Diagnostic and therapeutic applications of phthiocerol dimycocerosate (PDIM)-based methods and materials

Abstract

This invention provides methods, materials and systems for high throughput screening of Mtb for PDIM production. In some aspects, the invention provides culture media and kits that maintain, stimulate and / or augment PDIM production and / or prevents PDIM loss in mycobacterial culture in vitro, which in some aspects provides unexpectedly for clinically meaningful methods and materials for the rapid diagnosis of Mtb infection and antibiotic sensitivity of Mtb strains. In still other aspects, the invention provides methods / materials / kits and other means to exploit the link between propionate metabolism and PDIM production and applications of same for therapeutic efficacy, dosage efficacy in Mtb therapy, in addition to providing a high throughput means to assess Mtb drug resistance, for clinically meaningful applications.

Description

PA158379WO BERNEY7941PC Diagnostic and Therapeutic Applications of Phthiocerol Dimycocerosate (PDIM)-Based Methods and Materials CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Patent Application Serial Number 63 / 527,831, filed July 20, 2023, which is fully incorporated herein by reference. STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under R01AI175972 andR01AI139465 awarded by the National Institute of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] One-third of the world’s population is estimated to suffer from Mycobacterium tuberculosis (Mtb), the organism responsible for tuberculosis (TB), infection. An estimated 10 million people developed TB disease, as recently as 2017, with an estimated 1.3 million deaths attributable to TB among HIV-negative people, and an additional 300,000 deaths among HIV- positive people. TB is the leading cause of death from a single infectious agent in humans.

[0004] Mtb is characterized by a very complex cell wall, which is essential to its success as a pathogen. Phthiocerol dimycocerosates (PDIMs) are long apolar lipids found in the outermost layer of the cell wall of Mtb and other pathogenic slow-growing mycobacteria. PDIMs are involved in several key steps of Mtb pathogenesis and are an important component of the permeability barrier. Virulence attenuation has been shown for various Mtb mutants deficient in either PDIM biosynthesis or transport, demonstrating the crucial role of these lipids in Mtb pathogenesis.

[0005] Paradoxically, despite its important functions in vivo, Mtb is prone to losing the ability to produce PDIM in vitro due to spontaneous mutation of PDIM biosynthesis genes. PDIM production confers a growth disadvantage in the current standard culture media, resulting in PDIM-deficient mutants dominating in vitro cultures with successive passage. As PDIM deficiency leads to virulence attenuation and increased cell wall permeability, spontaneous PDIM loss can produce misleading results in infection experiments and genetic manipulations, presenting a major problem in TB research.PA158379WO BERNEY7941PC

[0006] Given PDIM’s role as a virulence factor, and its potential importance intherapeutics, the ability to track PDIM in cultured strains would seem to be paramount, yet routine PDIM screening efforts have been stymied by the current approaches suffering from various limitations.

[0007] There remains a need to both develop a simpler PDIM screening assay, and to develop culture methods that select for and maintain PDIM positivity in in vitro cultures, as part of the wider goal of developing new diagnostics and therapeutics against Mtb and developing better vaccines, as well. SUMMARY OF THE INVENTION

[0008] This invention provides, in some aspects methods, materials and systems for high throughput screening of Mtb for PDIM production.

[0009] This invention also provides, in other aspects, culture media and kits that maintain, stimulate and / or augment PDIM production and / or prevents PDIM loss in mycobacterial culture in vitro.

[0010] This invention also provides unexpectedly for clinically meaningful methods and materials for the rapid diagnosis of Mtb infection and antibiotic sensitivity of Mtb strains.

[0011] This invention also provides, in still other aspects, methods / materials / kits andother means to exploit the link between propionate metabolism and PDIM production and applications of same for therapeutic efficacy, dosage efficacy in Mtb therapy, in addition to providing a high throughput means to assess Mtb drug resistance, for clinically meaningful applications.

[0012] In one embodiment, this invention provides a method of rapid diagnosis of Mtb in a subject, the method comprising: (a) culturing a mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media, wherein said culturing is for a period of at least 5 days and establishing growth;PA158379WO BERNEY7941PC wherein growth is detected more rapidly in cultures conducted in the presence of propionate, thereby providing a more rapid diagnosis for Mtb infection in the subject.

[0013] In some embodiments, the method further constitutes a method of rapid determination of antibiotic sensitivity of Mtb in a subject, the method comprising the steps of: (b) contacting the cultured mycobacterial strain in (a) with at least one antibiotic for a period of from about 4 to 14 days; and (c) parallel culturing a second mycobacterial strain in a culture media supportive of mycobacterial growth in the absence of propionate and in the presence of the antibiotic; and (d) separately determining the MIC90 values for the mycobacterial strains in (b) and (c); wherein when the minimum inhibitory concentration (MIC) value can be distinguished at an earlier timepoint in the strain obtained in (b) as compared to the strain obtained in (c), the method serves to provide a more rapid determination of antibiotic sensitivity of the mycobacterial strain

[0014] In one embodiment, this invention provides a method of high throughput determination of PDIM production from a mycobacterial strain, said method comprising: · Culturing a PDIM reference strain set for a period of at least 5 days in mycobacteria growth supportive media wherein the PDIM reference strain set comprises a combination of at least one PDIM(+) and at least one PDIM(-) Mtb strain selected from the group of the following strains: mc26230 AE1601, mc26230 AE1611, H37Rv AE1028, H37Rv DppsD, mc26206, mc26209, mc27901, mc27902 or mc28398, wherein at least one of the PDIM(+) Mtb strains is optionally selected from the group consisting of mc26230 AE1601 and H37Rv AE1028, and at least one of the PDIM(-) Mtb strains is optionally selected from the group consisting of mc26230 AE1611 and H37Rv DppsD; · Culturing a strain of interest for a period of at least 5 days; · Further culturing parallel samples of said PDIM reference strain set and said strain of interest in media in the presence and absence of 5 - 50 mg / mlPA158379WO BERNEY7941PC vancomycin, for a period of at least from 5 to 14 days and measuring the optical density at 590 – 600 nm; · Establishing relative growth of the PDIM reference strain set and the strain of interest in vancomycin as compared to drug-free controls, through the following formula: (VAN10 growth / VAN0 growth ´ 100 = VAN10 growth%); Wherein a VAN10 growth% value of below 10% is indicative of a strain which does not appreciably produce PDIM.

[0015] In other embodiments, the invention provides a method of high throughput determination of PDIM production from a mycobacterial strain, said method comprising: · Culturing a PDIM reference strain set for a period of at least 5 days in mycobacteria growth supportive media wherein the PDIM reference strain set comprises a combination of at least one PDIM(+) and at least one PDIM(-) Mtb strain selected from the group of the following strains: mc26230 AE1601, mc26230 AE1611, H37Rv AE1028, H37Rv DppsD, mc26206, mc26209, mc27901, mc27902 or mc28398, wherein at least one of the PDIM(+) Mtb strains is optionally selected from the group consisting of mc26230 AE1601 and H37Rv AE1028, and at least one of the PDIM(-) Mtb strains is optionally selected from the group consisting of mc26230 AE1611 and H37Rv DppsD; · Culturing a strain of interest for a period of at least 5 days; · Further culturing parallel samples of said PDIM reference strain set and said strain of interest in media in the presence and absence of 5 - 50 mg / ml vancomycin, for a period of at least from 5 to 14 days and assessing the PDIM reference strain set and strain of interest growth via any appropriate means; · Establishing relative growth of the PDIM reference strain set and the strain of interest in vancomycin as compared to drug-free controls, through the following formula: (VAN10 growth / VAN0 growth ´ 100 = VAN10 growth%);PA158379WO BERNEY7941PC Wherein a VAN10 growth% value of below 10% is indicative of a strain which does not appreciably produce PDIM.

[0016] According to this aspect, and in some embodiments, optical density (OD) is one means of assessing strain growth, but it should be understood that any number of additional means can be utilized in accordance with the invention, such as, for example, Resazurin assay and others as will be appreciated by the skilled artisan.

[0017] In some embodiments this invention provides a kit for culture media supplementation for promoting, augmenting or maintaining PDIM production in a mycobacterial strain in vitro, wherein said kit comprises concentrated liquid or powdered propionate and instructions for reconstituting said culture media for supplementation to arrive at a final concentration of from 0.1 – 1.0 mM in the supplemented culture media.

[0018] According to this aspect and in some embodiments, the kit further comprises additional components, which may include vitamin B12and instructions for preparing a final desired concentration of 2-50 mg / ml of vitamin B12 in a final desired volume of culture media for use, and in some embodiments, the kit further comprises additional components, which may include valerate, cholesterol, pantothenate, arginine, leucine and methionine media components, and in some embodiments, the kit further comprises additional components, which may include tyloxapol. In some embodiments, the kit may comprise additional components, which may include glycerol, dimethylsulfoxide, or other cryoprotectants. In some embodiments, the kit comprises various combinations of these additional components, in any possible combination of same.

[0019] In some embodiments, this invention provides a ready to use culture media for promoting, augmenting or maintaining PDIM production in a mycobacterial strain in vitro, wherein said culture media is supplemented with 0.1 – 1.0 mM propionate. Such ready to use culture media may comprise additional components, which may include valerate, cholesterol, pantothenate, arginine, leucine and methionine media components, and in some embodiments, the kit further comprises additional components, which may include tyloxapol.

[0020] In other embodiments, this invention provides a method for preparing media for promoting, maintaining or augmenting PDIM production in a mycobacterial strain in culture, said method comprising culturing a mycobacterial strain in a culture media supportive of mycobacterial growth in culture for a period of at least 5 days, wherein said culture media hasPA158379WO BERNEY7941PC been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media. Various embodied and considered aspects of such method are described further hereinbelow.

[0021] In other embodiments, the invention provides a method for improvingmycobacterial strain PDIM production in vitro, the method comprising culturing the mycobacterial strain in a culture media supportive of mycobacterial growth for a period of at least 5 days, wherein said culture media has been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media.

[0022] In other embodiments, the invention provides method of improving protective immune responses to mycobacterial vaccine strains in a host against mycobacterial infection and / or disease, the method comprising: (i) Culturing a mycobacterial vaccine strain in a culture media supportive of mycobacterial growth in culture for a period of at least 5 days, wherein said culture media has been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; (ii) Preparing a vaccine from the mycobacterial vaccine strain in (i) following the culture period; and (iii)Administering the vaccine in (ii) to a subject, wherein immune responses in the subject are improved as compared to immune responses achieved in the subject following administration with the initial vaccine strain in (i), administered without further culture in media supplemented with propionate.

[0023] In further embodiments, the invention provides a method of improving immunogenicity of a vaccine in a host, the method comprising: (i) Culturing a mycobacterial vaccine strain in a culture media supportive of mycobacterial growth in culture for a period of at least 5 days, wherein said culture media has been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media;PA158379WO BERNEY7941PC (ii) Preparing an adjuvant from the mycobacterial vaccine strain cultured according to (i); (iii) Combining a vaccine of interest with the adjuvant of (ii); and (iv) Administering the vaccine and adjuvant combination of (iii) to a subject; wherein immune responses in the subject are improved as compared to immune responses achieved in the subject following administration with the vaccine, administered in the absence of the adjuvant.

[0024] In other embodiments, the invention provides a method of improved determination of antibiotic sensitivity of a mycobacterial strain, improved antibiotic dosage determination for a subject, or a combination thereof, the method comprising: (a) culturing a first mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media and separately similarly culturing a second identical mycobacterial strain in the absence of the propionate; (b) further contacting said first mycobacterial strain and said second identical mycobacterial strain with an antibiotic for a period of from about 5 to 10 days; (c) determining the minimum inhibitory concentration (MIC) values for MIC50, MIC90or MIC99for the first and second mycobacterial strains in (b), wherein when any of the MIC values in the first mycobacterial strain are obtained more rapidly or reliably than in the second mycobacterial strain, the method serves to improve determination of antibiotic sensitivity of the mycobacterial strain, or the method serves to improve antibiotic dosage determination for the subject infected with the mycobacterial strain, or a combination thereof.

[0025] In other embodiments, the invention provides a method of more rapid determination of antibiotic sensitivity or determination of inhibitory antibiotic dosage for mycobacterial infection in a subject, or a combination thereof, the method comprising:PA158379WO BERNEY7941PC (a) culturing a first mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; and (b) separately similarly culturing a second identical mycobacterial strain in the absence of the propionate; and (c) further contacting said first mycobacterial strain and said second identical mycobacterial strain with an antibiotic for a period of time; and (d) separately determining the minimum inhibitory concentration (MIC) values for MIC50, MIC90 or MIC99 for the first and second mycobacterial strains; wherein the MIC values in the first mycobacterial strain are obtained in at least 20% of the time necessary to obtain reliable MIC values in the second mycobacterial strain, thereby more rapidly determining antibiotic sensitivity or determining inhibitory antibiotic dosage for the subject infected with the mycobacterial strain, or a combination thereof.

[0026] In other embodiments, the invention provides a method of earlier diagnosis of mycobacterial infection in a subject, the method comprising: (a) culturing a suspected mycobacterial strain isolated from a subject in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; and (b) separately culturing the identical mycobacterial strain isolated from a subject in a culture media supportive of mycobacterial growth in culture in the absence of propionate; wherein mycobacterial strain growth is obtained more rapidly from said culture in (a) than in the culture in (b), thereby providing for earlier diagnosis of mycobacterial infection in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Various embodiments of the methods / kits / media / applications of this invention aredescribed herein with reference to the figures wherein:

[0028] Figure 1 depicts vancomycin resistance correlates with PDIM production in Mtb.PA158379WO BERNEY7941PC

[0029] Figure 2 depicts drug resistance and permeability of PDIM(+) and PDIM(-) Mtb.

[0030] Figure 3A Schematically depicts a vancomycin PDIM screening assay and Figure 3B depicts development of a single-concentration vancomycin PDIM screening assay.

[0031] Figure 4 depicts propionate supplementation improving assay robustness.

[0032] Figure 5 depicts propionate supplementation shortens assay duration, in addition to improving robustness and Vitamin B12also improves robustness, as shown herein.

[0033] Figure 6A depicts VAN-P assays accurately predict PDIM status during genetic manipulations and Figure 6B depicts VAN-P assays accurately predict PDIM status across different Mtb strains and lineages.

[0034] Figure 7 depicts propionate and vitamin B12 prevent PDIM loss in Mtb.

[0035] Figure 8 depicts propionate and vitamin B12supplementation increase rifampicin andbedaquiline resistance of PDIM(+) Mtb.

[0036] Figure 9 depicts propionate supplementation does not broadly increase drug resistance of Mtb.

[0037] Figure 10 depicts Detergent influence on the recapitulation in VAN-P assays or restoring growth of PDIM(+) Mtb with propionate or vitamin B12 supplementation.

[0038] Figure 11 depicts odd-chain short fatty acids, cholesterol and vitamin B12restore growth of PDIM(+) Mtb.

[0039] Figure 12 depicts propionate and vitamin B12supplementation restore intracellularpools of methylmalonyl-CoA (MMCoA) in PDIM(+) Mtb.

[0040] Figure 13 depicts propionate supplementation positively and expeditiously increases the growth rate of Mtb strains across different lineages.

[0041] Figure 14 depicts propionate supplementation reduces time to result for the assessment of antibiotic sensitivity Mtb strains across different lineages. DETAILED DESCRIPTION OF THE INVENTION

[0042] This invention provides, in some aspects, methods, materials and systems forhigh throughput screening of Mtb for PDIM production and applications of same.

[0043] This invention also provides, in other aspects, culture media and kits that maintain, stimulate and / or augment PDIM production and / or prevents PDIM loss in mycobacterial culture in vitro.PA158379WO BERNEY7941PC

[0044] This invention also provides, in still other aspects, methods / materials / kits andother means to exploit the link between propionate metabolism and PDIM production and applications of same for therapeutic efficacy, dosage efficacy in Mtb therapy, in addition to providing a high throughput means to assess Mtb drug resistance, in a clinically meaningful setting.

[0045] This invention also provides, in still other aspects, methods / materials / kits and other means to rapidly diagnose patients with Mtb infection, when the strains obtained in samples from such patients is cultured in the presence of propionate as herein described.

[0046] This invention also provides, in still other aspects, methods / materials / kits and other means to rapidly assess antibiotic sensitivity of Mtb strains infecting a patient, when the strains obtained in samples from such patients are cultured in the presence of propionate as herein described and further cultured in appropriate concentrations of one or more antibiotics for a period of about 5-7 days, optionally further assessing the MIC following up to 14 days of exposure of the strain to one or more antibiotics, whereby earlier determination of the antibiotic sensitivity can be assessed in samples grown in propionate supplemented cultures.

[0047] In still other embodiments, as described and exemplified herein, and representing embodied aspects of this invention, is the first provision of a highly straightforward, cost-effective, functional assay for high throughput screening of Mtb for PDIM production.

[0048] In some aspects, the invention provides a method of high throughput determination of PDIM production from a mycobacterial strain, the method comprising: · Culturing a PDIM reference strain set for a period of at least 5 days in mycobacteria growth supportive media wherein the PDIM reference strain set comprises a combination of at least one PDIM(+) and at least one PDIM(-) Mtb strain selected from the group of the following strains: mc26230 AE1601, mc26230 AE1611, H37Rv AE1028, H37Rv DppsD, mc26206, mc26209, mc27901, mc27902 or mc28398, wherein at least one of the PDIM(+) Mtb strains is optionally selected from the group consisting of mc26230 AE1601 and H37Rv AE1028, and at least one of the PDIM(-) Mtb strains is optionally selected from the group consisting of mc26230 AE1611 and H37Rv DppsD; · Culturing a strain of interest for a period of at least 5 days;PA158379WO BERNEY7941PC · Further culturing parallel samples of said PDIM reference strain set and said strain of interest in media in the presence and absence of 5 - 50 mg / ml vancomycin, optionally further in the presence and in the absence of 0.1 – 1.0 mM propionate, or 2 – 50 mg / ml vitamin B12, or 0.1 mM valerate, or 0.1 mM cholesterol for a period of at least from 5 to 14 days and measuring the optical density at 590 – 600 nm; · Establishing relative growth of the PDIM reference strain set and the strain of interest in vancomycin as compared to drug-free controls, through the following formula: (VAN10 growth / VAN0 growth ´ 100 = VAN10 growth%); Wherein a VAN10 growth% value of below 10% is indicative of a strain which does not appreciably produce PDIM. · Culturing a strain of interest for a period of at least about 5 days; · Further culturing parallel samples of said PDIM reference strain set and said strain of interest in media in the presence and absence of 5 – 50 mg / ml vancomycin, optionally further in the presence and in the absence of 0.1 – 1.0 mM propionate, or 2 – 50 mg / ml vitamin B12, or 0.1 – 0.5 mM valerate, or 0.1 - 0.5 mM cholesterol for from at least about 5 to about 14 days and measuring the optical density at 590 – 600 nm; · Establishing relative growth of the PDIM reference strain set and the strain of interest in vancomycin as compared to drug-free controls, through the following formula: (VAN10 growth / VAN0 growth ´ 100 = VAN10 growth%); Wherein a VAN10 growth% value of below 10% is indicative of a strain which does not appreciably produce PDIM.

[0049] In some aspects, such method is a high throughput method of determination of PDIM production from a mycobacterial strain, which is an Mtb strain, and in some embodiments, any PDIM producing mycobacteria can be thereby screened.

[0050] According to this aspect, and in some embodiments, Mtb reference strains are selected from the group consisting of at least mc26230 AE1601 and mc26230 AE1611, or H37Rv AE1028 and H37Rv DppsD.

[0051] In some embodiments, the culturing of the strain of interest and, optionally,culturing the PDIM reference strain set is for a period of up to 14 days.PA158379WO BERNEY7941PC

[0052] In other embodiments, the culturing of the strain of interest optionally furthercomprises sample culture in the absence and in the presence of 0.1 – 1.0 mM propionate, or 0.1 mM valerate, or 0.1 mM cholesterol, or 10 mg / ml vitamin B12.

[0053] In some aspects, the increased VAN10 growth% value indicates the strain of interest is PDIM(+). According to this aspect, and in some embodiments, a VAN10 growth% of more than 50% is indicative that the strain in question is a PDIM-positive strain.

[0054] It will be appreciated that strains exhibiting a VAN10 growth% value of between 10 – 50 % would represent intermediate values for PDIM expression, although it is noted that further analysis in the context of the parallel values obtained for the control strains will also provide a context for the specific PDIM values in the assay setting.

[0055] PDIM deletion mutants of Mtb and M. bovis BCG are more susceptible to vancomycin than PDIM(+) wildtype strains. As described further herein, in Example 1, this differential vancomycin sensitivity could be exploited as a means / method for assessment of PDIM production. As demonstrated herein, a reference set of Mtb strains were shown to produce varying levels of PDIM as determined by thin layer chromatography (TLC) and a correlation between vancomycin MIC50 and MIC90 and PDIM band intensities in TLC. As further described herein, uniquely, a single concentration ‘VAN10’ assay was then developed that effectively differentiates between PDIM(+) and PDIM(-) strains.

[0056] In some embodiments, the PDIM-deficient strains exhibit a vancomycin MIC90 below 10 mg / ml, while PDIM(+) strains showed intermediate growth around this concentration. The relative growth in 10 mg / ml vancomycin compared to no-drug controls (VAN10 growth%) was significantly correlated with PDIM levels at all measured time points, which assay could be conducted using simple, low-cost materials, such as inkwell bottles and a simple spectrophotometer so that same is appropriate even in low-resource settings.

[0057] The VAN10 assay as described herein provides a simple, cost-effective assaysystem to assess PDIM production in mycobacterial strains, however, it is noted, as described herein, for example, in regard to applying a similar methodology while making use of rifampicin, that the same principle of PDIM-deficient strains exhibiting an MIC50or MIC90of much lower values than when assessing PDIM(+) strains, which show greater growth around the same concentrations.

[0058] In some aspects, this may be particularly relevant in low resource settings where a high throughput assay is desired, yet one antibiotic may be more readily obtained than another.PA158379WO BERNEY7941PC

[0059] It will be understood that any of the large antibiotics of the glycopeptide (e.g.vancomycin, teicoplanin, ramoplanin) and macrolide (e.g. azithromycin and erythromycin) families, as well as rifampicin can be used, in a similar method / means as the VAN10 assay as described herein to arrive at a high throughput assay to assess PDIM production and any and all applications of same are to be considered embodied aspects of the invention.

[0060] In some aspects, as described herein, the high throughput determination of PDIM production from a mycobacterial strain relies upon culturing a PDIM(+) and PDIM(-) strain, including inter alia, the PDIM reference strains described herein, where the strains are cultured in control media for a period of at least 5 days to serve as a reference set of PDIM(+) and PDIM(-) strains, as comparators and to validate cutoff values for assays assessing PDIM production.

[0061] It will be appreciated that by “reference strain” it is intended that any PDIM(+) or PDIM(-) ascertained strain can be used, in accordance with the methods and materials of this invention, when same demonstrates the described activity reproducibly, so that it may serve as a comparator strain as described.

[0062] According to this aspect, and in some embodiments, the PDIM(-) strain set comprises a combination of at least one PDIM(+) and at least one PDIM(-) Mtb strain selected from the group of the following strains: mc26230 AE1601, mc26230 AE1611, H37Rv AE1028, H37Rv DppsD, mc26206, mc26209, mc27901, mc27902 or mc28398.

[0063] In some aspects at least one of said PDIM(+)Mtb strains is preferably selectedfrom the group consisting of mc26230 AE1601 and H37Rv AE1028, and one of said PDIM(-) Mtb strains is preferably selected from the group consisting of mc26230 AE1611and H37Rv DppsD.

[0064] In other aspects, the methods and kits may make use of any standard / pre- determined PDIM(+) and PDIM(-) strain, for example, being selected from the group consisting of mc26206, mc26209, mc27901, mc27902 or mc28398, or any others, as will be appreciated by the skilled artisan.

[0065] Importantly, for use as a reference strain the PDIM positivity or negativity will be known and maintained as long as the strain is used as a reference strain for the purposes as described herein.

[0066] In further aspects of these embodied methods, a strain of interest can be culturedin parallel to the PDIM reference strain set in media in the presence and absence of a definedPA158379WO BERNEY7941PC concentration of the listed antibiotics, and the impact of same will be assessed as a function of optical density, measured, for example, in a plate reader or using even a simple spectrophotometer, as described.

[0067] In some aspects, the growth may be measured by any appropriate means, for example, making use of the Resazurin assay, and others, as will be appreciated by the skilled artisan.

[0068] Still further, according to this aspect, is the establishment of the relative growth of the PDIM reference strain set and the strain of interest in the antibiotic as compared to drug- free controls and making use of the logic as described for the VAN10 growth % derivation, a growth% level in other antibiotics can be derived and validated so that same can also then provide additional high throughput PDIM production methods.

[0069] As noted in Example 1 herein, a more than 2-fold difference in MIC betweenPDIM(+) and PDIM(-) strains was observed for the indicated antibiotics, and thus same can be used to support methods to distinguish differences between PDIM(+) and PDIM(-) strains.

[0070] As also described and exemplified herein, PDIM precursors increasevancomycin resistance. Propionate, valerate, cholesterol and vitamin B12supplementation increase vancomycin resistance of PDIM(+) but not PDIM(-) Mtb, which is also applicable in further improving assay robustness and potentially reducing time to result for PDIM production and antibiotic sensitivity assays.

[0071] This invention therefore provides not only further methods / applications of these findings, but even kits for culture media supplementation for promoting, augmenting, or maintaining PDIM production in a mycobacterial strain in vitro.

[0072] According to this aspect, and in some embodiments, the kits of this invention may comprise, inter alia, concentrated liquid or powdered propionate, valerate or cholesterol and instructions for reconstituting culture media for supplementation to contain the propionate, valerate or cholesterol.

[0073] In some aspects, the desired final concentration of propionate is from about 0.1 –1.0 mM in the supplemented culture media and the kit may provide standardized and optimized methods and materials to produce a final culture media for use including instructions to achieve same. In some aspects, the desired final concentration of valerate or cholesterol is from about 0.01 – 0.5 mM.

[0074] In some embodiments, the kit may further comprise liquid or powdered 7H9 media components and instructions for reconstituting or preparing a final desired volume ofPA158379WO BERNEY7941PC 7H9 media for use, however, it will be appreciated that any media appropriate for mycobacterial culture is envisioned for use with the propionate supplementation and the kits can be adjusted to provide components and instructions for producing same.

[0075] In some aspects, it is contemplated that the media does not contain Tween 80and according to some aspects of the invention, the media may make use of an alternate detergent, such as tyloxapol. According to this aspect, and in some embodiments, the choice of detergent will be reflected in its incorporation in the culture media and assay media, and in some embodiments, better results are obtained when tyloxapol is incorporated. In other aspects, better results are obtained when Tween 80 is specifically not included in either the culture or the assay media.

[0076] In some embodiments, additional strains may be evaluated, wherein inclusion of Tween 80 as the detergent may not interfere with the ability to assay PDIM production in a given strain and / or to promote PDIM production in culture. Thus, while it is preferable to make use of media not containing Tween 80 when first assessing a given mycobacterial strain, this preference should not constitute a limitation of the invention and inclusion of Tween 80 in the media is not disavowed herein.

[0077] In still other embodied aspects, the kit may further comprise vitamin B12and instructions for preparing a final desired concentration of 10 mg / ml of the vitamin B12 in the final desired volume, e.g. of mycobacterial growth supportive media for use, for example, 7H9 media.

[0078] In some aspects, inclusion of both propionate and vitamin B12in the media, as described hereinabove, may provide certain beneficial effects in certain strains in terms of maintaining their PDIM positivity.

[0079] In some aspects, inclusion of odd-chain short fatty acids and / or cholesterol in the media, as described herein, may provide certain beneficial effects in certain strains in terms of maintaining their PDIM positivity.

[0080] In some embodiments, any embodied media supplementation or condition as described herein will be understood to be applicable and useful in the invention, in terms of the methods, kits, culture media, supplementation protocols, and methods of this invention and are to be considered embodied aspects of same.

[0081] In still other embodied aspects, the kit may further comprise pantothenate, arginine, leucine and methionine media components or other components that promote best mycobacterial growth.PA158379WO BERNEY7941PC

[0082] In still other aspects, this invention provides a culture media promoting,augmenting or maintaining PDIM production in a mycobacterial strain in vitro, wherein the culture media is supplemented with propionate according to the instructions and making use of the kits as described herein. Thus, the kits as described herein can be directly marketed to, for example, clinical laboratories or in some aspects, the kits can be provided to suppliers to produce a culture media for use for example, by a clinical laboratory.

[0083] According to this aspect and in some embodiments, the culture media comprises7H9 media, which in some aspects is further supplemented with OADC (oleic acid, albumin, dextrose, catalase) and glycerol and / or in some aspects may comprise or further comprise pantothenate, arginine, leucine and methionine.

[0084] In still other aspects, this invention provides a ready to use culture media for promoting, augmenting or maintaining PDIM production in a mycobacterial strain in vitro, wherein the culture media is supplemented with from 0.1 – 1.0 mM propionate. Such media may be any appropriate media for promoting mycobacterial growth, and in some aspects, the ready to use culture media will be based on 7H9 media long in use in the culture of mycobacteria. In other aspects, the ready to use culture media will further comprise OADC and glycerol and / or the ready to use culture media will further comprise pantothenate, arginine, leucine and methionine. In other aspects, the ready to use culture media will further comprise a detergent, such as tyloxapol, or other related detergents. In some aspects, it is specifically contemplated not to use Tween 80 as a detergent in the assay and / or culture media.

[0085] It will be appreciated that the invention contemplates provision of kits and prepared concentrated media for dilution and / or prepared, ready to use media, that is supplemented with propionate to ensure a final concentration of from 0.1 – 1.0 mM propionate in the media for use in culture of mycobacteria, to provide consistent and reliable PDIM production when culturing the organisms.

[0086] In some embodiments, the invention provides a method for preparing media for promoting, maintaining or augmenting PDIM production in a mycobacterial strain in culture, said method comprising culturing a mycobacterial strain in a culture media supportive of mycobacterial growth in culture for a period of at least 7 days, wherein said culture media has been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media.

[0087] In some aspects according to this embodiment, the culture period is from at least 5 to 14 days.PA158379WO BERNEY7941PC

[0088] In other embodiments, according to this aspect, the culture media is furthersupplemented with vitamin B12 achieving a final concentration of 10 mg / ml of the vitamin B12 in said culture media. In still other embodiments, the culture media comprises 7H9 media and in some embodiments, this may be supplemented with OADC and glycerol and in still further embodiments, the culture media may be further supplemented with pantothenate, arginine, leucine and methionine.

[0089] In other embodiments, the method for preparing media for promoting, maintaining or augmenting PDIM production in a mycobacterial strain in culture may further comprise the step of culturing the mycobacteria strain for a period of at least 5 days in parallel, in the presence and absence of propionate. According to this aspect, and in some embodiments such method may still further comprise the step of culturing in parallel a PDIM reference strain set in mycobacterial growth supportive control media for a period of at least 5 days, wherein the PDIM reference strain set comprises a combination of at least one PDIM positive and at least one PDIM negative Mtb strain selected from the group of the following strains: mc26230 AE1601, mc26230 AE1611, H37Rv AE1028, H37Rv DppsD, mc26206, mc26209, mc27901, mc27902 or mc28398, wherein the at least one PDIM positive Mtb strain is preferably selected from the group consisting of mc26230 AE1601 and H37Rv AE1028, and the at least one PDIM negative Mtb strain is preferably selected from the group consisting of mc26230 AE1611and H37Rv DppsD.

[0090] In still further aspects, such method may still further comprise the steps of:· Culturing the PDIM reference strain set and the mycobacteria strain in the presence and absence of 10 mg / ml vancomycin and measuring the optical density at 590 – 600 nm; · Establishing relative growth of the PDIM reference strain set in vancomycin as compared to drug-free controls, through the following formula: (VAN10 growth / VAN0 growth ´ 100 = VAN10 growth%); · Establishing the growth of the mycobacterial strain cultured in the presence and absence of propionate, in vancomycin as compared to drug-free controls, through the following formula: (VAN10 growth / VAN0 growth ´ 100 = VAN10 growth%);

[0091] wherein a VAN10 growth% value of significantly below that obtained atbaseline, and in comparison to the reference strain, for example, below 10%, or a comparablyPA158379WO BERNEY7941PC low value in comparison, as noted, will serve as an indicator that the media did not appreciably promote, maintain or augment PDIM production in the mycobacterial strain.

[0092] As described and exemplified herein, propionate supplementation prevents PDIM loss in vitro and the embodied methods / assays / kits / reagents of this invention provide a reliable and effective method for PDIM production assessment. As further described herein as well, Mtb drug efficacy and / or dosage can be assessed, in terms of the impact of propionate supplementation on same, in the context of PDIM production.

[0093] Accordingly, this invention provides a method for improving mycobacterial strain PDIM production in vitro, the method comprising culturing the mycobacterial strain in a culture media supportive of mycobacterial growth for a period of at least 7 days, wherein the culture media has been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media.

[0094] According to this aspect and in some embodiments, the culture period is from atleast about 5 to about 14 days and in some embodiments, the culture media is further supplemented with vitamin B12 achieving a final concentration of 2 – 50 mg / ml of the vitamin B12 in said culture media.

[0095] In still further embodiments the culture media comprises 7H9 media and in stillfurther embodiments, the culture media is further supplemented with OADC and glycerol. In still further embodiments the culture media comprises a detergent, such as tyloxapol, or related detergents. In other aspects, exclusion of Tween 80 from the growth and / or culture media is specifically contemplated.

[0096] As described an exemplified herein, uniquely, herein is provided a straightforward and scalable approach to routine PDIM screening using vancomycin and propionate that outperforms current methods.

[0097] In some aspects, surprisingly, sub-toxic concentrations of propionate or vitamin B12were found to alleviate an apparent metabolic deficiency that impairs the growth of PDIM(+) strains.

[0098] In still further aspects, propionate supplementation was shown to prevent PDIMloss, and propionate-grown Mtb displays increased resistance to rifampicin, bedaquiline, and glycopeptides in a PDIM-dependent manner.

[0099] The invention therefore also provides, in another embodiment, a method ofimproved determination of antibiotic sensitivity of a mycobacterial strain, improved antibiotic dosage determination for a subject, or a combination thereof, the method comprising:PA158379WO BERNEY7941PC (a) culturing a mycobacterial strain in a culture media supportive of mycobacterial growth in culture for a period of at least 5 days; (b) further culturing a first mycobacteria strain in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media and culturing a second mycobacteria strain in the absence of the propionate, then further contacting the first and second mycobacteria strains with an antibiotic for at least about 7 days; and (c) determining the MIC50, MIC90and MIC99values for the first and second mycobacterial strains in (b). wherein when any of MIC values in the second mycobacterial strain are greater than in the first mycobacterial strain, the method serves to improve determination of antibiotic sensitivity of the mycobacterial strain, or the method serves to improve antibiotic dosage determination for the subject infected with the mycobacterial strain, or a combination thereof. According to this aspect, and in some embodiments, the treatment regimen for the subject will comprise increasing the antibiotic dosage administered to the subject.

[0100] In some embodiments, the method may further comprise culturing the first and second mycobacteria strains in the presence of at least a second antibiotic in parallel and differentially determining the MIC values for the first and second mycobacterial strains following exposure to the first and second antibiotics.

[0101] According to this aspect and in other embodiments, the method comprises administering the second antibiotic to the subject, assuming the MIC values are improved for the second antibiotic as compared to the first antibiotic.

[0102] In some embodiments, of these methods of improved determination of antibiotic sensitivity of a mycobacterial strain, improved antibiotic dosage determination for a subject, or a combination thereof, the culture period is from at least 7 to 14 days and in other embodiments, the culture media is further supplemented with vitamin B12 achieving a final concentration of 10 mg / ml of the vitamin B12in said culture media and / or in other aspects, the culture media comprises 7H9 media, which may be further supplemented with OADC and glycerol.

[0103] As described and exemplified herein, the invention provides methods and materials to promote maintaining PDIM production in Mtb strains that can exhibit diminished PDIM production when passaged / cultured in vitro over time.PA158379WO BERNEY7941PC

[0104] PDIM is a potential virulence factor for Mtb and ensuring PDIM production infor example, vaccine strains, is expected to improve / elicit protective immune responses to mycobacterial vaccine strains in a host against mycobacterial infection and / or disease.

[0105] Moreover, PDIM may contribute to phagosomal escape and host cell exit of mycobacterial strains, which in turn can promote the generation of cytotoxic T lymphocyte specific responses.

[0106] Accordingly, in some aspects this invention provides a method of improving protective immune responses to mycobacterial vaccine strains in a host against mycobacterial infection and / or disease, the method comprising: (i) Culturing a mycobacterial vaccine strain in a culture media supportive of mycobacterial growth in culture for a period of at least 7 days, wherein said culture media has been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; (ii) Preparing a vaccine from the mycobacterial vaccine strain in (i) following the culture period; and (iii)Administering the vaccine in (ii) to a subject, wherein immune responses in the subject are improved as compared to immune responses achieved in the subject following administration with the initial vaccine strain in (i), administered without further culture in media supplemented with propionate.

[0107] In some aspects, according to this embodiment the culture period may be from atleast 5 to 14 days and in some aspects, the culture media is further supplemented with vitamin B12, achieving a final concentration of 2 – 50 mg / ml of the vitamin B12 in said culture media. In still further aspects, the culture media comprises 7H9 media, which in some aspects, is further supplemented with OADC and glycerol.

[0108] In some embodiments, the methods of improving protective immune responses specifically contemplate improving the responses following use of the mycobacterial vaccine strain BCG.

[0109] In some embodiments, the improved protective immune response improves cytotoxic T lymphocyte (CTL) responses in particular.

[0110] In some aspects, such method of improving protective immune responses in asubject may be improving same against mycobacterial infection, and in some embodiments,PA158379WO BERNEY7941PC such method of improving protective immune responses in a subject may include methods making use of mycobacterial vaccines to provoke non-specific, anti-tumor responses, for example, such as in bladder cancer.

[0111] This invention also provides a method of improving immunogenicity of avaccine in a host, the method comprising: (i) Culturing a mycobacterial vaccine strain in a culture media supportive of mycobacterial growth in culture for a period of at least 5 days, wherein said culture media has been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; (ii) Preparing an adjuvant from the mycobacterial vaccine strain cultured according to (i); (iii) Combining a vaccine of interest with the adjuvant of (ii); and (iv) Administering the vaccine and adjuvant combination of (iii) to a subject; wherein immune responses in the subject are improved as compared to immune responses achieved in the subject following administration with the vaccine, administered in the absence of the adjuvant.

[0112] Surprisingly, as described and demonstrated herein, propionate supplementation improves the growth of Mtb strains belonging to various lineages therebv providing a method or use for the media supplements / kits, etc. for rapid diagnosis of Mtb infection in a subject. Any appropriate source material can be used for such detection, which may include typical sputum samples and others, as will be known by the skilled artisan.

[0113] Thus, this invention provides a method of rapid diagnosis of Mtb in a subject, the method comprising: (a) culturing a mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media, wherein said culturing is for a period of at least 5 days and establishing growth; wherein growth is detected more rapidly in cultures conducted in the presence of propionate, thereby providing a more rapid diagnosis for Mtb infection in the subject.

[0114] According to this aspect and in some embodiments, the culturing is for a periodof from at least about 5 days, or in some embodiments, from at least about 6 days, or in somePA158379WO BERNEY7941PC embodiments, from at least about 7 days, or in some embodiments, from at least about 8 days, or in some embodiments, from at least about 9 days, or in some embodiments, from at least about 10 days, or in some embodiments, from at least about 4-6 days, or in some embodiments, from at least about 5-7 days, or in some embodiments, from at least about 5-8 days, or in some embodiments, from at least about 5-9 days, or in some embodiments, from at least about 6-10 days, or in some embodiments, from at least about 7-11 days, or in some embodiments, from at least about 7-12 days, or in some embodiments, from at least about 8-13 days, or in some embodiments, from at least about 5-14 days, or any subrange from the ranges as herein described.

[0115] In still other embodiments, the shortening of the culture period may lengthen or shorten, in part, in propionate supplemented cultures, as a function of how slow growing the mycobacterial strains, in general, or in some embodiments, how growth may be slower due to a very low inoculum being present. According to this aspect, however, the growth in propionate supplemented media will necessarily be more rapid for a given strain than in supportive growth media that is not supplemented with propionate.

[0116] In still other embodiments, according to this aspect, the method may reflectculturing a mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media, and when compared to culture of the same mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the absence of propionate, growth is more rapidly established in the propionate supplemented media as compared to the parallel culture lacking propionate. According to this aspect, and in some embodiments, growth in propionate supplemented media will be detectable in a time frame of at least 20% of the time frame needed for detectable growth in the non-propionate supplemented media for the parallel culture. In other embodiments, growth in propionate supplemented media will be detectable in a time frame of at least from about 65% to 40% of the time frame needed for detectable growth in the non-propionate supplemented media for the parallel culture. In some embodiments, growth in propionate supplemented media will be detectable in a time frame of at least from about 85% to 20% of the time frame, or in some embodiments, in a time frame of at least from about 70% to 30% of the time frame, or in some embodiments, in a time frame of at least from about 50% to 70% of the time frame, needed for detectable growth in the non- propionate supplemented media for the parallel culture.PA158379WO BERNEY7941PC

[0117] It will be appreciated that methods of establishing bacterial growth are wellknown and any such method is envisioned in regard to the description provided herein, including culture turbidity measurements (e.g. by optical density assessment), cell counting by manual or automated means, etc. and any other method accomplishing assessment of bacterial growth and represents an embodied aspect of the invention.

[0118] In some embodiments, reference herein to detection of bacterial growth as a result of the compositions / kits / methods of this invention is intended to include earliest detectable growth by any means, or, in other embodiments, rapid increased growth by any means, or in some embodiments, relative changes in growth rate or quantity as opposed to an identical strain similarly treated, but without the benefit of propionate supplementation as herein described.

[0119] In some embodiments, as referred to herein, antibiotic sensitivity is described in terms of indicated MIC values. Determination of MIC values is well known in the art, and represents a standard industry assay, and is also described and exemplified herein (see for example, Kowalska-Krochmal B, et.al. Pathogens. 2021 Feb 4;10(2):165. doi: 10.3390 / pathogens10020165. PMID: 33557078; PMCID: PMC7913839, fully incorporated by reference herein).

[0120] In other embodiments, the invention provides a method of earlier diagnosis of mycobacterial infection in a subject, the method comprising: (c) culturing a suspected mycobacterial strain isolated from a subject in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; and (d) separately culturing the identical mycobacterial strain isolated from a subject in a culture media supportive of mycobacterial growth in culture in the absence of propionate; wherein mycobacterial strain growth is obtained more rapidly from said culture in (a) than in the culture in (b), thereby providing for earlier diagnosis of mycobacterial infection in a subject.

[0121] A method of rapid diagnosis of Mtb in a subject, the method comprising:PA158379WO BERNEY7941PC (a) culturing a mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media, wherein said culturing is for a period of at least from about 5 to about 14 days and establishing growth; wherein growth is detected more rapidly in cultures conducted in the presence of propionate, thereby providing a more rapid diagnosis for Mtb infection in the subject.

[0122] In other aspects, and also as surprisingly demonstrated herein, the propionatesupplementation in culture medium of Mtb in isolated samples reduces time to result and assay robustness for drug sensitivity testing of Mtb.

[0123] According to this aspect, therefore, the invention further provides a method ofrapid and / or reliable determination of antibiotic sensitivity of Mtb in a subject, the method comprising the steps of: (b) contacting mycobacterial strains cultured in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media for a period of at least 5 days to establish growth and further contacting the culture with at least one antibiotic for a period of 7 to 14 days; and (c) parallel culturing a second mycobacterial strain in a culture media supportive of mycobacterial growth in the absence of propionate and in the presence of the antibiotic; and (d) separately determining the MIC90values for the mycobacterial strains in (b) and (c); wherein when the MIC value differs in the strain obtained in (b) as compared to the strain obtained in (c), and optionally the result is obtained more quickly, then the method serves to provide a more rapid and reliable determination of antibiotic sensitivity of the mycobacterial strain.

[0124] In some aspects, the MIC50, MIC90 or MIC99 values may be similarly assessed for the mycobacterial strains, and be equally informative.

[0125] In other embodiments, the invention provides a method of improveddetermination of antibiotic sensitivity of a mycobacterial strain, improved antibiotic dosage determination for a subject, or a combination thereof, the method comprising: (a) culturing a first mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a finalPA158379WO BERNEY7941PC concentration of 0.1 – 1.0 mM in the supplemented culture media and separately similarly culturing a second identical mycobacterial strain in the absence of the propionate; (b) further contacting said first mycobacterial strain and said second identical mycobacterial strain with an antibiotic for a period of from about 5 to 10 days; (ci) determining the minimum inhibitory concentration (MIC) values for MIC50, MIC90 or MIC99 for the first and second mycobacterial strains in (b), wherein when any of the MIC values in the first mycobacterial strain are obtained more rapidly or reliably than in the second mycobacterial strain, the method serves to improve determination of antibiotic sensitivity of the mycobacterial strain, or the method serves to improve antibiotic dosage determination for the subject infected with the mycobacterial strain, or a combination thereof.

[0126] In other embodiments, the invention provides a method of more rapid determination of antibiotic sensitivity or determination of inhibitory antibiotic dosage for mycobacterial infection in a subject, or a combination thereof, the method comprising: (e) culturing a first mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; and (f) separately similarly culturing a second identical mycobacterial strain in the absence of the propionate; and (g) further contacting said first mycobacterial strain and said second identical mycobacterial strain with an antibiotic for a period of time; and (h) separately determining the minimum inhibitory concentration (MIC) values for MIC50, MIC90 or MIC99 for the first and second mycobacterial strains; wherein the MIC values in the first mycobacterial strain are obtained in at least 20% of the time necessary to obtain reliable MIC values in the second mycobacterial strain, thereby more rapidly determining antibiotic sensitivity or determining inhibitory antibiotic dosage for the subject infected with the mycobacterial strain, or a combination thereof.PA158379WO BERNEY7941PC

[0127] In some embodied aspects the culture media may contain any of the additivesdescribed herein regarding media supplementation and methods involving propionate supplemented culture of Mtb.

[0128] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

[0129] It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as set forth in the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed in the scope of the claims.

[0130] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of a conflict between the specification and an incorporated reference, the specification shall control. Where number ranges are given in this document, endpoints are included within the range. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges, optionally including or excluding either or both endpoints, in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Where a percentage is recited in reference to a value that intrinsically has units that are whole numbers, any resulting fraction may be rounded to the nearest whole number.

[0131] In the claims articles such as "a,", "an" and "the" mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" or "and / or" between members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in,PA158379WO BERNEY7941PC employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides, in various embodiments, all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g. in Markush group format or the like, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in haec verba herein. Certain claims are presented in dependent form for the sake of convenience, but Applicant reserves the right to rewrite any dependent claim in independent format to include the elements or limitations of the independent claim and any other claim(s) on which such claim depends, and such rewritten claim is to be considered equivalent in all respects to the dependent claim in whatever form it is in (either amended or unamended) prior to being rewritten in independent format.

[0132] The following examples describe certain embodiments of the invention andshould not be construed as limiting the scope of what is encompassed by the invention in any way. EXAMPLES EXAMPLE 1: DEVELOPMENT OF HIGH THROUGPUT ASSAYS AND METHODS FOR PDIM PRODUCTION ASSESSMENT

[0133] Embodied methods and materials of this invention include and rely on use of a PDIM reference strain set for applications in the assays of this invention. Toward this end, six Mtb strains with an H37Rv genetic background as listed in Table 1 below were assessed for their PDIM-content determined by lipid analysis using TLC autoradiography (Fig. 1A).

[0134] Table 1: Mtb PDIM reference strains.PA158379WO BERNEY7941PC https: / / doi.org / 10.1016 / j.vaccine.2006.05.097 2 Jain et al. (2014) mc 6206 ΔpanCD ΔleuCD https: / / doi.org / 10.1128 / mbio.01245-14 mc26209 ΔpanCD ΔleuCD ΔsecA2 Jain et al. (2014) ibid. mc27901 ΔpanCD ΔleuCD ΔmetAVilchèze et al. (2018)https: / / doi.org / 10.1128 / mBio.00938-18 mc27902 ΔpanCD ΔleuCD ΔargB Vilchèze et al. (2018) ibid. mc28398 ΔmetA ΔargB This study mc26230 ΔRD1 ΔpanCD This study, only pantothenate AE1601 mc26230 ΔRD1 ΔpanCD This study, only pantothenate AE1611 ppsC c.2685(C)7→8 H37Rv wildtype This study, no PALM AE1028 H37Rv AE1028 ∆Rv2934::hygR This study, no PALM ΔppsD

[0135] The listed strains have an H37Rv genetic background and were supplemented with ‘PALM’ supplements (pantothenate, arginine, leucine and methionine) in in vitro culture, unless otherwise indicated. In this context, it is noted that the two H37Rv strains do not require PALM supplementation and the two mc26230 AE strains require only pantothenate supplementation.

[0136] Three of these strains – mc26206, mc26209, and mc27902 produced moderate tohigh levels of PDIMs, while the remaining strains – mc28398, mc27901, and mc26230, were all PDIM deficient, producing either no or very minimal PDIM, as determined by TLC lipid analysis of the PDIM reference strain set (Fig 1A).

[0137] Whole genome sequencing (WGS) and PDIM mutation analysis of these and other strains included in this study were then conducted to further extend these findings (Table 2).

[0138] Sequencing was performed using the Illumina MiSeq platform, with the exception of mc26230 AE1601 and the parent mc26230 stock which was additionally sequenced on Illumina NextSeq. Short-read data were mapped to the following reference genomes according to the strain: H37Rv NC_000962.3, Erdman NC_020559.2, KZN 4207 NC_016768.1, HN878 NZ_CM001043.1, and CDC1551 NC_002755.2. Non-synonymous variants in genes involved in PDIM biosynthesis, assembly, processing, or transport are described at the protein level for amino acid substitutions (p.) and at the coding DNA level for frameshift / INDEL mutations (c.). Variants were identified by variant calling using thePA158379WO BERNEY7941PC software tool Pilon, with the exception of ppsC c.2668 frameshift mutations which were analyzed by PCR and Sanger sequencing and are indicated with †. For mutations with mixed coverage (<90%) the mutation frequency is given in brackets. ‘n.d.’ = no PDIM mutations detected.

[0139] WGS sequencing identified mutations in PDIM biosynthetic genes, inter alia, for mc28398 (ppsC c.2475delC) and mc27901 (fadD28 p.Arg5662Gly) explaining their PDIM defects (Table 2).

[0140] Table 2: PDIM mutations in Mtb strains included in this study. Strain Genome coverage (´) PDIM mutations mc26206 66.3 n.d. mc26209 56.8 n.d. mc26230 187.0 n.d. mc26230 (NextSeq) 134.2 ppsC c.2685(C)7à8(66%) mc26230 AE1601 (NextSeq) 171.1 n.d mc26230 AE1611 30.5 ppsC c.2685(C)7à8†mc27901 26.7 fadD28 p.Arg562Gly mc27902 47.8 n.d. mc28398 62.1 ppsC c.2475delC H37Rv-A 93.5 n.d. H37Rv-B 60.5 n.d. H37Rv AE1028 57.1 n.d. H37Rv ΔppsD 58.7 ΔppsD H37Rv Δtgs1-1 95.6 fadD28 p.Leu241Pro H37Rv Δtgs1-2 90.3 ppsA p.Trp968* H37Rv Δtgs1-3 17.2 ppsD p.Ser247Pro H37Rv Δtgs1-4 86.3 ppsE p.Asp74Asn H37Rv Δtgs1-5 119.4 ppsC c.2668(C)7à6†H37Rv Δtgs1-7 184.5 n.d. H37Rv Δtgs1-8 58.2 ppsC c.2668 (C)7à8†H37Rv Δtgs1-9 69.7 ppsC c.2668(C)7à6†Erdman 84.7 ppsA c.74dupA (72%) Erdman-SC 91.2 n.d. HN878 76.1 n.d. HN878-SC 65.9 n.d. KZN 4207 86.7 n.d. CDC1551-A 106.5 n.d. CDC1551-B 54.7 n.d. CDC1551-SC 69.3 n.d. CDC1551 Δmas 43.6 Dmas

[0141] Intriguingly, no PDIM mutations were detected in mc26230.PA158379WO BERNEY7941PC

[0142] PDIM(-) Mtb and M. bovis BCG have been shown to be more susceptible tovancomycin than the corresponding PDIM(+) wildtype strains, although vancomycin, is not normally used for the treatment of Mtb stains due to poor penetration.

[0143] To test if this can be exploited as a useful diagnostic tool, and other applications, vancomycin minimum inhibitory concentration (MIC) assays of the strains in Fig. 1A were conducted (Fig. 1B-D). MICs were performed using the broth microdilution method in standard 7H9 / OADC / glycerol / tyloxapol + PALM media. Bacterial growth was measured by optical density after 7, 10 and 14 days, respectively, of incubation and normalized to drug-free controls. Mean ± s.d., n = 2 biological replicates. Simple linear regression analysis shows the correlation between PDIM band intensity and vancomycin MIC50and MIC90, with the error bars representing the 95% CI.

[0144] Given the interesting finding regarding vancomycin responsiveness, it was of interest to determine the MICs of a range of large antibiotics of the glycopeptide (vancomycin, teicoplanin, ramoplanin) and macrolide (azithromycin and erythromycin) families as well as the two frontline antibiotics rifampicin and isoniazid for Mtb mc28398 (PDIM(-)) and mc27902 (PDIM(+)) strains to understand if there was a broader impact related to PDIM.

[0145] Fig. 2 depicts the results of MIC assays of Mtb mc27902 and mc28398 to A, ramoplanin (RAM), B, teicoplanin (TEC), C, vancomycin (VAN), D, rifampicin (RIF), E, azithromycin (AZM), F, erythromycin (ERY), and G, isoniazid (INH). Compounds were arranged by descending molecular weight, which is shown on the MIC plot for each. Bacterial growth was measured by optical density after 10 days of incubation and normalized to drug- free control wells. Data are shown as mean ± s.d., n = 2 duplicate wells. H, Ethidium bromide uptake of mc27902 and mc28398. Uptake in whole cell suspensions was monitored by fluorescence (Ex 355 nm / Em 590 nm). Data are shown as mean ± s.d., n = 4 biological replicates measured as n = 5 technical replicates. MIC assays are representative of a single experiment and uptake assays of two independent experiments.

[0146] While most of the high molecular weight compounds exhibited a greater than 2-fold difference in MIC between PDIM(+) and PDIM(-) strains, the greatest difference was observed for vancomycin with a 13.5- and 9.5-fold difference in MIC90and MIC50, respectively (Fig. 2).

[0147] This indicated that permeability differences are responsible for the increasedsusceptibility of PDIM(-) mc28398 to high molecular weight drugs. Indeed, the commonlyPA158379WO BERNEY7941PC used ethidium bromide permeability assay revealed a significant EtBr uptake in PDIM(-) mc28398, while in PDIM(+) mc27902 only very limited uptake was detected (panel H).

[0148] Based also on these findings, it was desired to establish if differential vancomycin sensitivity could be exploited as a general screen for PDIM production in mycobacterial strains.

[0149] Toward this end, it was found as described hereinabove, when assessing vancomycin MICs for the reference strain set (Fig. 1A), that the PDIM levels measured by TLC were highly correlated with vancomycin MIC90 and MIC50 after 10 and 14 days of incubation (Fig. 1B-D, and additional data not shown).

[0150] Once the differential vancomycin sensitivity was determined, the developmentof an embodied method / kit / assay was then developed that provides for high-throughput assessment of PDIM production.

[0151] It was determined that all PDIM-deficient strains exhibited an MIC90below 10 mg / ml while PDIM(+) strains showed intermediate growth around this concentration (Fig. 1C-D, Fig. 2C, Fig. 3B, A-C). Relative growth in 10 mg / ml vancomycin compared to no-drug controls (VAN10 growth%) significantly correlated with PDIM levels, at all measured time points with the strongest relationship observed after 10 to 14 days (Fig. 3B, D-E). Growth curves in Fig. 3B, A-C are shown as mean ± s.d., n = 3 for biological replicates, and VAN10 growth% in Fig. 3B, D-E as mean ± s.d., n = 2 for two experiments, each performed in triplicate.

[0152] The embodied methods / kits / assays are also therefore amenable to use in low-resource settings, since consistent reliable results were obtained using simple equipment, such as inkwell bottles and a simple spectrophotometer (Fig. 3B, E).

[0153] This is the first demonstration that PDIM production can be readily determined, through the embodied methods / kits / assays of this invention, using the developed vancomycin resistance screening (MIC or single concentration assays) as an effective approach to assess PDIM production in Mtb.

[0154] Thus, high throughput assays / methods / materials for PDIM production assessment was achieved, applicable in any appropriate laboratory setting, including low- resource settings.PA158379WO BERNEY7941PC EXAMPLE 2: PDIM PRECURSORS INCREASE VANCOMYCIN RESISTANCE AND IMPROVE SCREENING ASSAYS

[0155] It is known that PDIM production is not only dependent on the activity of the biosynthetic pathway but also on the availability of the PDIM precursor MMCoA. MMCoA is produced from propionyl-CoA by propionyl-CoA carboxylase or from succinyl-CoA by vitamin B12-dependent MMCoA mutase (Fig. 4A). Cholesterol, a host carbon source favored by Mtb, provides a major source of propionyl-CoA during infection. Propionyl-CoA can also be generated via beta-oxidation of odd-chain fatty acids and catabolism of branched-chain amino acids (Fig. 4A).

[0156] Standard 7H9 culture media used to cultivate mycobacterial strains (7H9 / OADC / glycerol), however, contains neither cholesterol, odd-chain fatty acids, nor vitamin B12. Propionate supplementation and growth on cholesterol as a sole carbon source have previously been shown to increase PDIM production. To validate this and to further improve the PDIM high throughput production assay, we assessed whether propionate supplementation would impact the assay system in Example 1.

[0157] The mycobacterial strains indicated in Fig. 4B-C, were cultured in standard 7H9 / OADC / glycerol / tyloxapol media + PALM supplementation. MICs to vancomycin were then determined in the same media and additionally supplemented with 0.1 mM propionate. Bacterial growth was measured after 7 days of incubation and normalized to drug-free controls. Data are shown as mean ± s.d., n = 4 for biological replicates from two independent experiments.

[0158] These findings were extended to assess the mycobacterial strains indicated in Fig. 4D, following culture in standard 7H9 / OADC / glycerol / tyloxapol media + PALM supplement and then further exposed to 10 mg / ml vancomycin in the same media and additionally supplemented with either 0.1 or 1.0 mM propionate or 10 mg / ml vitamin B12and VAN10 growth% determined after 10 days of incubation. Data are shown as mean ± s.d., n = 3 for replicate experiments, each performed in triplicate.

[0159] To further validate our approach, we next tested isogenic PDIM(+) (AE1028wildtype and DppsD::comp) and PDIM(-) (DppsD) and H37Rv strains. These strains werePA158379WO BERNEY7941PC constructed by specialized transduction using a PDIM(+) clone derived from a single colony (H37Rv AE1028 wildtype). The results are shown in Fig. 4F-H, comparing vancomyin MIC assays in standard 7H9 / OADC / glycerol / tyloxapol media (panel F), and the same media additionally supplemented with either 0.1 mM propionate (panel G) or 10 mg / ml vitamin B12(panel H). We observed a ~30-fold difference in MIC90 between PDIM(+) and PDIM(-) H37Rv with 0.1 mM propionate (Fig. 4G). Growth was measured after 10 days and normalized to drug-free controls. Data are shown as mean ± s.d., n = 4 for biological replicates from two independent experiments.

[0160] Further validation of these improved results was conducted using the VAN10 assay developed in Example 1, assessing the indicated isogenic H37Rv strains after growth in media supplemented with 0.1 mM propionate Fig. 4E. Growth was measured after 10 days. Data are shown as mean ± s.d., n = 3 for replicate experiments, each performed in triplicate. Differences were evaluated by one-way ANOVA with Tukey’s multiple comparison test. ****P < 0.0001.

[0161] Propionate and vitamin B12supplementation increase vancomycin resistance of PDIM(+) but not PDIM(-) Mtb, improving assay robustness and reducing time to result (Fig. 5). Fig. 5A-F plots the vancomycin MICs for the PDIM reference strain set in standard 7H9 / OADC / glycerol / tyloxapol + PALM media and media supplemented with either 0.1 mM propionate, 1.0 mM propionate, or 10 mg / ml vitamin B12, as indicated. Bacterial growth was measured by optical density after 10 days of incubation. Strains assessed are as follows: mc28398 (PDIM(-)) (panel A); mc26230 (PDIM(-)) (panel B); mc27901 (PDIM(-)) (panel C); mc26209 (PDIM(+)) (panel D); mc26206 (PDIM(+)) (panel E); and mc27902 (PDIM(+)) (panel F). Fig. 5G-H plot vancomycin MICs for the reference strain set in standard 7H9 / OADC / glycerol / tyloxapol + PALM media (panel G) and the same media additionally supplemented with 0.1 mM propionate (panel H) measuring growth after 7, 10, and 14 days of incubation. Data were normalized to drug-free controls and are shown as mean ± s.d., n = 4 for biological replicates from two independent experiments. The data in A-F includes one of the same experiments shown in G-H, together with data from an independent experiment. The day seven data in G-H is the same as that shown in Fig. 4B-C alongside additional time points.

[0162] As can be readily seen in these results, the addition of low-concentration propionate (0.1 or 1.0 mM), preferentially increased vancomycin resistance of the PDIM(+) strains, improved the differentiation between PDIM(+) and PDIM(-) strains, and reduced assayPA158379WO BERNEY7941PC duration (Fig. 4B-D and G-H, Fig. 5). Vitamin B12also increased the vancomycin resistance of PDIM(+) strains similar to propionate (Fig. 4D and H, Fig. 5A-F).

[0163] We term the measurement of the vancomycin MIC in the presence of 0.1 mM propionate the ‘VAN-P’ MIC assay and the VAN10 assay in the presence of 0.1 mM propionate the ‘VAN10-P’ assay.

[0164] Even more surprisingly, the PDIM-deficient strain mc26230 began to show a somewhat delayed increase in vancomycin resistance after 10 to 14 days with propionate or vitamin B12 supplementation (Fig. 4D, Fig. 5H).

[0165] Upon conducting further analysis, it was found that the mc26230 stock previously determined to be PDIM(-) in fact comprised a PDIM(-)-dominated mixed population (data not shown). This is indicated in the re-sequencing where using a different Illumina platform (NextSeq) we detect a ppsC mutation at 66% frequency. Following a single passage in 10 mg / ml vancomycin we were able to enrich for PDIM(+) cells (data not shown). Using the VAN10-P assay to screen single colonies for PDIM we isolated pure PDIM(+) and PDIM(-) mc26230 clones (AE1601 and AE1611, respectively).

[0166] Uniquely, the methods / assays / reagents / kits of this invention therefore also provided a means for selection of low-representation PDIM(+) clones in a mixed sample, which supports a number of valuable applications, including, for example, a means to re- isolate PDIM(+) clones from mixed population stocks.

[0167] Propionate and vitamin B12supplementation selectively enhanced the vancomycin resistance of the PDIM(+) strains, with no effect on PDIM(-), indicating that optimal PDIM-production in Mtb is dependent on exogenous sources and that PDIM precursor availability influences vancomycin resistance of PDIM(+) Mtb, and presumably other large antibiotics of the glycopeptide (teicoplanin, ramoplanin) and macrolide (azithromycin and erythromycin) families, in addition to the known Mtb antibiotics, such as rifampicin and isoniazid. EXAMPLE 3: PDIM SCREENING REVEALS ERRORS IN THERAPEUTICS AND VACCINE DEVELOPMENT WHICH ARE PDIM BIAS RELATEDPA158379WO BERNEY7941PC

[0168] Genetic engineering of Mtb strains may be affected by PDIM loss. Usingspecialized transduction, following methods as described in Jain et. al. (2014) (https: / / doi.org / 10.1128 / mbio.01245-14), knockout mutants of the non-PDIM-related gene tgs1 (Rv3130c) were generated from a mouse-passaged H37Rv stock (H37Rv-B). tgs1 encodes triacylglycerol synthase Tgs1 and is involved in carbon storage. Only one out of eight tgs1 mutants obtained – Dtgs1-7, was determined to be fully PDIM(+) by VAN-P MICs, TLC, and WGS (Fig. 6A).

[0169] VAN-P MIC and VAN10-P assays conducted predicted PDIM status duringgenetic manipulations and across different strains and lineages. Fig. 6A panel A demonstrates the vancomycin MICs obtained with 0.1 mM propionate (VAN-P MIC) for eight confirmed Dtgs1 mutants and the parent strain, H37Rv-B. Bacterial growth was measured after 10 days and normalized to drug-free controls. Mean ± s.d., n = 4 for biological replicates from two independent experiments. Panel B shows TLC lipid analysis of four Dtgs1 mutants and H37Rv-B, and panel C shows the mutations in PDIM biosynthetic genes for all eight Dtgs1 mutants and H37Rv-B. Mutations are described at the protein level for amino acid substitutions (p.), and at the coding DNA level for frameshift / INDEL mutations (c.).

[0170] Fig. 6B shows the results of TLC lipid analysis, WGS, VAN-P MIC andVAN10-P assays for different laboratory stocks of a range of virulent Mtb strains. In the VAN10-P assay in panel A, mc27902 and mc28398 were included as PDIM(+) and PDIM(-) controls. Growth was measured after 10 days. Data show mean ± s.d., n = 9 for all pairwise comparisons between triplicate replicate wells and are representative of two independent experiments. Panel C shows VAN10-P assay results for single colonies isolated from two different H37Rv stocks; H37Rv-A (n = 38) and H37Rv-B (n = 37). VAN10-P growth% was measured after 14 days. Data points show mean VAN10-P growth% from triplicate wells assessed for each colony and a solid line indicates the median. Differences were evaluated using the Mann-Whitney test (unpaired, two-tailed). ****P < 0.0001.

[0171] Fig. 6A panels F-H depict the VAN-P MICs for the Erdman (panel F), HN878(panel G), and CDC1551 (panel H) strains compared to MICs for PDIM(+) clones of each strain isolated by VAN10-P screening of single colonies. Data shown are from panel E together with independently measured MICs for single PDIM(+) clones (SC). MIC data were measured on day 10 and normalized to drug-free control wells. Mean ± s.d., n = 4 for biological replicates from two independent experiments. These results demonstrate the applicability of assay in different strains and for re-isolation of PDIM(+) clones.PA158379WO BERNEY7941PC

[0172] VAN-P MIC assays detected PDIM defects in seven of the eight Dtgs1 mutantsobtained by specialized transduction, three of which were additionally validated by TLC (Fig. 6A panel B). WGS and Sanger sequencing identified six different mutations in five different PDIM genes across the seven PDIM(-) Dtgs1 mutants (Fig. 6A panel C, Table 2), emphasizing the genetic heterogeneity in the PDIM gene cluster in mixed populations. Amongst the six different PDIM mutations detected were two unique frameshift mutations in a 7-cytosine homopolymeric tract in ppsC (c.2668(C)7à6and c.2668(C)7à8) (Fig. 6A panel C, Table 2). We additionally found a ppsC frameshift in an adjacent homopolymeric tract mc26230 (c.2685(C)7à8) (Table 2) and other groups have also reported frameshift mutations in these sites, collectively pointing to this region as a potential ‘hotspot’ for mutation and PDIM loss. Frameshift mutations can occur at elevated frequencies in homopolymeric tracts and provide a mechanism for bacteria to rapidly adapt to changes in their environment.

[0173] The ppsC frameshift mutations were not reliably detected by Illumina MiSeqdue to poor sequencing coverage over this genomic region. This shows that additional care is needed when analyzing these sites because of their high mutation rate in standard culture media and potentially low coverage due to sequencing bias. The fact that we find such a broad range of mutations in these strains indicates an evolutionary sandbox with a high propensity for PDIM mutations, which may be further augmented by homopolymeric tracts due to their high mutation frequency.

[0174] Next, we determined the predictive power of our approach in different Mtb strains and compared VAN-P MIC and VAN10-P assays, TLC, and WGS for virulent Mtb strains belonging to different lineages: CDC1551 (Lineage 4.1.1.3), Erdman (L4.1.2.1, Haarlem), KZN 4207 (L4.3.3, LAM), and HN878 (L2.2.1.2, Beijing), as well as two different H37Rv stocks (H37Rv-B and H37Rv-A) (L4) (Fig. 6B). For all these strains VAN-P MIC and VAN10-P assays reliably predicted PDIM levels as determined by TLC (panels A, D and E). Furthermore, VAN-P MIC and VAN10-P assays outperformed WGS at diagnosing PDIM deficiencies caused by culture heterogeneity. H37Rv-A, Erdman, and CDC1551-A were all found to be highly PDIM-deficient by VAN-P MIC and VAN10-P assays and TLC, however, variant calling failed to identify PDIM mutations in H37Rv-A or CDC1551-A, and identified a ppsA frameshift mutation with 72% frequency in Erdman (Table 2). We therefore searched for low-frequency mutations and identified one or two low-frequency (10-13%) non-synonymous PDIM mutations in each of these strains, as well as two mutations with 14% and 19% frequency in the moderate PDIM producer HN878 (Fig. 6B panel B). The presence of low-PA158379WO BERNEY7941PC frequency mutations in these stocks rather than single fixed mutations indicate that they comprise a mixture of different PDIM mutants. Low-frequency mutations are thus not a good predictor of PDIM levels as heterogeneous populations may comprise an array of different PDIM mutants which can be difficult to detect by WGS, making WGS a poor predictor of PDIM levels in mixed populations.

[0175] Historically, mouse passaging was the only procedure known to select for PDIM(+) Mtb. Using our VAN10-P assay, we found that while mouse passage enriched for PDIM(+) cells, this did not produce a fully PDIM(+) stock. H37Rv-A was previously passaged through a C57BL / 6 mouse and all colonies from the lung were harvested generating H37Rv-B. While we observed markedly higher PDIM lipid levels and vancomycin resistance in H37Rv-B compared to H37Rv-A (Fig. 6B, panels A and D), H37Rv-B still had lower PDIM levels and vancomycin resistance than mc27902 (Fig. 6B, panel A), Dtgs1-7 (Fig. 6A, panels A-B), and the PDIM(+) H37Rv clone used for DppsD mutant construction (H37Rv AE1028) (Fig. 6B panel D). VAN10-P screening of single colonies isolated from H37Rv-A and H37Rv-B determined that H37Rv-A comprised 95% PDIM(-) clones (36 / 38). Conversely, H37Rv-B comprised only 5% PDIM(-) clones (2 / 37), however, most of the population appeared to have an intermediate PDIM phenotype (Fig. 6B panel C). This confirms that H37Rv-A comprises a mostly PDIM(-) population and animal passage selected for PDIM(+) clones, however, PDIM-deficient strains were not completely removed. EXAMPLE 4: PROPIONATE SUPPLEMENTATION PREVENTS PDIM LOSS IN VITRO

[0176] Examples 1-3 demonstrate the reliability of the embodied methods / assays / kits / reagents of this invention as a reliable and effective method for PDIM production assessment. It was of interest to apply this, as well, to assessing PDIM loss.

[0177] In Mtb infection, lipid production is tightly coupled to its metabolism and propionyl-CoA, an upstream precursor of PDIM, may be inhibitory to bacterial growth, though the mechanism for same is unclear.

[0178] The major pathways for propionyl-CoA detoxification are the methylcitrate cycle, the vitamin B12-dependent methylmalonyl pathway, and the incorporation into PDIM and other virulence-associated lipids (Fig. 4A).PA158379WO BERNEY7941PC

[0179] It was of interest to assess whether PDIM-deficient strains are more sensitive topropionate toxicity due to the absence of PDIM as a sink for propionyl-CoA detoxification and whether same could be exploited to create a PDIM(+) selective medium.

[0180] PDIM(-) strains in our reference strain set were more sensitive to propionate than the PDIM(+) strains (Fig. 7A). Surprisingly, we also observed that PDIM(+) strains reached higher density at low propionate concentrations, suggesting sub-toxic propionate may provide a growth advantage to PDIM(+) Mtb. Data are shown as mean ± s.d., n = 3 for biological replicates and are representative of two independent experiments.

[0181] To further investigate this, we measured the growth of isogenic PDIM(+) and PDIM(-) H37Rv in standard media with and without propionate supplementation. PDIM(-) Mtb had a growth advantage over PDIM(+) in standard 7H9 / OADC / glycerol / tyloxapol media (Fig. 7B), whereas the addition of sub-toxic propionate (0.1 or 1.0 mM) restored the growth of PDIM(+) strains to that of PDIM(-) (Fig. 7C-D). Vitamin B12also rescued the growth of PDIM(+) strains (Fig. 7E), indicating PDIM(+) growth is impaired in standard media due to a deficiency of MMCoA precursors. Data are shown as mean ± s.d., n = 3 for biological replicates and are representative of at least two independent experiments.

[0182] Following this result, we reasoned that propionate would be selective for PDIM(+) cells by eliminating the growth advantage conferred by PDIM loss in standard media. To assess this, we performed experimental evolution experiments using strains with different PDIM(+) to PDIM(-) ratios. First, we serially passaged H37Rv-B – a moderately PDIM(+) mixed population (Fig. 6H), by weekly subculture in 7H9 / OADC / glycerol / tyloxapol media with and without propionate supplementation and then assessed PDIM levels by TLC and VAN10-P assays. Significant PDIM loss was observed after six passages in standard media, whereas the addition of 0.1 or 1.0 mM propionate maintained full PDIM levels (Fig. 7F-G). Next, we repeated the experiment starting from a PDIM(+) clone (H37Rv AE1028). A marked decline in PDIM levels was observed after only 5 – 8 passages, while 0.1 mM propionate maintained PDIM for the duration of the experiment (Fig. 7H). Starting from H37Rv-A – a predominantly PDIM(-) population (Fig. 6H), we observed a gradual increase in PDIM with successive passaging in 0.1 mM propionate (Fig. 7I). Single colony screening confirmed enrichment of PDIM(+) cells following propionate passage (Fig. 7J), demonstrating that propionate positively selects for PDIM(+) Mtb. Data are shown as mean ± s.d., n = 3 for biological replicates each assayed in triplicate. ***P < 0.001, ****P < 0.0001, two-wayPA158379WO BERNEY7941PC ANOVA with Tukey’s (Fig. 7G-H) or Šidák’s (Fig. 7I) multiple comparison test. Differences between media are shown in Fig. 7G and H, and between consecutive passages for each media in Fig. 7I. Data points in Fig. 7J show mean VAN10 growth% for single colonies each assayed in triplicate and a line indicates the median. For P0, n = 38 (same data as Fig. 6F) and P9, n = 30. **P < 0.01; unpaired two-tailed Mann-Whitney test.

[0183] We next assessed the effect of vitamin B12 supplementation on the maintenance of PDIM production in Mtb. Serial passage of H37Rv-B in media supplemented with 10 mg / ml vitamin B12shows that vitamin B12also maintains PDIM production (Fig. 7K). Mean ± s.d., n = 3 for biological replicates each assayed in triplicate. *P < 0.05, differences were evaluated using two-way ANOVA with Tukey’s multiple comparison test.

[0184] We speculated that while advantageous for the growth of PDIM(+) Mtb,propionate selects against PDIM(-) cells owing to propionyl-CoA toxicity in the absence of a functional PDIM biosynthetic pathway. Indeed, the addition of vitamin B12 to alleviate propionyl-CoA toxicity via activation of the methylmalonyl pathway considerably slowed the selection process and resembled more cultures with vitamin B12alone. (Fig. 7L). Mean ± s.d., n = 3 for biological replicates each assayed in triplicate. Significant differences compared with +0.1 mM propionate are indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA with Tukey’s multiple-comparison test. P > 0.05 for vitamin B12versus vitamin B12 + 0.1 mM propionate, and P < 0.0001 for the standard medium versus each supplemented condition at all timepoints.

[0185] These data support that small quantities of propionate give a selective advantage to PDIM(+) over PDIM(-) bacilli, which can be exploited to support the methods / systems / materials of this invention. EXAMPLE 5: PROPIONATE-ENHANCED PDIM PRODUCTION INCREASES RESISTANCE TO SELECT ANTIBIOTICS

[0186] Propionate provides a source of three-carbon precursors otherwise missing from standard culture media, mimicking host nutrient conditions during growth on cholesterol and fatty acids. As propionate had such a dramatic effect on vancomycin resistance of PDIM(+) Mtb (as described in Example 1 hereinabove), these results were extended to evaluate any impact in terms of other antibiotic sensitivity changes in Mtb.PA158379WO BERNEY7941PC

[0187] Toward this end, it was specifically of interest to evaluate further, other knownMtb drugs and assess any impact related to propionate supplementation.

[0188] Propionate markedly increased rifampicin resistance of PDIM(+) but not PDIM(-) Mtb (Fig. 8A-B). A smaller but reproducible propionate-induced MIC shift was additionally found for bedaquiline (Fig. 8C), however, propionate did not induce MIC shifts for smaller inhibitors (Fig. 9), including capreomycin (CAP) (panel A); moxifloxacin (MFX) (panel B); pretomanid (PMD) (panel C); linezolid (LNZ) (panel D); ethambutol (EMB) (panel E); and isoniazid (panel F). Supplementation with 10 mg / ml vitamin B12 mirrored the propionate-induced MIC increase for rifampicin and bedaquiline in PDIM(+) Mtb (Fig. 8A and C). The compounds with PDIM-dependent propionate-induced MIC shifts all have a high molecular weight (³555 g / mol). Data are shown as mean ± s.d., n = 4 for biological replicates from two independent experiments for Fig. 8A and C, and for n = 2 for biological replicates from a single experiment for Fig. 8B and Fig. 9.

[0189] It has long been accepted that PDIM loss correlates with virulence attenuation in Mtb.

[0190] The instant disclosure is the first to demonstrate that simple addition of propionate to Mtb cultures selects for PDIM(+) strains. In the host, Mtb has access to propionyl-CoA-generating carbon sources. Current culture media however lacks a three- carbon source, imposing a metabolic burden on Mtb to maintain sufficient propionyl-CoA pools for PDIM production.

[0191] The current disclosure therefore supports one embodied aspect of this invention,providing improved culture conditions for mycobacterial strains that promote PDIM(+) phenotype maintenance

[0192] Vitamin B12supplementation also positively impacted restoring full growth of PDIM(+) Mtb in vitro. EXAMPLE 6: DETERGENT INFLUENCES ON PDIM PRODUCTION AND RESISTANCE TO SELECT ANTIBIOTICS

[0193] To assess the effect of detergents on PDIM(+) Mtb, including with propionate or vitamin B12supplementation, and to assess the effect on VAN-P MIC and VAN10-P assays, VAN-P MIC and VAN10-P assays of isogenic PDIM(+) and PDIM(-) H37Rv strains werePA158379WO BERNEY7941PC determined in 7H9 / OADC / glycerol + 0.1 mM propionate, differentially assessing the results when two different detergents were assessed, either tyloxapol or Tween 80 (Fig. 10A-B). Data are shown as mean ± s.d., n = 3 for independent experiments each performed in triplicate for VAN10-P assays (Fig. 10A), and n = 4 for biological replicates from two independent experiments for VAN-P MICs (Fig. 10B). Growth of isogenic H37Rv strains (Fig. 10C-E) where in C. 7H9 / OADC / glycerol / tween and D. the same media supplemented with 0.1 mM propionate, were assessed or, E. 10 mg / ml vitamin B12was added. Data are shown as mean ± s.d., for n = 3 biological replicates and are representative of at least two independent experiments. EXAMPLE 7: PDIM PRODUCTION CAN BE POSITIVELY AFFECTED BY A NUMBER OF AGENTS

[0194] To assess whether other agents could positively impact PDIM(+) growth, growth curves of isogenic PDIM(+)and PDIM(-) H37Rv strains were assessed (Fig. 11) under the following conditions: A. 7H9 / OADC / glycerol / tyloxapol and the same media supplemented with B. 0.1 mM propionate (3 carbon fatty acid), C. 10 mg / ml vitamin B12, D. 0.1 mM propionate and 10 mg / ml vitamin B12, E. 0.1 mM acetate (2 carbon fatty acid), F. 0.1 mM butyrate (4 carbon fatty acid), G. 0.1 mM valerate (5 carbon fatty acid), and H. 0.1 mM cholesterol. Data are shown as mean ± s.d., n = 3 for biological replicates. All data are representative of at least two independent experiments. As can be seen from the data, odd- chain short fatty acids (propionate, valerate), were impactful, while even-chain fatty acids (acetate, butyrate) did not appreciably restore growth of PDIM(+) Mtb. In this study, combining both propionate and vitamin B12did not seem to result in a greater effect than either agent alone, however, these results are preliminary. Furthermore, cholesterol not only restores growth of PDIM(+) Mtb, but additionally has an inhibitory effect on PDIM(-).

[0195] Furthermore, intracellular MMCoA levels can be positively affected bypropionate and vitamin B12 supplementation. Intracellular MMCoA levels were measured by liquid chromatography–mass spectrometry (LC-MS) (Fig. 12), under the following conditions: A. Isogenic PDIM(+) and PDIM(-) H37Rv in 7H9 / OADC / glycerol / tyloxapol ± 0.1 mM propionate. B. PDIM(+) H37Rv AE1028 wildtype in 7H9 / OADC / glycerol / tyloxapol and media supplemented with either 0.1 mM propionate, 1.0 mM propionate, or 10 mg / ml vitamin B12. Data are shown as area under the curve (AUC) for the [M-H]- ion normalized to total ionPA158379WO BERNEY7941PC abundance for a larger panel of metabolites to correct for differences in extraction efficiency. Mean ± s.d., n = 6 for biological replicates from two independent experiments. Differences were evaluated using one way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0196] As can be seen from these data, propionate increases MMCoA levels to that of PDIM(-) levels in standard media, while vitamin B12 increases MMCoA but to a lesser degree than propionate supplementation.

[0197] Most of the Mtb culture media in use today were optimized to promote rapid planktonic growth, and following observations that stearic and oleic acid esters favored “submerged and rapid” growth of mycobacteria in liquid culture, oleic acid-albumin media was adopted. As a result, the standard media in use is devoid of odd-chain fatty acids and despite the widely accepted importance of propionyl-CoA metabolism in Mtb virulence, even- chain fatty acid media has prevailed.

[0198] Mtb is highly efficient at cholesterol utilization and employs the methylcitrate cycle for propionyl-CoA metabolism, which is essential for in vitro growth on three-carbon generating nutrients and in macrophages. In the absence of a functional methylcitrate cycle propionyl-CoA intoxication can be abrogated by supplementing vitamin B12, which activates the methylmalonyl pathway.

[0199] Propionyl-CoA is also a key precursor to PDIM and other methyl-branchedlipids and in fact, PDIM production appears to be the preferred route for propionyl-CoA flux in standard culture media even in the presence of a functional methylcitrate cycle.

[0200] As demonstrated herein, the maintenance of an intact PDIM pathway in vitro isdependent on sufficient MMCoA pools supporting that Mtb benefits from low levels of propionyl-CoA to reach its full growth potential on glycolytic substrates.

[0201] The discrepancy between in vitro culture and the host environment can also impact drug discovery efforts and has implications for drug susceptibility testing. An up to ~30-fold increase in MIC in PDIM(+) Mtb grown with propionate to that using standard media was demonstrated herein, and thus the in vivo potency of rifampicin and other drugs might be drastically overestimated. Inadequate drug levels are associated with poorer treatment outcomes and even very small increases (1.5-fold) in rifampicin MIC in pre-treatment Mtb isolates are predictive of patient relapse. Sub-optimal drug doses can also lead to the selection of low-level antibiotic resistance, providing a gateway for the development of high-level resistant mutants.PA158379WO BERNEY7941PC

[0202] As observed from the data presented herein, our is the first direct link ofpropionyl-CoA pools and PDIM production with enhanced rifampicin resistance. Emerging from these results is the importance in using PDIM(+) strains when evaluating Mtb growth and pathogenesis and the importance in working with propionate-containing medium. Our results enable, for the first time, the selection of fully-PDIM(+) strains and constant maintenance of that status throughout drug discovery experiments. Since Mtb exists in a PDIM enriched state during host infection the PDIM maintaining medium described here mimicks the host environment better than other media, and hence should be used the new standard media for Mtb cultivation in all aspects of TB research.

[0203] The PDIM analysis tools developed and described herein enable address of anumber of previously unaddressed limitations in Mtb tools and therapeutics development. Since heterogenous PDIM(+ / -) populations comprise an array of different low-frequency PDIM mutations, such defects are difficult to detect by WGS. WGS is therefore not informative of PDIM levels in mixed populations.

[0204] A 7-cytosine homopolymeric tract in ppsC was herein identified that is a hotspot for mutation and PDIM loss. Homopolymeric tracts are prone to mutations caused by slipped- strand mispairing and since Mtb lacks a DNA mismatch repair system this may lead to a higher rate of homopolymeric tract mutation. Such mutation-prone regions may potentiate PDIM loss in standard culture media.

[0205] Another unique embodied aspect of the invention is the development of the VAN10-P assay, which can be performed in accordance with the requirements for a high- throughput assay, i.e. being amenable to a 96-well format enabling PDIM heterogeneity assessment with much higher resolution.

[0206] For example, as described herein, Mtb mc26230, although appearing PDIM(-) in TLC, has around 10% PDIM(+) cells remaining. Growth in vancomycin facilitated selection for PDIM(+) clones, even when present at very low frequency, offering a means / method / system to “clean up” mixed population stocks, which represents additional embodied aspects of the invention.

[0207] Taken together, the embodied methods / reagents / kits / assays of this invention uniquely relate to the perceived decreased virulence and increased drug susceptibility of PDIM(-) Mtb as being related to artifacts of in vitro culture systems currently employed. Moreover, the embodied methods / reagents / kits / assays suggest new therapeutic avenues toPA158379WO BERNEY7941PC utilize inhibitors of PDIM biosynthesis to increase the in vivo potency of current drug regimens.

[0208] Still further, the embodied methods / reagents / kits / assays of this invention uniquely provide an opportunity to introduce standardized cultivation and strain maintenance of Mtb strains for use in research and vaccine development, on the one hand, and clinical evaluation tests on the other hand, and these methods / reagents / kits / assays are appropriate in low-resource settings.

[0209] From the examples presented herein it can be seen that sub-toxic concentrations of propionate or vitamin B12 alleviate a metabolic deficiency that impairs the growth of PDIM- producing strains. Propionate supplementation prevented PDIM loss by enabling PDIM(+) cells to outcompete PDIM(-) cells and propionate-grown Mtb displays increased resistance to rifampicin, bedaquiline, and glycopeptides in a PDIM-dependent manner, the findings of which support the embodied methods / reagents / kits / assays of this invention. It is also noted that vitamin B12also increases resistance to rifampicin and bedaquiline, as well as maintaining PDIM production, but does not select against PDIM(-) cells like propionate. EXAMPLE 8: PROPIONATE SUPPLEMENTATION REDUCES TIME TO RESULT AND ASSAY ROBUSTNESS FOR DRUG SENSITIVITY TESTING OF M. TUBERCULOSIS

[0210] It is of interest to assess whether propionate supplementation improved the growth of different strains of Mtb, since if it broadly improves growth, then this has the potential to improve clinical diagnostics.

[0211] The growth of Mtb strains belonging to the four main lineages – lineage 1 (N0072, Fig. 13, panel A), lineage 2 (HN878, panel B), lineage 3 (N0054, panel C), and lineage 4 (H37Rv, panel D), were assessed for their growth in 7H9 / OADC / glycerol / tyloxapol media with and without 0.1 mM propionate, by methods as described herein. Two of these isolates – N0072 and N0054, represent clinical isolates from a previously described collection (Borrell et. al.,(2019)Reference set of Mycobacterium tuberculosis clinical strains: A tool for research and product development. PLoS ONE 14(3): e0214088. https: / / doi.org / 10.1371 / journal.pone.0214088). The H37Rv and HN878 strains used were single PDIM(+) clones isolated following passage through a C57BL / 6 mouse (H37Rv AE1001 and HN878 AE8001). n = 3, with the data presented as mean ± s.d., in Fig. 13. For some data points the s.d. is smaller than the data symbols.PA158379WO BERNEY7941PC

[0212] Propionate substantially increased the growth of all strains tested, as is seen inthe Figure, indicating that propionate supplementation is useful in a clinical setting, reducing diagnostic turnaround time.

[0213] Since propionate supplementation substantially increased the growth of all strains tested, it was of interest to determine whether propionate supplementation would improve the turnaround time for drug sensitivity testing and if propionate affected resistance to bedaquiline and rifampicin in other lineages.

[0214] Toward this end, MICs for four strains from different lineages were assessed according to the methods described in Example 1 herein. Optical density was measured after 7 and 14 days.

[0215] Referring to Fig. 14, Panels (A) demonstrate growth with and without propionate supplementation with further administration of rifampicin, for 7 days incubation, in the indicated strains; or Panels (B) where rifampicin treatment with 14 days is shown, Panels (C) where bedaquiline treatment for 7 days effects on growth with and without propionate supplementation is shown and in panels (D) bedaquiline treatment for 14 days effects on growth with and without propionate supplementation is shown. The dashed line indicates the baseline (no growth), with n = 2, data reflecting mean ± s.d. values. In each of these panels, for some data points the s.d. is smaller than the data symbols.

[0216] After only 7 days, remarkably, a robust MIC result could be determined for the propionate-supplemented media for all strains evaluated. In marked contrast, robust MIC results could not be determined as readily in the non-propionate-supplemented media due to poor bacterial growth.

[0217] This effect was most pronounced for the lineage 2 and lineage 4 strains, which are the most widely distributed lineages globally.

[0218] Propionate supplementation also increased rifampicin resistance of all strains (4- to 16-fold change in MIC90 on day 14).

[0219] The unique compositions, kits, and methods of this invention, therefore, have thepotential to reduce turnaround time for drug sensitivity testing and improve the reliability and robustness of results.

[0220] Furthermore, remarkably, the present invention supports results a more host-relevant result as Mtb has access to propionyl-CoA-generating carbon sources during infection.

Claims

PA158379WO BERNEY7941PC [00221]WHAT IS CLAIMED IS:

1. A method of rapid diagnosis of M. tuberculosis (Mtb) in a subject, the method comprising: (a) culturing a mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media, wherein said culturing is for a period of at least about 5 days and establishing growth of said mycobacterial strain; wherein growth is detected more rapidly in cultures conducted in the presence of propionate, thereby providing a more rapid diagnosis for Mtb infection in the subject.

2. The method of claim 1, further constituting a method of rapid determination of antibiotic sensitivity of Mtb in a subject, the method comprising the steps of: (b) contacting the cultured mycobacterial strain in (a) with at least one antibiotic for a period of from about 4 to 14 days; and (c) parallel culturing a second identical mycobacterial strain in a culture media supportive of mycobacterial growth in the absence of propionate and in the presence of the antibiotic; and (d) separately determining the MIC90 values for the mycobacterial strains in (b) and (c); wherein when the MIC value can be distinguished at an earlier timepoint in the strain obtained in (b) as compared to the strain obtained in (c), the method serves to provide a more rapid determination of antibiotic sensitivity of the mycobacterial strain.

3. The method of claim 1 or 2, wherein said mycobacterial strain is obtained from a sputum specimen.

4. The method of any one of claims 1-3, wherein said culture media is further supplemented with OADC and glycerol.

5. The method of any one of claims 1-4, wherein said culture media is further supplemented with pantothenate, arginine, leucine and methionine.

6. The method of any one of claims 1-5, wherein culturing said strain of interest comprises sample culture in the presence of 2 - 50 mg / ml vitamin B12.PA158379WO BERNEY7941PC 7. The method of any one of claims 1-6, wherein culturing said strain of interest comprises further sample culture in the presence of 0.1 mM cholesterol or 0.1 mM valerate.

8. The method of any one of claims 1-7, wherein culturing said strain of interest comprises sample culture in the presence of odd chain fatty acids.

9. A method of high throughput determination of PDIM production from a mycobacterial strain, said method comprising: · Culturing a PDIM reference strain set for a period of at least 5 days in mycobacteria growth supportive media wherein the PDIM reference strain set comprises a combination of at least one PDIM(+) and at least one PDIM(-) Mtb strain selected from the group of the following strains: mc26230 AE1601, mc26230 AE1611, H37Rv AE1028, H37Rv DppsD, mc26206, mc26209, mc27901, mc27902 or mc28398, wherein at least one of the PDIM(+) Mtb strains is optionally selected from the group consisting of mc26230 AE1601 and H37Rv AE1028, and at least one of the PDIM(-) Mtb strains is optionally selected from the group consisting of mc26230 AE1611 and H37Rv DppsD; · Culturing a strain of interest for a period of at least 5 days; · Further culturing parallel samples of said PDIM reference strain set and said strain of interest in media in the presence and absence of 5 - 50 mg / ml vancomycin, for a period of at least from 5 to 14 days and measuring the optical density at 590 – 600 nm; · Establishing relative growth of the PDIM reference strain set and the strain of interest in vancomycin as compared to drug-free controls, through the following formula: (VAN10 growth / VAN0 growth ´ 100 = VAN10 growth%); Wherein a VAN10 growth% value of below 10% is indicative of a strain which does not appreciably produce PDIM.

10. The method of claim 9, wherein said Mtb strains of said PDIM reference strain set may comprise a PDIM(+) Mtb strain selected from the group consisting of mc27902, mc26206 and mc26209 and a PDIM(-) Mtb strain selected from the group of mc28398 or mc27901.PA158379WO BERNEY7941PC 11. The method of claim 9, wherein culturing said strain of interest and culturing said PDIM reference strain set is for a period of at up to 14 days.

12. The method of claim 9, wherein culturing said strain of interest comprises further optional sample culture in the presence of 0.1 – 1.0 mM propionate.

13. The method of claim 9, wherein culturing said strain of interest comprises sample culture in the presence of 2 – 50 mg / ml vitamin B12.

14. The method of claim 9, wherein culturing said strain of interest comprises further sample culture in the presence of 0.1 mM cholesterol or 0.1 mM valerate.

15. The method of claim 9, wherein culturing said strain of interest comprises sample culture in the presence of odd chain fatty acids.

16. The method of claim9, wherein an increased VAN10 growth% value indicates the strain of interest is PDIM (+).

17. A kit for culture media supplementation for promoting, augmenting or maintaining PDIM production in a mycobacterial strain in vitro, wherein said kit comprises concentrated liquid or powdered propionate and instructions for reconstituting said culture media for supplementation to arrive at a final concentration of from 0.1 – 1.0 mM in the supplemented culture media.

18. The kit of claim 17, wherein said kit further comprises liquid or powdered 7H9 media components and instructions for reconstituting or preparing a final desired volume of 7H9 media for use.

19. The kit of claim 17, wherein said kit further comprises 7H10 media components and instructions for preparing solid 7H10 agar media for use.

20. The kit of claim 17, wherein said kit further comprises vitamin B12and instructions for preparing a final desired concentration of 10 mg / ml of the vitamin B12 in a final desired volume of culture media for use.

21. The kit of claim 17, wherein said kit further comprises pantothenate, arginine, leucine and methionine media components.

22. The kit of claim 17, wherein said kit further comprises tyloxapol.

23. The kit of any one of claims 17-22, further comprising glycerol, dimethylsulfoxide, or other cryoprotectants.PA158379WO BERNEY7941PC 24. A culture media promoting, augmenting or maintaining PDIM production in a mycobacterial strain in vitro, wherein said culture media is supplemented with propionate according to the instructions and making use of the kit of claim 8.

25. The culture media of claim 24, wherein said culture media comprises 7H9 liquid media.

26. The culture media of claim 24, wherein said culture media comprises 7H10 solid media.

27. The culture media of claim 24, wherein said culture media is further supplemented with OADC and glycerol.

28. The culture media of claim 24, wherein said media further comprises pantothenate, arginine, leucine and methionine.

29. A ready to use culture media for promoting, augmenting or maintaining PDIM production in a mycobacterial strain in vitro, wherein said culture media is supplemented with from 0.1 – 1.0 mM propionate.

30. The ready to use culture media of claim 29, wherein said culture media is further supplemented with OADC and glycerol.

31. The ready to use culture media of claim 29, wherein said culture media is further supplemented with pantothenate, arginine, leucine and methionine.

32. A method for preparing media for promoting, maintaining or augmenting PDIM production in a mycobacterial strain in culture, said method comprising culturing a mycobacterial strain in a culture media supportive of mycobacterial growth in culture for a period of at least 5 days, wherein said culture media has been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media.

33. The method of claim 32, wherein said culture period is from 5 – 14 days.

34. The method of claim 32, wherein said culture media is further supplemented with vitamin B12 achieving a final concentration of 2 – 50 mg / ml of vitamin B12 in said culture media.

35. The method of claim 32, wherein said culture media is further supplemented with 0.1 mM cholesterol or 0.1 mM valerate.

36. The method of claim 32, wherein said culture media comprises 7H9 media.

37. The method of claim 32, wherein said culture media is further supplemented with OADC and glycerol.PA158379WO BERNEY7941PC 38. The method of claim 32, wherein said culture media is further supplemented with pantothenate, arginine, leucine and methionine.

39. The method of claim 32, wherein said culture media is a liquid media.

40. The method of claim 32, wherein said culture media is a solid media.

41. The method of any one of claims 32-40, further comprising the step of culturing in parallel a PDIM reference strain set in media supportive of mycobacterial growth for a period of at least 5, wherein the PDIM reference strain set comprises a combination of at least one PDIM(+) and at least one PDIM(-) Mtb strain selected from the group consisting of the following strains: mc26230 AE1601, mc26230 AE1611, H37Rv AE1028, H37Rv DppsD, mc26206, mc26209, mc27901, mc27902 or mc28398, wherein at least one of the PDIM(+) Mtb strains is optionally selected from the group consisting of mc26230 AE1601 and H37Rv AE1028, and at least one of the PDIM(-) Mtb strains is optionally selected from the group consisting of mc26230 AE1611 and H37Rv DppsD.

42. The method of claim 41, further comprising the steps of: · Culturing the PDIM reference strain set and the mycobacteria strain for a period of at least 5 days in the presence and absence of 5 – 50 mg / ml vancomycin or 2 – 50 mg / ml vitamin B12, or 0.1 mM valerate, or 0.1 mM cholesterol and measuring the optical density at 590 – 600 nm; · Establishing relative growth of the PDIM reference strain set in vancomycin as compared to drug-free controls, through the following formula: (VAN10 growth / VAN0 growth ´ 100 = VAN10 growth%); · Establishing the growth of the mycobacterial strain cultured in the presence and absence of propionate, in vancomycin as compared to drug-free controls, through the following formula: (VAN10 growth / VAN0 growth ´ 100 = VAN10 growth%); wherein a VAN10 growth% value of below 10% is indicative that the media did not appreciably promote, maintain or augment PDIM production in the mycobacterial strain.

43. A method for improving mycobacterial strain PDIM production in vitro, the method comprising culturing the mycobacterial strain in a culture media supportive of mycobacterial growth for a period of at least 7 days, wherein said culture media has beenPA158379WO BERNEY7941PC supplemented with propionate to achieve a final concentration of 0.1 – 1 mM in the supplemented culture media.

44. The method of claim 43, wherein said culture period is from at least 7 to 14 days.

45. The method of claim 43, wherein said culture media is further supplemented with vitamin B12achieving a final concentration of 2 – 50 mg / ml of the vitamin B12in said culture media.

46. The method of claim 43, wherein said culture media comprises 7H9 liquid media.

47. The method of claim 43, wherein the culture media comprises 7H10 solid agar media.

48. The method of any one of claims 43-47, wherein said culture media is further supplemented with OADC and glycerol.

49. method of improving protective immune responses to mycobacterial vaccine strains in a host against mycobacterial infection and / or disease, the method comprising: (i) Culturing a mycobacterial vaccine strain in a culture media supportive of mycobacterial growth in culture for a period of at least 7 days, wherein said culture media has been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; (ii) Preparing a vaccine from the mycobacterial vaccine strain in (i) following the culture period; and (iii)Administering the vaccine in (ii) to a subject, wherein immune responses in the subject are improved as compared to immune responses achieved in the subject following administration with the initial vaccine strain in (i), administered without further culture in media supplemented with propionate.

50. The method of claim 49, wherein the culture period is from at least 7 to 14 days.

51. The method of claim 49, wherein the culture media is further supplemented with vitamin B12, achieving a final concentration of 2 – 50 mg / ml of the vitamin B12in said culture media.

52. The method of claim 49, wherein said culture media comprises 7H9 media.PA158379WO BERNEY7941PC 53. The method of claim 49, wherein said culture media is further supplemented with OADC and glycerol.

54. The method of claim 49, wherein the mycobacterial vaccine strain is BCG.

55. The method of claim 49, wherein the improved protective immune response improves cytotoxic T lymphocyte (CTL) responses.

56. A method of improving immunogenicity of a vaccine in a host, the method comprising: (i) Culturing a mycobacterial vaccine strain in a culture media supportive of mycobacterial growth in culture for a period of at least 5 to 14 days, wherein said culture media has been supplemented with propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; (ii) Preparing an adjuvant from the mycobacterial vaccine strain cultured according to (i); (iii) Combining a vaccine of interest with the adjuvant of (ii); and (iv) Administering the vaccine and adjuvant combination of (iii) to a subject; wherein immune responses in the subject are improved as compared to immune responses achieved in the subject following administration with the vaccine, administered in the absence of the adjuvant.

57. The method of claim 56, wherein said method improves CTL generation in the subject.

58. A method of improved determination of antibiotic sensitivity of a mycobacterial strain, improved antibiotic dosage determination for a subject, or a combination thereof, the method comprising: (a) culturing a first mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media and separately similarly culturing a second mycobacteria strain in the absence of the propionate, wherein said culturing is for a period of at least 5 days;PA158379WO BERNEY7941PC (b) further contacting said first mycobacteria strain and and said second mycobacteria strain with an antibiotic for a period of 7 to 14 days; (c) determining the MIC50, MIC90and MIC99values for the first and second mycobacterial strains in (b); wherein when any of the MIC values in the second mycobacterial strain are greater than in the first mycobacterial strain, the method serves to improve determination of antibiotic sensitivity of the mycobacterial strain, or the method serves to improve antibiotic dosage determination for the subject infected with the mycobacterial strain, or a combination thereof.

59. The method of claim 58, wherein the treatment regimen for the subject will comprise increasing the antibiotic dosage administered to the subject.

60. The method of claim 58, further comprising culturing the first and second mycobacteria strains in the presence of at least a second antibiotic in parallel and differentially determining the MIC values for the first and second mycobacterial strains following exposure to the first and second antibiotics.

61. The method of claim 58, wherein the method comprises administering the second antibiotic to the subject, assuming the MIC values are improved for the second antibiotic as compared to the first antibiotic.

62. The method of claim 58, wherein said culture period is from at least 7 to 14 days.

63. The method of claim 58, wherein said culture media is further supplemented with vitamin B12 achieving a final concentration of 10 mg / ml of the vitamin B12 in said culture media.

64. The method of claim 58, wherein said culture media comprises 7H9 media.

65. The method of claim 58, wherein said culture media is further supplemented with OADC and glycerol.

66. A method of improved determination of antibiotic sensitivity of a mycobacterial strain, improved antibiotic dosage determination for a subject, or a combination thereof, the method comprising: (a) culturing a first mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media and separatelyPA158379WO BERNEY7941PC similarly culturing a second identical mycobacterial strain in the absence of the propionate; (b) further contacting said first mycobacterial strain and said second identical mycobacterial strain with an antibiotic for a period of from about 5 to 10 days; (c) determining the minimum inhibitory concentration (MIC) values for MIC50, MIC90or MIC99for the first and second mycobacterial strains in (b), wherein when any of the MIC values in the first mycobacterial strain are obtained more rapidly or reliably than in the second mycobacterial strain, the method serves to improve determination of antibiotic sensitivity of the mycobacterial strain, or the method serves to improve antibiotic dosage determination for the subject infected with the mycobacterial strain, or a combination thereof.

67. method of more rapid determination of antibiotic sensitivity or determination of inhibitory antibiotic dosage for mycobacterial infection in a subject, or a combination thereof, the method comprising: (i) culturing a first mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; and (j) separately similarly culturing a second identical mycobacterial strain in the absence of the propionate; and (k) further contacting said first mycobacterial strain and said second identical mycobacterial strain with an antibiotic for a period of time; and (l) separately determining the minimum inhibitory concentration (MIC) values for MIC50, MIC90or MIC99for the first and second mycobacterial strains; wherein the MIC values in the first mycobacterial strain are obtained in at least 20% of the time necessary to obtain reliable MIC values in the second mycobacterial strain, thereby more rapidly determining antibiotic sensitivity or determining inhibitory antibiotic dosage for the subject infected with the mycobacterial strain, or a combination thereof.

68. A method of earlier diagnosis of mycobacterial infection in a subject, the method comprising:PA158379WO BERNEY7941PC (e) culturing a suspected mycobacterial strain isolated from a subject in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media; and (f) separately culturing the identical mycobacterial strain isolated from a subject in a culture media supportive of mycobacterial growth in culture in the absence of propionate; wherein mycobacterial strain growth is obtained more rapidly from said culture in (a) than in the culture in (b), thereby providing for earlier diagnosis of mycobacterial infection in a subject.

69. A method of rapid diagnosis of Mtb in a subject, the method comprising: (a) culturing a mycobacterial strain in a culture media supportive of mycobacterial growth in culture in the presence of propionate to achieve a final concentration of 0.1 – 1.0 mM in the supplemented culture media, wherein said culturing is for a period of at least from about 5 to about 14 days and establishing growth; wherein growth is detected more rapidly in cultures conducted in the presence of propionate, thereby providing a more rapid diagnosis for Mtb infection in the subject.

Images