Methods for determining siderophore beta-lactam susceptibility

Abstract

Disclosed herein are methods useful for determining the susceptibility of a bacterial strain to a siderophore beta-lactam in combination with a beta-lactamase inhibitor in an iron-containing growth medium. Disclosed herein are methods for determining the susceptibility of a bacterial strain to a siderophore beta-lactam in combination with a beta-lactamase inhibitor. In some embodiments, the method for determining the susceptibility of a bacterial strain to a siderophore beta-lactam comprises growing the bacterial strain in a growth medium having an iron concentration greater than 1 μM, treating the bacterial strain with a beta-lactamase inhibitor and a siderophore, and determining the minimum inhibitory concentration of the siderophore for the bacterial strain.

Description

[Technical Field]

[0001] Field of Disclosure The present disclosure relates to the fields of chemistry and medicine. More specifically, the present disclosure generally relates to methods for determining the susceptibility of siderophores in combination with beta-lactamase inhibitors. [Background technology]

[0002] background Iron in the growth medium can make it difficult to accurately measure the efficacy of siderophore beta-lactams. Some beta-lactams, such as cefiderocol, are transported into bacteria by siderophore transporters. Increased uptake increases drug concentrations at the target site and may overcome beta-lactamase-mediated resistance in some strains. Upregulation of siderophore transporters in bacteria under low iron conditions can increase the uptake of siderophore compounds, such as cefiderocol. When in vitro susceptibility testing of these drugs is performed under low iron conditions, minimum inhibitory concentration (MIC) values ​​determined in iron-restricted media are generally lower than those determined using standard in vitro test media. Studies in animal models of infection have shown that MIC values ​​measured under iron-restricted conditions correlate best with in vivo efficacy. Therefore, in vitro testing to determine the efficacy of siderophore beta-lactams (e.g., cefiderocol) in microbiology laboratories is often performed using iron-deficient test media.

[0003] However, the use of iron-restricted media is not standardized and can increase costs for clinical laboratories. Discrepancies have been reported even when quality control (QC) strains are used. Furthermore, some questions have been raised about whether the iron-deficient conditions under these test conditions mimic all in vivo situations.

[0004] Therefore, there is a need for an accurate siderophore susceptibility test that does not rely on the use of iron-deficient media.

[0005] Importantly, the effect of iron-restricted medium on siderophore beta-lactam (e.g., cefiderocol) susceptibility is particularly evident for beta-lactamase-producing bacterial strains and less pronounced for beta-lactamase-negative strains. For beta-lactamase-producing strains, the cefiderocol MIC in iron-restricted medium is nearly as low as that for strains lacking beta-lactamase. The rate of cefiderocol uptake into cells appears to be high enough to overwhelm the beta-lactamase and minimize its effect on the cefiderocol MIC. However, in standard medium, the cefiderocol MIC for beta-lactamase-producing strains is significantly higher than that for strains lacking beta-lactamase. In this case, the rate of cefiderocol entry is slower than the rate of hydrolysis by beta-lactamase, allowing the beta-lactamase to hydrolyze cefiderocol molecules as soon as they enter the cells, ensuring their effect on the cefiderocol MIC. As a result, for beta-lactamase-producing strains, the cefiderocol MIC in iron-deficient medium is significantly lower compared to standard medium. In contrast, in the absence of beta-lactamase, the cefiderocol MIC values ​​in iron-restricted medium and standard medium are very similar, despite the faster uptake rate of cefiderocol in iron-restricted medium.

[0006] Therefore, under conditions of inhibition of beta-lactamase activity (mimicking the absence of beta-lactamase), siderophore susceptibility testing may not depend on the use of iron-deficient media. Summary of the Invention [Means for solving the problem]

[0007] Disclosure Overview Disclosed herein is a method for determining the susceptibility of a bacterial strain to a siderophore beta-lactam in combination with a beta-lactamase inhibitor. In some embodiments, the method for determining the susceptibility of a bacterial strain to a siderophore beta-lactam includes growing the bacterial strain in a growth medium having an iron concentration greater than 1 μM, treating the bacterial strain with a beta-lactamase inhibitor and a siderophore, and determining the minimum inhibitory concentration of the siderophore for the bacterial strain.

[0008] In some embodiments, the beta-lactamase inhibitor is selected from the group consisting of zerborbactam, clavulanate, sulbactam, tazobactam, avibactam, vaborbactam, taniborbactam, ledaborbactam, relebactam, durrobactam, and zidebactam. In some embodiments, the beta-lactamase inhibitor is zerborbactam. In some embodiments, the beta-lactamase inhibitor is taniborbactam.

[0009] In some embodiments, the beta-lactamase inhibitor is [ka] In some embodiments, the beta-lactamase inhibitor is selected from the group consisting of: [ka] is.

[0010] In some embodiments, the siderophore is a cephalosporin. In some embodiments, the siderophore is cefiderocol.

[0011] In some embodiments, the bacterial strain is Enterobacterales, A. baumannii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Stenotrophomonas maltophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae、Neisseria meningitidis、Moraxella、Bacteroides fragilis、Bacteroides vulgatus、Bacteroides ovalus、Bacteroides thetaiotaomicron、Bacteroides uniformis、Bacteroideseggerthii, Bacteroides splanchnicus, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Burkholderia cepacia, Aeromonas hydrophilia, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter baumannii, Bordetella pertussis, Bordetella para pertussis, Bordetella bronchiseptica, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Borrelia burgdorferi, Kingella, Gardnerella vaginalis, Bacteroides distasonis, Bacteroides 3452A homologous group, Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes、Enterococcus faecalis、Enterococcus faecium、Staphylococcus aureus、Staphylococcus epidermidis、Staphylococcus saprophyticus、Staphylococcus intermedius、Staphylococcus hyicus subsp.hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus saccharolyticus.

[0012] In some embodiments, the growth medium is selected from the group consisting of tryptic soy, Mueller-Hinton, Ca-Mueller-Hinton, MacConkey, M9, Brucella broth, and Luria-Bertani (LB).

[0013] In some embodiments, the concentration of the siderophore ranges from about 0.05 μg / ml to about 32 μg / ml. In some embodiments, the concentration of the siderophore ranges from about 1 μg / ml to about 16 μg / ml. In some embodiments, the concentration of the beta-lactamase inhibitor ranges from about 2.0 mg / L to about 32 mg / L.

[0014] In some embodiments, the bacteria are grown in a growth medium at a temperature ranging from approximately 32°C to approximately 37°C.

[0015] In some embodiments, the bacteria are grown in a growth medium for at least about 24 hours before the MIC of the siderophore with the beta-lactamase inhibitor is determined. In some embodiments, the bacteria are grown in a growth medium for at least about 36 hours before the MIC of the siderophore with the beta-lactamase inhibitor is determined. In some embodiments, the bacteria are grown in a growth medium for at least about 48 hours before the MIC of the siderophore with the beta-lactamase inhibitor is determined.

[0016] In some embodiments, the iron concentration in the growth medium ranges from greater than 1 μM to approximately 50 μM, hi some embodiments, the iron concentration in the growth medium ranges from approximately 5 μM to approximately 20 μM.

[0017] In some embodiments, a method of treating a bacterial infection includes obtaining a sample comprising a bacterial strain from a subject having a bacterial infection; growing the bacterial strain in the presence of a growth medium having an iron concentration greater than 1 μM; treating the bacterial strain with a beta-lactamase inhibitor and a siderophore beta-lactam; determining a minimum inhibitory concentration of the siderophore beta-lactam for the bacterial strain; identifying whether the bacterial strain is susceptible to treatment with the beta-lactamase inhibitor and the siderophore beta-lactam based on the minimum inhibitory concentration; and if the bacterial strain is identified as susceptible, administering the beta-lactamase inhibitor and the siderophore beta-lactam to the subject. Includes:

[0018] Not all objectives set forth herein may necessarily be achieved in all embodiments disclosed and / or claimed herein. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description The following description provides context and examples, but should not be construed as limiting the scope of the present disclosure, which is covered by the claims following this specification or any other application claiming priority hereto. No single component or collection of components is required or essential. For example, in some embodiments, one or more variables may be omitted. Any feature, structure, component, material, step, or method described and / or illustrated in any embodiment herein can be used in conjunction with or in place of any feature, structure, component, material, step, or method described and / or illustrated in any other embodiment herein.

[0020] definition As used herein, and unless otherwise stated or implied by context, terms have the meanings defined below. The descriptions of the described embodiments and examples illustrate the present disclosure and are not intended to limit the disclosure in any way. Unless otherwise contraindicated or implied, for example, by including mutually exclusive elements or alternatives, in these definitions and throughout this specification, the terms "a" and "an" mean one or more, and the term "or" means and / or.

[0021] As used herein, "subject," "host," "patient," and "individual" are used interchangeably and shall have their normal meaning in the art, and also refer to organisms with cancer and / or leukemia. This includes mammals, such as humans, non-human primates, ungulates, dogs, cats, horses, mice, rats, etc. The term "mammal" includes both human and non-human mammals.

[0022] The terms "therapeutically effective amount" and "effective amount" refer to the amount of an active pharmaceutical ingredient required to provide the desired pharmacological result. In practice, the therapeutically effective amount will vary widely depending on the severity of the disease state, the age of the subject, and the desired therapeutic effect.

[0023] The terms "treatment," "treating," "treating," and the like shall have their ordinary meanings and, as used herein, shall encompass a general reference to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partial or complete stabilization or cure of the disease and / or adverse effects resulting from the disease. The term "treatment," as used herein, shall have its ordinary meaning and also encompasses any treatment of disease in a mammal, particularly a human, including (a) preventing a disease or condition from occurring in a subject who may be predisposed to the disease or condition but has not yet been diagnosed as having the disease or condition, (b) inhibiting a disease symptom, e.g., halting its development, and / or (c) alleviating a disease symptom, e.g., causing regression of the disease or condition.

[0024] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, with the acceptable error range depending in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, according to practice in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, and up to 1% of a given value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0025] All literature and similar materials cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. Where definitions of terms in incorporated references appear to differ from definitions provided in the present teachings, the definitions provided in the present teachings shall control. It is understood that the implicit use of "about" precedes temperatures, concentrations, times, etc. discussed in the present teachings, so that minor and insignificant deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "comprise," "comprises," "including," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is understood that both the general description and the following detailed description are exemplary and explanatory only and are not restrictive. The term "and / or" indicates that the provided possibilities can be used together or alternatively. Thus, the term "and / or" indicates that both options exist for the set of possibilities.

[0026] Terms and phrases used in this application, and particularly variations thereof in the appended claims, should be construed as open-ended rather than limiting, unless expressly stated otherwise. As examples of the foregoing, the term "including" should be interpreted to mean "including, but not limited to," "including, but not limited to," and the like; the term "including," as used herein, is synonymous with "including," "containing," or "featuring," is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps; the term "having" should be interpreted as "having at least," and the term "including" should be interpreted as "including, but not limited to," the term "examples" is used to provide illustrative instances of the items being discussed and is not intended to be exhaustive or limiting of the list; and the use of terms such as "preferably," "preferred," "desired," or "desirable," and words of similar import, should not be understood to imply that a particular feature is critical, essential, or even important to the structure or function of the invention, but instead is merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present disclosure. Furthermore, the term "comprising" should be interpreted synonymously with the phrases "having at least" or "at least including." When used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components. Similarly, a group of items joined by the conjunction "and" should not be construed as requiring every item in the group, but should be construed as "and / or" unless expressly stated otherwise.Similarly, groups of items joined by the conjunction "or" should not be construed as requiring mutual exclusivity between the groups, but should be construed as "and / or" unless expressly stated otherwise.

[0027] With respect to the use of virtually any plural and / or singular term herein, those skilled in the art can substitute plural for singular and / or singular for plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein. The indefinite article "a" or "an" does not exclude plural. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be advantageously used. Any reference signs in the claims should not be construed as limiting the scope.

[0028] Methods for determining minimum inhibitory concentrations Aspects of the present disclosure relate to methods for determining the susceptibility of a bacterial strain to a siderophore in combination with a beta-lactamase inhibitor. In some embodiments, the method for determining the susceptibility of a bacterial strain to a siderophore includes growing the bacterial strain in the presence of a growth medium having an iron concentration greater than 1.0 μM, treating the bacterial strain with a beta-lactamase inhibitor and a siderophore, and determining the minimum inhibitory concentration of the siderophore for the bacterial strain.

[0029] In some embodiments, the method may further include isolating a bacterial strain from a patient. In some embodiments, the method may further include isolating a bacterial strain from a patient, determining the siderophore susceptibility of the bacterial strain in combination with a beta-lactamase inhibitor, and administering a therapeutically effective amount of the siderophore and the beta-lactamase inhibitor to the patient.

[0030] It has been discovered that the need to use iron-deficient media to determine in vitro potency may be unnecessary for drugs that are combinations of a siderophore, such as the cephalosporin (SC) cefiderocol, and a beta-lactamase inhibitor (BLI), such as zerborbactam. Without being bound by any particular theory, the BLI inhibits low-efficiency beta-lactamases, thus reducing the dependency on increased drug uptake to overcome the effect of the beta-lactamase and the need for iron-deficient media to maximize transport. For resistant strains harboring highly efficient beta-lactamases, the MIC cannot be reduced by high uptake anyway, and therefore iron-deficient media is not necessary to start with such strains.

[0031] Thus, in some embodiments, for in vitro testing of the efficacy of SC BLI combinations, it is not necessary to use iron-deficient media, but rather standard test media can be used.

[0032] In some embodiments, the iron concentration in the growth medium is approximately 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 3.0 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10.0 μM, 11.0 μM, 12.0 μM, 13.0 μM, 14.0 μM, 15.0 μM, 16.0 μM, 17.0 μM, 18.0 μM, 19.0 μM, 20.0 μM, 21.0 μM, 22.0 μM, 23.0 μM, 24.0 μM, 25.0 μM, 26.0 μM, 27.0 μM, 28.0 μM, 29.0 μM, 30.0 μM, 31.0 μM, 32.0 μM, 33.0 μM, 34.0 μM, 35.0 μM, 36.0 μM, 37.0 μM, 38.0 μM, 39.0 μM, 40.0 μM, 41.0 μM, 42.0 μM, 43.0 μM, 44.0 μM, 45.0 μM, The iron concentration may be equal to or greater than 1.0 μM, 14.0 μM, 15.0 μM, 16.0 μM, 17.0 μM, 18.0 μM, 19.0 μM, 20.0 μM, 22.0 μM, 24.0 μM, 26.0 μM, 28.0 μM, 30.0 μM, 32.0 μM, 34.0 μM, 36.0 μM, 38.0 μM, 40.0 μM, 42.0 μM, 45.0 μM, 50.0 μM, 55.0 μM, 60.0 μM, 65.0 μM, 70.0 μM, 75.0 μM, or a range including and / or spanning the aforementioned values. For example, the iron concentration in the growth medium may range from approximately 1.0 μM to 50.0 μM.

[0033] In some embodiments, the siderophore is present in a concentration of approximately 0.025 μg / ml, 0.05 μg / ml, 0.1 μg / ml, 0.2 μg / ml, 0.3 μg / ml, 0.4 μg / ml, 0.5 μg / ml, 0.6 μg / ml, 0.7 μg / ml, 0.8 μg / ml, 0.9 μg / ml, 1.0 μg / ml, 1.1 μg / ml, 1.2 μg / ml, 1.3 μg / ml, 1.4 μg / ml, 1. 5μg / ml, 1.6μg / ml, 1.7μg / ml, 1.8μg / ml, 1.9μg / ml, 2.0μg / ml, 2.1μg / ml, 2.2μg / ml, 2.3μg / ml, 2.4μg / ml ml, 2.5μg / ml, 3.0μg / ml, 3.5μg / ml, 4.0μg / ml, 4.5μg / ml, 5.0μg / ml, 5.5μg / ml, 6.0μg / ml, 6.5μg / ml, 7 .0μg / ml, 7.5μg / ml, 8.0μg / ml, 8.5μg / ml, 9.0μg / ml, 9.5μg / ml, 10.0μg / ml, 11.0μg / ml, 12.0μg / ml, 13 .0μg / ml, 14.0μg / ml, 15.0μg / ml, 16.0μg / ml, 17.0μg / ml, 18.0μg / ml, 19.0μg / ml, 20.0μg / ml, 22.0μg / The siderophore may be present at a concentration equal to or ranging from approximately 0.05 μg / mL to approximately 32.0 μg / mL, 25.0 μg / mL, 30.0 μg / mL, 31.0 μg / mL, 32.0 μg / mL, 33.0 μg / mL, 2.5 μg / mL, 35.0 μg / mL, 40 μg / mL, 45.0 μg / mL, 50.0 μg / mL, 55.0 μg / mL, 60.0 μg / mL, 65.0 μg / mL, or a range including and / or spanning the aforementioned values. For example, the siderophore may be present at a concentration ranging from approximately 0.05 μg / mL to approximately 32.0 μg / mL.

[0034] In some embodiments, the siderophore is present at a concentration of approximately 1.0 μg / ml. In some embodiments, the siderophore is present at a concentration of approximately 2.0 μg / ml. In some embodiments, the siderophore is present at a concentration of approximately 4.0 μg / ml. In some embodiments, the siderophore is present at a concentration of approximately 8.0 μg / ml. In some embodiments, the siderophore is present at a concentration of approximately 16.0 μg / ml.

[0035] In some embodiments, the beta-lactamase inhibitor is at or below approximately 0.025 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 μg / ml, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2.0 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, 2.5 mg / L, 2.6 mg / L, 2.7 mg / L, 2 mg / L, 1.5mg / L, 1.6mg / L, 1.7mg / L, 1.8mg / L, 1.9mg / L, 2.0mg / L, 2.1mg / L, 2.2mg / L, 2.3mg / L, 2 .4mg / L, 2.5mg / L, 3.0mg / L, 3.5mg / L, 4.0mg / L, 4.5mg / L, 5.0mg / L, 5.5mg / L, 6.0mg / L, 6.5mg / L , 7.0mg / L, 7.5mg / L, 8.0mg / L, 8.5mg / L, 9.0mg / L, 9.5mg / L, 10.0mg / L, 11.0mg / L, 12.0mg / L, 1 3.0mg / L, 14.0mg / L, 15.0mg / L, 16.0mg / L, 17.0mg / L, 18.0mg / L, 19.0mg / L, 20.0mg / L, 22.0mg / 2.0 mg / L to 32.0 mg / L. For example, a beta-lactamase inhibitor may be present at a concentration equal to 2.0 mg / L, 25.0 mg / L, 30.0 mg / L, 31.0 mg / L, 32.0 mg / L, 33.0 mg / L, 2.5 mg / L, 35.0 mg / L, 40 mg / L, 45.0 mg / L, 50.0 mg / L, 55.0 mg / L, 60.0 mg / L, 65.0 mg / L, or a range including and / or spanning the aforementioned values. For example, a beta-lactamase inhibitor may be present at a concentration ranging from approximately 2.0 mg / L to approximately 32.0 mg / L.

[0036] In some embodiments, the beta-lactamase inhibitor is present at a concentration of approximately 2.0 mg / L. In some embodiments, the beta-lactamase inhibitor is present at a concentration of approximately 4.0 mg / L. In some embodiments, the beta-lactamase inhibitor is present at a concentration of approximately 8.0 mg / L. In some embodiments, the beta-lactamase inhibitor is present at a concentration of approximately 16.0 mg / L.

[0037] In some embodiments, the bacterial strain is grown in a growth medium at a temperature equal to or ranging from approximately 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C. For example, the bacterial strain may be grown in a growth medium at a temperature range from approximately 32° C. to approximately 37° C.

[0038] In some embodiments, the beta-lactamase inhibitor is [ka] or a pharmaceutically acceptable salt thereof.

[0039] In some embodiments, the beta-lactamase inhibitor is [ka] , which is also known as zerborbactam.

[0040] In some embodiments, the beta-lactamase inhibitor is selected from the group consisting of compounds described in the following patents and applications: U.S. Pat. No. 8,680,136, entitled "Cyclic Boronic Acid Ester Derivatives and Therapeutic Uses Thereof," U.S. Pat. No. 9,012,491, entitled "Heterocyclic Boronic Acid Ester Derivatives and Therapeutic Uses Thereof," U.S. Pat. No. 9,101,638, entitled "Boronic Acid Derivatives and Therapeutic Uses Thereof," U.S. Pat. No. 9,241,947, entitled "Boronic Acid Derivatives and Therapeutic Uses Thereof," U.S. Pat. No. 10,294,249, entitled "Boronic Acid Derivatives and Therapeutic Uses Thereof," U.S. Pat. No. 10,294,249, entitled "Cyclic Boronic Acid Ester ... No. 10,662,205 entitled "Boronic Acid Derivatives and Therapeutic Uses Thereof," U.S. Patent No. 9,963,467 entitled "Boronic Acid Derivatives and Therapeutic Uses Thereof," U.S. Patent No. 10,618,918 entitled "Substituted Boronic Acids as Antimicrobials," U.S. Patent Application No. 17 / 906,340 entitled "Boronic Acid Derivatives and Therapeutic Uses Thereof," filed September 14, 2022, U.S. Patent Application No. 10,206,937 entitled "Boronic Acid Derivatives and Therapeutic Uses Thereof," and U.S. Patent Application No. 17 / 048 entitled "Boronic Acid Derivatives and Therapeutic Uses Thereof," filed October 15, 2020,The beta-lactamase inhibitors may be selected from those disclosed in any of US Pat. Nos. 5,052,052, 5 ...

[0041] In some embodiments, the beta-lactamase inhibitor is selected from the group consisting of zerborbactam, clavulanate, sulbactam, tazobactam, avibactam, vaborbactam, taniborbactam, relebactam, zidebactam, and durrobactam. In some embodiments, the beta-lactamase inhibitor is zerborbactam. In some embodiments, the beta-lactamase inhibitor is taniborbactam.

[0042] In some embodiments, the siderophore is a cephalosporin. In some embodiments, the siderophore is selected from the group consisting of ceftolozane, ceftazidime, and cefiderocol. In some embodiments, the siderophore is cefiderocol.

[0043] In some embodiments, the bacterial strain is: enterobacterales, A. baumannii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Stenotrophomonas maltophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae、Neisseria meningitidis、Moraxella、Bacteroides fragilis、Bacteroides vulgatus、Bacteroides ovalus、Bacteroides thetaiotaomicron、Bacteroides uniformis、Bacteroideseggerthii, Bacteroides splanchnicus, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Burkholderia cepacia, Aeromonas hydrophilia, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter baumannii, Bordetella pertussis, Bordetella para pertussis, Bordetella bronchiseptica, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Borrelia burgdorferi, Kingella, Gardnerella vaginalis, Bacteroides distasonis, Bacteroides 3452A homologous group, Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes、Enterococcus faecalis、Enterococcus faecium、Staphylococcus aureus、Staphylococcus epidermidis、Staphylococcus saprophyticus、Staphylococcus intermedius、Staphylococcus hyicus subsp.hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus saccharolyticus.

[0044] In some embodiments, the growth medium is selected from the group consisting of tryptic soy, Mueller-Hinton, Ca-Mueller-Hinton, MacConkey, M9, Brucella broth, and Luria-Bertani (LB).

[0045] In some embodiments, the bacteria are grown in the growth medium for approximately 24 hours. In some embodiments, the bacteria are grown in the growth medium for approximately 36 hours. In some embodiments, the bacteria are grown in the growth medium for approximately 48 hours. In some embodiments, the bacterial strain is grown in the growth medium for a period equal to or including and / or spanning approximately 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, 72 hours, 90 hours, 108 hours, 144 hours, or a range including and / or spanning the aforementioned values. For example, the bacterial strain may be grown in the growth medium for a period ranging from approximately 24 hours to approximately 48 hours.

[0046] Compositions include those suitable for the aforementioned routes of administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods known in the art.

[0047] In some embodiments, the composition contains less than about 3% by weight of impurities.

[0048] Methods for Treating Bacterial Infections In some embodiments, methods of treating a bacterial infection are provided. In some embodiments, the methods include obtaining a sample containing a bacterial strain from a subject with a bacterial infection. The bacterial sample can be obtained from the subject in a variety of ways. In some embodiments, the bacterial sample is obtained from a blood sample. In some embodiments, the bacterial sample is obtained from a swab of a site suspected of having a bacterial infection. In some embodiments, the swab is a skin swab. In other embodiments, the swab is a swab of a mucous membrane or lining, such as an oral swab, buccal swab, ear swab, eye swab, or rectal swab. In some embodiments, the bacterial sample can be obtained from any of the subject's bodily fluids, such as a blood sample, saliva sample, urine sample, or stool sample.

[0049] In some embodiments, the bacterial sample may be obtained from a blood sample, wherein the blood is drawn from a vein. The vein may be from the inside of the elbow or the back of the hand of the subject. For example, a needle may be inserted into the vein, and the blood is collected in an airtight vial or syringe.

[0050] In some embodiments, the method also includes growing the bacterial strain in the presence of a growth medium. The bacterial strain may be grown, for example, by inoculating the growth medium with a bacterial sample and maintaining the growth medium, for example, at a temperature suitable to support growth of the bacterial strain for a suitable period of time.

[0051] The growth medium can be any growth medium known to those skilled in the art. In some embodiments, the growth medium is an agar, such as nutrient agar, tryptic soy agar, chocolate agar, Sayer-Martin agar, lactose agar, eosin-methylene blue agar, Hektoen agar, mannitol salt agar, triple sugar iron agar, or brain heart infusion agar. In other embodiments, the growth medium is tryptic soy, Mueller-Hinton, Ca-Muller-Hinton, MacConkey, M9, Brucella broth, or Luria-Bertani (LB). In some embodiments, the growth medium is an enriched growth medium, such as an agar growth medium enriched by the addition of blood, serum, or other nutrients.

[0052] In some embodiments, the growth medium has an iron concentration greater than 1 μM. Non-limiting examples of iron concentrations contemplated herein include 1.01 μM, 1.05 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 3.0 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10.0 μM, 11.0 μM, 12.0 μM, 13.0 μM, 14.0 μM, 15.0 μM, 16.0 μM, 17.0 μM, 18.0 μM, 19.0 μM, 20.0 μM, 21.0 μM, 22.0 μM, 23.0 μM, 24.0 μM, 25.0 μM, 26.0 μM, 27.0 μM, 28.0 μM, 29.0 μM, 30.0 μM, 31.0 μM, 32.0 μM, 33.0 μM, 34.0 μM, 35.0 μM, 36.0 μM, 37.0 μM, 38.0 μM, 39.0 μM, 40.0 μM, 41.0 μM, 42.0 μM, 43.0 μM, 44.0 μM, 45.0 μM, 46.0 μM, 47.0 μM, 48.0 μ 1.0 μM, 15.0 μM, 16.0 μM, 17.0 μM, 18.0 μM, 19.0 μM, 20.0 μM, 22.0 μM, 24.0 μM, 26.0 μM, 28.0 μM, 30.0 μM, 32.0 μM, 34.0 μM, 36.0 μM, 38.0 μM, 40.0 μM, 42.0 μM, 45.0 μM, 50.0 μM, 55.0 μM, 60.0 μM, 65.0 μM, 70.0 μM, 75.0 μM, or a range including and / or spanning the aforementioned values. For example, the iron concentration in the growth medium can range from approximately 1.01 μM to 50.0 μM.

[0053] In some embodiments, the method also includes treating the bacterial strain with a beta-lactamase inhibitor and a siderophore beta-lactam. In some embodiments, the concentration of the siderophore beta-lactam ranges from about 0.05 μg / ml to about 32 μg / ml. In some embodiments, the concentration of the siderophore beta-lactam ranges from about 1 μg / ml to about 16 μg / ml. In some embodiments, the concentration of the beta-lactamase inhibitor ranges from about 2.0 mg / L to about 32 mg / L.

[0054] In some embodiments, the method also includes determining the minimum inhibitory concentration (MIC) of a siderophore beta-lactam for the bacterial strain. The MIC can be determined in a number of ways. In some embodiments, the concentration of the beta-lactamase inhibitor is constant, while the concentration of the siderophore beta-lactam is varied to determine its MIC. For example, in some embodiments, the concentration of the beta-lactamase inhibitor is fixed at 2 μg / ml, 4 μg / ml, or 8 μg / ml, while the concentration of the siderophore beta-lactam is varied from 0.016 μg / ml to 32 μg / ml or greater. In some embodiments, the MIC is determined using a microbroth dilution method, in which a beta-lactamase inhibitor and a siderophore beta-lactam are used in a specific ratio, and the concentrations of both are gradually reduced in a well of liquid medium containing bacterial cells. For example, in some embodiments, a beta-lactamase inhibitor and a siderophore beta-lactam are mixed in a 1:1 ratio, with the concentrations of both agents varying from 0.016 μg / ml to 32 μg / ml or greater than 32 μg / ml.

[0055] In other embodiments, gradient methods may be used to determine MICs, for example, by using strips impregnated with defined concentrations of a beta-lactamase inhibitor and a siderophore beta-lactam.

[0056] In some embodiments, the method also includes identifying whether the bacterial strain is susceptible to treatment with a beta-lactamase inhibitor and a siderophore beta-lactam, where the identification is based at least in part on the determined MIC. In some embodiments, the MIC is compared to an average MIC obtained from a standard population of patients. In some embodiments, the MIC is compared to an MIC obtained from the same patient but using a different composition, such as a panel of standard antibiotics.

[0057] Generally, identifying whether a bacterial strain is susceptible to treatment with a beta-lactamase inhibitor and a siderophore beta-lactam involves comparing the determined MIC with a threshold MIC for the siderophore beta-lactam, also known as a breakpoint. The lower the MIC determined using a beta-lactamase inhibitor and a siderophore beta-lactam, the more susceptible the bacterial strain is to the beta-lactamase inhibitor and the siderophore beta-lactam. In some embodiments, a bacterial strain is identified as susceptible to treatment with a beta-lactamase inhibitor (e.g., zerborbactam) and a siderophore beta-lactam (e.g., cefiderocol) if the MIC for the siderophore beta-lactam is less than 8 μg / ml, less than 4 μg / ml, less than 2 μg / ml, or less than 1 μg / ml.

[0058] In some embodiments, the method also includes administering a beta-lactamase inhibitor and a siderophore beta-lactam to the subject if the bacterial strain is identified as susceptible to the beta-lactamase inhibitor and the siderophore beta-lactam. Administration of the beta-lactamase inhibitor and the siderophore beta-lactam and / or pharmaceutically acceptable salts thereof can be via any of the accepted modes of administration for agents providing similar benefits, including, but not limited to, oral, subcutaneous, intravenous, intranasal, topical, transdermal, intraperitoneal, intramuscular, pulmonary, vaginal, rectal, or intraocular. Oral and parenteral administration are conventional in treating the indications that are the subject of preferred embodiments. [Example]

[0059] The following examples are given for the purpose of illustrating various embodiments of the present disclosure and are not intended to limit the disclosure in any way. Those skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the aims and advantages stated, as well as to carry out the objects and obtain the aims and advantages inherent therein. Those skilled in the art will recognize modifications and other uses of the present disclosure that are encompassed within the spirit of the disclosure as defined by the claims.

[0060] Example 1 In this example, the in vitro activity of cefiderocol was evaluated alone against clinical isolates of Enterobacterales (N=99) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB).

[0061] Isolates were tested against cefiderocol alone by reference broth microdilution. Isolates were grown in ID-MHB at concentrations of <0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and >64 mg / L. Isolates were grown separately in Ca-MHB at concentrations of <0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and >64 mg / L. The minimum inhibitory concentration (MIC) of cefiderocol was then determined in each of these samples.

[0062] The number of strains with the indicated combination of MICs for ID-MHB and Ca-MHB is shown in Table 1, and the relative error rates for the samples are shown in Table 2. The error rates in Table 2 were calculated using iron concentrations of 4 μg / ml or less, 8 μg / ml, and 16 μg / ml or more for susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB. A "very major error" (false susceptible, false negative) indicates that the isolate was classified as susceptible when tested in standard medium (Ca-MHB) and as resistant when tested in ID-MHB. A "major error" (false resistant, false positive) indicates that the isolate was classified as resistant when tested in standard medium (Ca-MHB) and as susceptible when tested in ID-MHB. A "minor error" indicates that the isolate was classified as intermediate by one method and as resistant or susceptible by the other. Essential agreement was defined as an MIC value for Ca-MHB within the log2 dilution of the MIC result obtained with ID-MHB. Categorical agreement was defined as an S, I, or R interpretation for Ca-MHB that agreed with the ID-MHB result. [Table 1] [Table 2]

[0063] The data show that the minimum inhibitory concentrations (MICs) for cefiderocol showed some significant errors when tested alone. When tested alone, some strains that are sensitive in ID-MHB medium, such as Enterobacterales, appear to be insensitive in Ca-MHB medium.

[0064] Example 2 In this example, the in vitro activity of cefiderocol was evaluated alone against clinical isolates of A. baumannii (N=174) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB).

[0065] Isolates were tested against cefiderocol alone by reference broth microdilution. Isolates were grown in ID-MHB at concentrations of <0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and >64 mg / L. Isolates were grown separately in Ca-MHB at concentrations of <0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and >64 mg / L, and the minimum inhibitory concentration (MIC) of cefiderocol was then determined in each of these samples.

[0066] The number of strains with the indicated combination of MICs for ID-MHB and Ca-MHB is shown in Table 3, and the relative error rates for the samples are shown in Table 4. The error rates in Table 4 were calculated using ≤1 μg / ml, 2 μg / ml, and ≥4 μg / ml for S / I / R for Ca-MHB versus ID-MHB. Error rate determination was as defined in Example 1. [Table 3] [Table 4]

[0067] When tested with cefiderocol alone, some strains that are sensitive in ID-MHB medium, such as A. baumannii, appear to be insensitive in Ca-MHB medium.

[0068] Example 3 In this example, the in vitro activity of cefiderocol was evaluated in combination with zerborbactam at a concentration of 8 mg / L against clinical isolates of Enterobacterales (N=99) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB).

[0069] Isolates were tested by reference broth microdilution against cefiderocol in combination with zervorbactam at a concentration of 8 mg / L. Isolates were grown in ID-MHB at concentrations of less than 0.03 mg / L, 0.06 mg / L, 0.12 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, 64 mg / L, and greater than 64 mg / L. Isolates were grown separately in Ca-MHB at concentrations of <0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and >64 mg / L, and the minimum inhibitory concentration (MIC) of cefiderocol was then determined in each of these samples.

[0070] The number of strains with the indicated combinations of MICs for ID-MHB and Ca-MHB are shown in Table 5, and the relative error rates for the samples are shown in Table 6. The error rates in Table 6 were calculated using iron concentrations of 4 μg / ml or less, 8 μg / ml, and 16 μg / ml or more for sensitive, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB. Error rate determination was as defined in Example 1. [Table 5] [Table 6]

[0071] When cefiderocol was tested with zerborbactam, all strains appeared susceptible, whether ID-MHB or Ca-MHB was used. Ca-MHB medium, used as the standard, accurately predicted susceptibility in Enterobacterales (N = 99), and there were no falsely resistant or falsely susceptible isolates when comparing the results on the two media.

[0072] Example 4 Embodiments of the present application are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way.

[0073] In this example, the in vitro activity of cefiderocol was evaluated in combination with zerborbactam at a concentration of 8 mg / L against clinical isolates of A. baumannii (N=174) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB).

[0074] Isolates were tested by reference broth microdilution against cefiderocol in combination with zervorbactam at a concentration of 8 mg / L. Isolates were grown in ID-MHB at concentrations of less than 0.03 mg / L, 0.06 mg / L, 0.12 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, 64 mg / L, and greater than 64 mg / L. Isolates were grown separately in Ca-MHB at concentrations of <0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and >64 mg / L, and the minimum inhibitory concentration (MIC) of cefiderocol was then determined in each of these samples.

[0075] The number of strains with the indicated combination of MICs for ID-MHB and Ca-MHB is shown in Table 7, and the relative error rates for the samples are shown in Table 8. The error rates in Table 8 were calculated using ≤1 μg / ml, 2 μg / ml, and ≥4 μg / ml for S / I / R for Ca-MHB versus ID-MHB. Error rate determination was as defined in Example 1. [Table 7] [Table 8]

[0076] When cefiderocol was tested with zerborbactam, all strains appeared susceptible, regardless of whether ID-MHB or Ca-MHB was used. Ca-MHB medium, used as the standard, accurately predicted susceptibility and resistance in A. baumannii (N = 174), and when comparing results from the two media, there were no falsely resistant (major error) or falsely susceptible (very major error) isolates. Ten isolates, approximately 5.75% of the total, fell into the so-called minor error category: four I (intermediate MIC) in ID-MHB were S (susceptible MIC) in CA-MHB, two S (S) in ID-MHB were I in CA-MHB, and four R (resistant) in ID-MHB were I in MHB. The low number of errors resulted in a high category agreement rate (accurate prediction of resistant, intermediate, and susceptible MICs) of approximately 94.25%.

[0077] Example 5 In this example, the in vitro activity of cefiderocol was evaluated alone and in combination with zerborbactam at a concentration of 8 mg / L against clinical isolates of the Enterobacterales (N=164) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB).

[0078] Isolates were tested by reference broth microdilution. Isolates were grown in ID-MHB at cefiderocol concentrations of 0.016 mg / L, 0.03 mg / L, 0.06 mg / L, 0.12 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, and greater than 32 mg / L. Isolates were grown separately in Ca-MHB at cefiderocol concentrations of 0.016 mg / L, 0.03 mg / L, 0.06 mg / L, 0.12 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, and greater than 32 mg / L. The minimum inhibitory concentration (MIC) of cefiderocol was then determined in each of these samples.

[0079] Table 9 shows the number of strains with the indicated combinations of MICs for ID-MHB and Ca-MHB for cefiderocol alone. Table 10 shows the relative error rates for samples calculated using susceptible, intermediate, or resistant (S / I / R) iron concentrations of 4 μg / ml or less, 8 μg / ml, and 16 μg / ml or more for Ca-MHB versus ID-MHB. Error rate determination was as defined in Example 1.

[0080] Table 11 shows the number of strains with the indicated combinations of MICs for ID-MHB and Ca-MHB for cefiderocol in combination with zervorbactam at a concentration of 8 mg / L. Table 12 shows the relative error rates for samples calculated using iron concentrations of 4 μg / ml or less, 8 μg / ml, and 16 μg / ml or more for susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB. Error rate determination was as defined in Example 1. [Table 9] [Table 10]

[0081] The data show that the minimum inhibitory concentrations (MICs) for cefiderocol alone showed some significant error: some strains that were sensitive in ID-MHB medium when tested with cefiderocol alone appeared to be insensitive in Ca-MHB medium. [Table 11] [Table 12]

[0082] However, when cefiderocol is tested together with zerborbactam, the error rate is substantially reduced. Notably, all strains identified as susceptible in ID-MHB are also susceptible when using Ca-MHB. Ca-MHB medium, used as a standard, accurately predicted susceptibility in Enterobacterales, and there were no falsely resistant or falsely susceptible isolates when comparing results on the two media.

[0083] Example 6 In this example, the in vitro activity of cefiderocol was evaluated against clinical isolates of A. baumannii (N=160) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB), alone and in combination with zerborbactam at a concentration of 8 mg / L.

[0084] Isolates were tested by reference broth microdilution. Isolates were grown in ID-MHB at cefiderocol concentrations of 0.016 mg / L, 0.03 mg / L, 0.06 mg / L, 0.12 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, and greater than 32 mg / L. Isolates were grown separately in Ca-MHB at cefiderocol concentrations of 0.016 mg / L, 0.03 mg / L, 0.06 mg / L, 0.12 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, and greater than 32 mg / L. The minimum inhibitory concentration (MIC) of cefiderocol was then determined in each of these samples.

[0085] Table 13 shows the number of strains with the indicated combinations of MICs for ID-MHB and Ca-MHB for cefiderocol alone. Table 14 shows the relative error rates for samples calculated using iron concentrations of 4 μg / ml or less, 8 μg / ml, and 16 μg / ml or more for susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB. Table 15 shows the relative error rates for samples calculated using iron concentrations of 2 μg / ml or less, 4 μg / ml, and 8 μg / ml or more for susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB. Error rate determination was as defined in Example 1.

[0086] Table 16 shows the number of strains with the indicated combinations of MICs for ID-MHB and Ca-MHB for cefiderocol in combination with zervorbactam at a concentration of 8 mg / L. Table 17 shows the relative error rates for samples calculated using iron concentrations of susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB of 4 μg / ml or less, 8 μg / ml, and 16 μg / ml or more. Table 18 shows the relative error rates for samples calculated using iron concentrations of susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB of 2 μg / ml or less, 4 μg / ml, and 8 μg / ml or more. Error rate determination was as defined in Example 1. [Table 13] [Table 14] [Table 15]

[0087] The data show that the minimum inhibitory concentrations (MICs) for cefiderocol alone showed some significant error: some strains that were sensitive in ID-MHB medium when tested with cefiderocol alone appeared to be insensitive in Ca-MHB medium. [Table 16] [Table 17] [Table 18]

[0088] When cefiderocol was tested with zerborbactam, and relative error rates were calculated using iron concentrations of ≤4 μg / ml, ≥8 μg / ml, and ≥16 μg / ml for susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB, Ca-MHB medium accurately predicted susceptibility in A. baumannii, and there was no error when comparing results on the two media.

[0089] When cefiderocol was tested with zerborbactam, and relative error rates were calculated for susceptible, intermediate, or resistant (S / I / R) iron concentrations for Ca-MHB versus ID-MHB at ≤2 μg / ml, ≥4 μg / ml, and ≥8 μg / ml, Ca-MHB medium accurately predicted susceptibility in A. baumannii. There were no falsely resistant (major error) or falsely susceptible (very major error) isolates when comparing the results for the two media. Two isolates, approximately 1.25% of the total number of isolates, fell into so-called minor errors. With a low number of errors, the categorical agreement rate for accurate prediction of resistant, intermediate, and susceptible MICs was high, approximately 98.8%.

[0090] Example 7 In this example, the in vitro activity of cefiderocol was evaluated against clinical isolates of Pseudomonas aeruginosa (N=156) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB), alone and in combination with zerborbactam at a concentration of 8 mg / L.

[0091] Isolates were tested by reference broth microdilution. Isolates were grown in ID-MHB at cefiderocol concentrations of 0.016 mg / L, 0.03 mg / L, 0.06 mg / L, 0.12 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, and greater than 32 mg / L. Isolates were grown separately in Ca-MHB at cefiderocol concentrations of 0.016 mg / L, 0.03 mg / L, 0.06 mg / L, 0.12 mg / L, 0.25 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, 32 mg / L, and greater than 32 mg / L. The minimum inhibitory concentration (MIC) of cefiderocol was then determined in each of these samples.

[0092] Table 19 shows the number of strains with the indicated combinations of MICs for ID-MHB and Ca-MHB for cefiderocol alone. Table 20 shows the relative error rates for samples calculated using iron concentrations of ≤4 μg / ml, ≤8 μg / ml, and ≥16 μg / ml for susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB. Table 21 shows the relative error rates for samples calculated using iron concentrations of ≤2 μg / ml, ≤4 μg / ml, and ≥8 μg / ml for susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB. Error rate determination was as defined in Example 1.

[0093] Table 22 shows the number of strains with the indicated combinations of MICs for ID-MHB and Ca-MHB for cefiderocol in combination with zervorbactam at a concentration of 8 mg / L. Table 23 shows the relative error rates for samples calculated using iron concentrations of susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB of 4 μg / ml or less, 8 μg / ml, and 16 μg / ml or more. Table 24 shows the relative error rates for samples calculated using iron concentrations of susceptible, intermediate, or resistant (S / I / R) for Ca-MHB versus ID-MHB of 2 μg / ml or less, 4 μg / ml, and 8 μg / ml or more. Error rate determination was as defined in Example 1. [Table 19] [Table 20] [Table 21]

[0094] The data show that the minimum inhibitory concentrations (MICs) for cefiderocol alone showed some significant error: some strains that were sensitive in ID-MHB medium when tested with cefiderocol alone appeared to be insensitive in Ca-MHB medium. [Table 22] [Table 23] [Table 24]

[0095] When cefiderocol was tested with zerborbactam, and relative error rates were calculated for susceptible, intermediate, or resistant (S / I / R) iron concentrations for Ca-MHB versus ID-MHB at ≤4 μg / ml, ≥8 μg / ml, and ≥16 μg / ml, Ca-MHB medium accurately predicted susceptibility in Pseudomonas aeruginosa. When comparing results for the two media, one falsely resistant (major error) isolate was found, and no falsely susceptible (very major error) isolates were found. One isolate, approximately 0.64% of the total number of isolates, fell into a so-called minor error category. With a low number of errors, the categorical agreement rate for accurate prediction of resistant, intermediate, and susceptible MICs was high, approximately 98.7%.

[0096] When cefiderocol was tested with zerborbactam, and relative error rates were calculated for susceptible, intermediate, or resistant (S / I / R) iron concentrations for Ca-MHB versus ID-MHB at ≤2 μg / ml, 4 μg / ml, and ≥8 μg / ml, Ca-MHB medium accurately predicted susceptibility in Pseudomonas aeruginosa. Comparing the results for the two media, there was one falsely resistant (major error) isolate and no falsely susceptible (very major error) isolates. Five isolates, approximately 5.13% of the total number of isolates, fell into so-called minor errors. With a low number of errors, the categorical agreement rate for accurate prediction of resistant, intermediate, and susceptible MICs was high, approximately 94.2%.

[0097] Example 8 In this example, the in vitro activity of cefiderocol was evaluated against carbapenem-resistant Enterobacterales clinical isolates (N=99) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB), both alone and in combination with zerborbactam at a concentration of 8 mg / L.

[0098] Clinical isolates were tested by reference broth microdilution against both cefiderocol alone and cefiderocol in combination with zerborbactam at a concentration of 8 mg / L. Clinical isolates were grown in ID-MHB and Ca-MHB, and the panel of isolates was represented by NDM (New Delhi metallo-beta-lactamase) beta-lactamase-producing strains (N = 55), NDM-negative strains (N = 44), and carbapenem-resistant Enterobacterales strains (N = 82) with cefiderocol MICs of 4 μg / mL or higher. The minimum inhibitory concentration (MIC) of cefiderocol was then determined for each of these samples.

[0099] Activity data for cefiderocol alone and in combination with zerborbactam are shown in Table 25.

[0100] [Table 25]

[0101] Zervorbactam significantly increased the efficacy of cefiderocol against cefiderocol-resistant isolates of carbapenem-resistant Enterobacterales in either standard or iron-supplemented media.

[0102] Example 9 In this example, the in vitro activity of cefiderocol was evaluated both alone and in combination with zerborbactam at a concentration of 8 mg / L against carbapenem-resistant A. baumannii clinical isolates (N=174) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB).

[0103] Clinical isolates were tested by reference broth microdilution against both cefiderocol alone and in combination with zerborbactam at a concentration of 8 mg / L. Clinical isolates were grown in ID-MHB and Ca-MHB with representative strains of NDM (N = 17), PER (pseudomonas extended resistant) (N = 36), and carbapenem-resistant A. baumannii (N = 131) with cefiderocol MICs of 1 μg / mL or higher, and without MBL (metallo-β-lactamase) (N = 157). The minimum inhibitory concentration (MIC) of cefiderocol was then determined for each of these samples.

[0104] Activity data for cefiderocol alone and in combination with zerborbactam are shown in Table 26. [Table 26]

[0105] Zervorbactam significantly increased the efficacy of cefiderocol against cefiderocol-resistant isolates of carbapenem-resistant A. baumannii. Cefiderocol efficacy was significantly increased by zervorbactam in standard or iron-supplemented medium.

[0106] Example 10 In this example, the in vitro activity of cefiderocol was evaluated both alone and in combination with various concentrations (0–8 mg / L) of zerborbactam (“xeru”) against clinical isolates of the Enterobacterales (N=164) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB).

[0107] Clinical isolates were tested by reference broth microdilution against cefiderocol alone and in combination with various concentrations of zerborbactam (0-8 mg / L). Clinical isolates were grown in ID-MHB and Ca-MHB, and a panel of isolates was represented with MBL (metallo-β-lactamase) (N = 74) and without MBL (N = 90) as representative strains. The minimum inhibitory concentration (MIC) of cefiderocol was then determined in each of these samples.

[0108] Activity data for cefiderocol alone and in combination with zerborbactam are shown in Table 27. [Table 27]

[0109] Zervorbactam significantly increased the efficacy of cefiderocol against Enterobacterales. Cefiderocol efficacy was significantly increased by zervorbactam in both standard and iron-deficient media.

[0110] Example 11 In this example, the in vitro activity of cefiderocol was evaluated both alone and in combination with various concentrations (0–8 mg / L) of zerborbactam against clinical isolates of A. baumannii (N=160) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB).

[0111] Clinical isolates were tested by reference broth microdilution against cefiderocol alone and in combination with zerborbactam at various concentrations (0–8 mg / L). Clinical isolates were grown in ID-MHB and Ca-MHB. The panel of isolates was represented by representative strains: PER (Pseudomonas broad-spectrum resistant) (N = 58), NDM (New Delhi metallo-beta-lactamase) beta-lactamase producers (N = 22), NDM-negative strains (N = 138), and acquired OXA-carbapenemase (PER, NDM, VEB, no GES) (N = 41) (OXA = oxacillinase, VEB = Vietnamese extended-spectrum beta-lactamase). The minimum inhibitory concentration (MIC) of cefiderocol was then determined for each of these samples.

[0112] Activity data for cefiderocol alone and in combination with zerborbactam are shown in Table 28. [Table 28]

[0113] The efficacy of cefiderocol was significantly increased by zerborbactam in both standard and iron-deficient media.

[0114] Example 12 In this example, the in vitro activity of cefiderocol was evaluated both alone and in combination with various concentrations (0–8 mg / L) of zerborbactam against clinical isolates of Pseudomonas aeruginosa (N=156) in both iron-deficient Mueller-Hinton broth (ID-MHB) and reference Mueller-Hinton broth (Ca-MHB).

[0115] Clinical isolates were tested by reference broth microdilution against cefiderocol alone and in combination with zerborbactam at various concentrations (0–8 mg / L). Clinical isolates were grown in ID-MHB and Ca-MHB, and the panel of isolates was represented by representative strains: PER (Pseudomonas broad-spectrum resistant) (N = 21), VEB (Vietnam-type extended-spectrum beta-lactamase) (N = 21), MBL (metallo-beta-lactamase) (N = 80), VIM (Verona integron-encoded metallo-beta-lactamase) (N = 53), and IMP (imipenemase) (N = 23). The minimum inhibitory concentration (MIC) of cefiderocol was then determined for each of these samples.

[0116] Activity data for cefiderocol alone and in combination with zerborbactam are shown in Table 29. [Table 29]

[0117] The efficacy of cefiderocol was significantly increased by zerborbactam in both standard and iron-deficient media.

[0118] Although some embodiments have been illustrated and described, one of ordinary skill in the art, after reading the foregoing specification, can effect variations, equivalent substitutions, and other types of modifications to the compounds of the present technology as described herein, or their salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers, or racemic mixtures. Also, each of the above-described aspects and embodiments may be encompassed or incorporated with variations or aspects as disclosed with respect to any or all of the other aspects and embodiments.

[0119] The present technology should also not be limited by the specific embodiments described herein, which are intended as separate descriptions of individual embodiments of the technology. Many modifications and variations of the present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the appended claims. It is understood that the present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds, or biological systems, which can, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Therefore, it is intended that the specification be considered merely exemplary, with the breadth, scope, and spirit of the technology being indicated only by the appended claims, the definitions therein, and any equivalents thereof.

[0120] The embodiments illustratively described herein may suitably be practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" should be interpreted broadly and not limitingly. Furthermore, the terms and expressions used herein are used as terms of description and not as terms of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or portions thereof, and it will be recognized that various modifications are possible within the scope of the claimed technology. Furthermore, the phrase "consisting essentially of" will be understood to include the specifically recited elements as well as additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase "consisting of" excludes any elements not specified.

[0121] Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, one of skill in the art will recognize that the present disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings included within the generic disclosure also form part of the present technology. This includes the generic description of the technology with a condition or negative limitation removing any subject matter from the genus, regardless of whether the deleted material is specifically recited herein.

[0122] All publications, patent applications, issued patents, and other documents (e.g., journals, articles, and / or texts) referenced herein are incorporated by reference herein as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in text incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

[0123] Other embodiments are set forth in the following claims, to which such claims are entitled, along with their full range of equivalents.

[0124] While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and detail can be made in the embodiments without departing from the spirit and scope of the invention.

[0125] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0126] While the present invention has been described with reference to embodiments and examples, it should be understood that numerous and various modifications can be made without departing from the spirit of the invention. Accordingly, the present invention is limited only by the scope of the following claims.

Claims

1. 1. A method for determining the susceptibility of a bacterial strain to a siderophore beta-lactam, comprising: Growing the bacterial strain in the presence of a growth medium having an iron concentration greater than 1 μM; treating the bacterial strain with a beta-lactamase inhibitor and the siderophore beta-lactam; determining the minimum inhibitory concentration of said siderophore for said bacterial strain; A method comprising:

2. The beta-lactamase inhibitor is 【Transformation 5】 10. The method of claim 1, wherein the compound is selected from the group consisting of:

3. The beta-lactamase inhibitor is 【Transformation 6】 The method of claim 1, wherein

4. 2. The method of claim 1, wherein the beta-lactamase inhibitor is selected from the group consisting of zerborbactam, clavulanate, sulbactam, tazobactam, avibactam, vaborbactam, taniborbactam, relebactam, durrobactam, and didebactam.

5. 2. The method of claim 1, wherein the beta-lactamase inhibitor is taniborbactam.

6. The method of any one of claims 1 to 5, wherein the siderophore beta-lactam is a cephalosporin.

7. 6. The method of any one of claims 1 to 5, wherein the siderophore is cefiderocol.

8. The bacterial strain is from Enterobacterales, A. baumannii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Stenotrophomonas maltophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahemolyticus, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii、Acteroides splanchnicus、Pseudomonas acidovorans、Pseudomonas alcaligenes、Pseudomonas putida、Burkholderia cepacia、Aeromonas hydreophilia、Francisella tularensis、Morganella morganii、Proteus mirabilis、Proteus vulgaris、Proteus vulgaris alcalifaciens、Providentia rettgeri、rovidentia stuartii、Acinetobacter baumannii、Bordetella pertussis、Bordetella para pertussis、Bordetella bronchiseptica、Haemophilus ducreyi、Pasteurella multocida、Pasteurella haemolytica、Branhamella Cataracts, diseases burgdorferi, Kingella, Gardnerella vaginalis, Bacteroides distasonis, Bacteroides 3452A-like, Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium、Sta･ylococcus aureus、Staombococcus epidermidis、Staomboccus saprooyticus、Staomboccus intermediate Subs.

8. The method of any one of claims 1 to 7, wherein the bacterial strain is selected from the group consisting of Staphylococcus hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus saccharolyticus.

9. 9. The method of any one of claims 1 to 8, wherein the growth medium is selected from the group consisting of tryptic soy, Mueller-Hinton, Ca-Mueller-Hinton, MacConkey, M9, Brucella broth, and Luria-Bertani (LB).

10. 10. The method of any one of claims 1 to 9, wherein the concentration of the siderophore ranges from approximately 0.05 μg / ml to approximately 32 μg / ml.

11. 11. The method of any one of claims 1 to 10, wherein the concentration of the siderophore ranges from approximately 1 μg / ml to approximately 16 μg / ml.

12. 12. The method of any one of claims 1 to 11, wherein the concentration of the beta-lactamase inhibitor ranges from approximately 2.0 mg / L to approximately 32 mg / L.

13. 13. The method of any one of claims 1 to 12, wherein the bacteria is grown in the growth medium at a temperature in the range of approximately 32°C to approximately 37°C.

14. 14. The method of any one of claims 1 to 13, wherein the bacteria is allowed to grow in the growth medium for at least approximately 24 hours before the MIC is determined.

15. 15. The method of any one of claims 1 to 14, wherein the bacteria is allowed to grow in the growth medium for at least approximately 36 hours before the MIC is determined.

16. 16. The method of any one of claims 1 to 15, wherein the bacteria is allowed to grow in the growth medium for at least approximately 48 hours before the MIC is determined.

17. 17. The method of any one of claims 1 to 16, wherein the iron concentration in the growth medium ranges from greater than 1 μM to approximately 50 μM.

18. 18. The method of any one of claims 1 to 17, wherein the iron concentration in the growth medium ranges from approximately 5 μM to approximately 20 μM.

19. 1. A method of treating a bacterial infection, comprising: obtaining a sample comprising a bacterial strain from a subject having said bacterial infection; Growing the bacterial strain in the presence of a growth medium having an iron concentration greater than 1 μM; treating the bacterial strain with a beta-lactamase inhibitor and a siderophore beta-lactam; determining the minimum inhibitory concentration of the siderophore beta-lactam for the bacterial strain; Identifying whether the bacterial strain is susceptible to treatment with the beta-lactamase inhibitor and the siderophore beta-lactam based on the minimum inhibitory concentration; If the bacterial strain is identified as susceptible, administering the beta-lactamase inhibitor and the siderophore beta-lactam to the subject. A method comprising:

20. The beta-lactamase inhibitor is 【Transformation 7】 20. The method of claim 19, wherein the compound is selected from the group consisting of:

21. The beta-lactamase inhibitor is 【Transformation 8】 20. The method of claim 19, wherein:

22. 20. The method of claim 19, wherein the beta-lactamase inhibitor is selected from the group consisting of zerborbactam, clavulanate, sulbactam, tazobactam, avibactam, vaborbactam, taniborbactam, relebactam, durrobactam, and didebactam.

23. 20. The method of claim 19, wherein the beta-lactamase inhibitor is taniborbactam.

24. 24. The method of any one of claims 19 to 23, wherein the siderophore beta-lactam is a cephalosporin.

25. 25. The method of any one of claims 19 to 24, wherein the siderophore is cefiderocol.

26. The bacterial strain is Enterobacterales, A. baumannii, Pseudomonas aeruginosa, Pseudomonas fluorescens, Stenotrophomonas maltophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahemolyticus, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii、Acteroides splanchnicus、Pseudomonas acidovorans、Pseudomonas alcaligenes、Pseudomonas putida、Burkholderia cepacia、Aeromonas hydreophilia、Francisella tularensis、Morganella morganii、Proteus mirabilis、Proteus vulgaris、Proteus vulgaris alcalifaciens、Providentia rettgeri、rovidentia stuartii、Acinetobacter baumannii、Bordetella pertussis、Bordetella para pertussis、Bordetella bronchiseptica、Haemophilus ducreyi、Pasteurella multocida、Pasteurella haemolytica、Branhamella Cataracts, diseases burgdorferi, Kingella, Gardnerella vaginalis, Bacteroides distasonis, Bacteroides 3452A-like, Clostridium difficile, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium、Sta･ylococcus aureus、Staombococcus epidermidis、Staomboccus saprooyticus、Staomboccus intermediate Subs.

26. The method of any one of claims 19 to 25, wherein the bacterial strain is selected from the group consisting of Staphylococcus hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus saccharolyticus.

27. 27. The method of any one of claims 19 to 26, wherein the growth medium is selected from the group consisting of tryptic soy, Mueller-Hinton, Ca-Mueller-Hinton, MacConkey, M9, Brucella broth, and Luria-Bertani (LB).

28. 28. The method of any one of claims 19 to 27, wherein the concentration of the siderophore ranges from approximately 0.05 μg / ml to approximately 32 μg / ml.

29. 29. The method of any one of claims 19 to 28, wherein the concentration of the siderophore ranges from approximately 1 μg / ml to approximately 16 μg / ml.

30. 30. The method of any one of claims 19 to 29, wherein the concentration of the beta-lactamase inhibitor ranges from approximately 2.0 mg / L to approximately 32 mg / L.

31. 31. The method of any one of claims 19 to 30, wherein the bacteria is grown in the growth medium at a temperature in the range of approximately 32°C to approximately 37°C.

32. 32. The method of any one of claims 19 to 31, wherein the bacteria is allowed to grow in the growth medium for at least approximately 24 hours before the MIC is determined.

33. 33. The method of any one of claims 19 to 32, wherein the bacteria is allowed to grow in the growth medium for at least approximately 36 hours before the MIC is determined.

34. 34. The method of any one of claims 19 to 33, wherein the bacteria is allowed to grow in the growth medium for at least approximately 48 hours before the MIC is determined.

35. 35. The method of any one of claims 19 to 34, wherein the iron concentration in the growth medium ranges from greater than 1 μM to approximately 50 μM.

36. 36. The method of any one of claims 19 to 35, wherein the iron concentration in the growth medium ranges from approximately 5 μM to approximately 20 μM.

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