substantially non-toxic to cell growth. The concentration of the enhancing agents, CE, may be set such that CE≥CR; CE≥2×CR; CE≥5×CR; CE≥10×CR. The conditions that promote microorganism growth may comprise a temperature in the range of 33-37° C. The fluorescence measurement of resorufin may be used to determine antimicrobial susceptibility to one or more antimicrobials.
In another aspect of this disclosure, one or more antimicrobial compounds may be present during incubation with the metabolic probe formulation. The incubation with the metabolic probe formulation may follow an incubation of the microorganism and the one or more antimicrobials in nutrient broth without the metabolic probe formulation under conditions that promote microorganism growth. Further, a 30-120-minute incubation with the metabolic probe formulation may follow a 3-9 hour incubation of the microorganism and the one or more antimicrobials in nutrient broth without the metabolic probe formulation under conditions that promote microorganism growth. The resorufin fluorescence from the metabolic probe formulation assay may be compared with data from one or more growth assays. The one or more growth assays may comprise optical density measurements, measurements of growth media pH, alternate metabolic probe measurements, ATP measurements, NADH measurements, DNA or RNA measurements, protein measurements, and/or enzymatic assays. The alternate metabolic probe may comprise resazurin at concentration CR2, methylene blue at concentration CB2, potassium ferricyanide and potassium ferrocyanide, such that CR2≥0.5×CB2. These assays may be used to indicate gram type of the microorganism. The resorufin fluorescence from the metabolic probe formulation may be measured after a 30-180-minute incubation. The microorganisms may derive from a clinical sample. The clinical sample may be selected from the list including, but not limited to, blood, cerebrospinal fluid, urine, stool, vaginal, sputum, bronchoalveolar lavage, throat, nasal/wound swabs, and combinations thereof. The microorganisms may be selected from the list including, but not limited to, Escherichia coli, Enterococcus spp., Staphylococcus spp., Klebsiella spp., Acinetobacter spp., Pseudomonas spp., Enterobacter spp., Streptococcus spp., Proteus spp., Aerococcus spp., Actinomyces spp., Bacillus spp., Bartonella spp., Bordetella spp., Brucella spp., Campylobacter spp., Chlamydia spp., Chlamydophila spp., Clostridium spp., Corynebacterium spp., Ehrlichia spp., Francisella spp., Gardenerella spp., Haemophilius spp., Helicobacter spp., Lactobacillus spp., Legionella spp., Leptospira spp., Listeria spp., Mycobacterium spp., Mycoplasma spp., Neisseria spp., Nocardia spp., Pasteurella spp., Rickettsia spp., Salmonella spp., Shigella spp., Stenotrophomonas spp., Treponema spp., Ureaplasma spp., Vibrio spp., Yersinia spp., Candida spp., Issatchenkia spp., Blastomyces spp., Coccidioides spp., Aspergillus spp., Cryptococcus spp., Histoplasma spp., Pneumocystis spp., Stachybotrys spp., Sporothrix, Exserohilum, Cladosporium, ringworm, mucormycetes, and combinations thereof. The inoculation steps may be automated. The assay steps may be automated. The resazurin may be a water-soluble salt.
In another embodiment, this disclosure describes a method for determining antimicrobial susceptibility of a microorganism comprising introducing a suspension of a microorganism to a cartridge comprising a plurality of chambers comprising one or more antimicrobials, incubating the cartridge under conditions promoting microorganism growth for an initial time period wherein the microorganism is in a reservoir comprising nutrient broth in the presence of a metabolic probe formulation, performing a checkpoint assay in at least a subset of chambers for determining whether a microorganism growth has achieved a threshold value, and upon microorganism growth achieving the threshold value, performing a surface area assay in a plurality of cartridge chambers and comparing surface area measurements between the plurality of cartridge chambers, thereby determining the susceptibility of the microorganism to a plurality of antimicrobials. The metabolic probe formulation comprises resazurin at a concentration CR, one or more stabilizing salts that maintain the potential of the growth media between +0.3 and +0.45 volts in the absence of cellular growth, and one or more enhancing agents that maintain the redox potential of the growth media above −0.1 volts and are present at a concentration CE, where CE≥0.5×CR.
In another embodiment of the disclosure, a method of assessing antimicrobial susceptibility comprises the steps of inoculating an AST panel with a patient sample, the AST panel comprising a plurality of serially diluted antimicrobials, incubating the AST panel under conditions favorable for microbial growth, performing a checkpoint assay to determine a level of microbial growth in a control well of the AST panel, if a level of microbial growth exceeds a predetermined threshold, performing a growth assay, and based on a result of the growth assay, determining the antimicrobial susceptibility of the microorganism wherein (a) the step of performing a growth assay comprises assessing a metabolic signal in each of the plurality of serially diluted antimicrobials, (b) the metabolic signal is a signal from a redox reaction, and (c) the redox reaction can be carried out by pseudomonas bacteria. In this embodiment, the step of performing a growth assay further comprises assessing the surface area of cells in each of the serially diluted antimicrobials.
Here we introduce the surprising finding that increasing the methylene blue “poising agent” to concentrations 5-50-fold, preferably 15-fold, greater than resazurin (w/w), while keeping iron ferricyanide and iron ferrocyanide concentrations similar to commercial Alamar Blue formulations (0.0.1-0.03% w/v), enables P. aeruginosa to metabolize the resazurin,
Methylene blue is included in commercial formulations to maintain the potential above −0.1 volts so as to inhibit the secondary reduction of resorufin to the non-fluorescent, uncolored dihydroresorufin. It may thus be surmised that P. aeruginosa fails to provide a strong fluorescent signal with commercial Alamar Blue formulations due to dual reductions of resazurin. To test this, resorufin alone was added to P. aeruginosa, with no methylene blue present,
Thus, it is surprising that the addition of high concentrations of methylene blue to the formulation described here enables P. aeruginosa reduction of resazurin to resorufin.
The high concentration of methylene blue in the formulation described here also enables the solution to be used for gram typing of bacteria. High concentrations of methylene blue are known to be tolerated by gram-negative organisms but inhibitory to gram-positive organisms [Fung and Miller. Appl. Microbiol. 25, 793-799 (1973)]. Studies with 10 species of non-fastidious bacteria showed that the new formulation developed here is metabolized much more rapidly by gram-negative species and negligibly by gram-positive species under the conditions of the test,
The Table below shows the formulation of an embodiment for this disclosure.
Additional agents that can be used in place of methylene blue include toluidine blue, azure I, and gallocyanine. The ferricyanide to ferrocyanide molar ratios are preferably 1:1 but may range from 4:1 to 1:4. Other suitable salt pairs include ferricenium/ferrocene and ferric/ferrous salts. Alternative salts include others from the same electrochemical series that can maintain media potentials between +0.3 and +0.45 volts in the absence of cellular growth, are non-toxic and soluble at the concentrations and pH used. The resazurin is preferably an aqueous-soluble salt. The preferred embodiment is purely aqueous.
AST Protocol: AST plates were removed from the −80° C. freezer and thawed. 0.5 McFarland dilutions were prepared. 10 μL of the bacteria dilution was added to the plate (except in well H12). 10 μL of Alamar Blue™ was added to wells G12 and H12. The plates were incubated for 3 hrs at 35° C. in the shaking incubator. After, 10 μL of Alamar Blue™, INT, or the preferred embodiment formulation was added to each well. The plate was incubated for another hour at 35° C. The plate was read at 560 nm/590 nm. Next, 150 μL of BLAST buffer was added to each well (CTAB for gram positive [or Proteus, Serratia, Morganella] or 1% PBST for gram negative). The plate was incubated at room temperature for 10 min on a shaker at 450 rpm. The plate was spun at 2,500×g for 2.5 min. The plate was aspirated and 100 μL of 1×PBST was added to each well. 10 μL of 20 ng Europium cryptate was added to each well. The plate was incubated at room temperature for 10 min on a shaker at 450 rpm. The plate was aspirated and 200 μL of 1×PBST was added to each well. The plate was spun at 2,500×g for 2.5 min. Steps 15 and 16 were repeated two more times. The plate was read at 330 nm/615 nm.
Protocol for Gram Determination: A 0.5 McFarland dilutions were prepared. The McFarlands were further diluted to 24,000 CFU/mL in 5 mL of Mueller Hinton Broth (MHB) and 150 μL was added to the plate. The first row of wells did not have any Alamar Blue™ added. Alamar Blue™ or the preferred embodiment formulation described here was added to the remaining rows. The plate was read every hour at absorbance 600 and 560 nm/590 nm. Ratios between Alamar Blue™ and the preferred embodiment formulation were calculated, and differences between the final and initial ratios were calculated.
This application claims priority to U.S. provisional application 62/648,826, filed on Mar. 27, 2018, and titled “Metabolic Assay for Bacterial Growth and Gram Typing.” The foregoing application is incorporated by reference herein in its entirety and for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 62648826 | Mar 2018 | US |
