Method to detect contamination with antibiotic metabolites and method to reduce or avoid development of antibiotic resistance

Abstract

A method to detect an antibiotic metabolite in the environment is provided, comprising assaying a sample from the environment for the metabolite. Further provided is a method to reduce or prevent the development of antibiotic resistance, the method comprising assaying a sample from a food or beverage product, or a sample from the environment, for the presence of one or more antibiotic or pharmaceutical metabolites, wherein when the assay indicates presence of the one or more metabolites, the method further comprises one or more of the following steps: removing or degrading the metabolite from the environment or the product source or environment, implementing metabolite capture system, and preventing entry of the metabolite into the environment or the product. Further provided is a method to validate an antibiotic or pharmaceutical metabolite capture system.

Description

[0001] Title of the Invention

[0002] METHOD TO DETECT CONTAMINATION WITH ANTIBIOTIC METABOLITES AND METHOD TO REDUCE OR AVOID DEVELOPMENT OF ANTIBIOTIC RESISTANCE

[0003] Field of the Invention

[0004] The current invention relates to a method to detect and / or monitor contamination by an antibiotic metabolite(s) in an environmental sample. To this end, the current invention further relates to a method to reduce, or avoid, development of antibiotic resistance in bacteria.

[0005] Background of the Invention

[0006] Bacterial antimicrobial resistance (AMR), also known as antibiotic resistance, occurs when bacteria develop the ability to resist, or combat, previously effective antibiotic treatments (World Health Organisation. Interagency Coordination Group on Antimicrobial Resistance. No Time to Wait: Securing the Future from Drug-Resistant Infections. World Health Organisation, 2019). AMR is one of the major global healthcare challenges. Bacterial AMR was attributed to over 1.2 million fatalities in 2019 (Murray, C, et al., The Lancet, 2022, 399 (10325), 629-655). AMR has the possibility of negating all of the huge medical advances made in the last 50 years, including organ transplantation, cancer treatments and joint replacement surgeries.

[0007] Bacteria exposed to sub-inhibitory concentrations (SIC) of antibiotics can develop resistance. There is a large amount of published literature on the effects of antibiotics at various concentration levels on the development of resistance and tolerance to antibiotics. For example, there are many literature reports on the development of resistance, by Gram-positive and Gram-negative bacteria, to the commonly used antibiotics ciprofloxacin and sulfamethoxazole (C. Sinel et al., Antimicrob Agents Chemother. 2017, 61 ; E. Griffith et al., Front. Microbiol, 2018).

[0008] When antibiotics are taken by humans or animals, many are metabolised in the body and these metabolites are excreted into wastewater systems and the wider environment, e.g. in water treatment plants, rivers, lakes, soils and on agricultural lands. For example, spreading of slurry on the land can allow the antibiotics and their veterinary metabolites to enter rivers and streams, etc. Furthermore, the breakdown of the antibiotics compounds in the environment leads to a larger set of metabolites called Environmental Transformation Products (ETPs).

[0009] By their very nature most of these metabolites are structurally related to the parent antibiotic from which they are derived. There are reports on the identification of metabolites of these antibiotics and whether they show any bioactivity, i.e. whether they also have any antibacterial activity (G. Suaifan, et al., Bioorg. Med. Chem, 2019, 27, 3005). Some of the metabolites are known to have antibacterial activities themselves while others have been described as inactive. Although it is known that sub-clinical doses of antibiotics causes the development of resistance in bacteria, the ability of metabolites to cause resistance to the antibiotic itself, i.e. , the parent antibiotic from which it is derived, has not been previously reported.

[0010] In Ireland, the entry of antibiotics and their metabolites into the environment is a crucial health issue. Many of the same antibiotics used in human health are also used as veterinary therapeutics, which exacerbates the problem. Although the levels of many antibiotics are monitored, there is currently no system in place for monitoring their metabolites.

[0011] For the dairy industry, the use of antibiotics is carefully controlled, where rigorous testing has to take place to make sure the milk being processed has antibiotic residue levels below the allowed concentrations. Within this testing regime there is currently no requirement for screening for levels of any bovine antibiotic metabolites.

[0012] Several groups have discussed measuring antibiotics and antibiotic metabolites in samples, such as milk samples and water, but no group reports the association between the existence of antibiotic metabolites in the environment and the development of resistance to the parent antibiotic in bacteria or any means for tackling this issue.

[0013] D Fabregat-Safont et al., (Chemosphere 2023, 337) discusses antibiotic metabolite identification in environmental waters. S. Han et al., (Sci Total Environ 2022, 826, 154116) discusses sampling of antibiotics and metabolites in Chinese rivers, and the relationship of the measurements to the levels of antibiotic consumption. F. Tasci et al., (Food Control 2021 , 127, 108147) measures antibiotics and metabolite levels in milk samples by LC-MS. D Wang., et al., (Env Sci Pollut Research 2019, 26, 33363) discusses occurrence of sulfonamide antibiotics and their metabolites in Chinese wastewater samples.

[0014] The current inventors have surprisingly discovered that known human metabolites of known and commonly used antibiotics contribute to AMR. Therefore, the current invention could influence the proper use, disposal and waste treatment of all antibiotic residues. Summary of the invention

[0015] The current inventors have surprisingly discovered that known human metabolites of known and used antibiotics contribute to AMR. There are no literature reports of this phenomenon to date.

[0016] The current invention will help to prevent the emergence of antibiotic metabolites into the environment and prevent the spread of antibiotic resistance caused by these metabolites. This will decrease the spread or emergence of antimicrobial resistant bacteria.

[0017] As shown in the Examples, the inventors have proven that the metabolites of antibiotics are responsible for the development of resistance by bacterial species to the parent antibiotic from which the metabolites are derived. For example, the inventors have shown that the main metabolites of ciprofloxacin, namely N-formyl and 2-oxo ciprofloxacin, have an effect on resistance development (Figure 6). As illustrated in Figure 4, the inventors have shown a statistically significant increase in biofilm formation for Pseudomonas aeruginosa passaged in the presence of N-formyl ciprofloxacin in comparison to the untreated control.

[0018] An aspect of the invention provides a method to detect, or monitor, an antibiotic metabolite and / or pharmaceutical metabolite in the environment, the method comprising assaying a sample from the environment for the presence or absence of the metabolite.

[0019] It will be appreciated that “an” antibiotic and / or pharmaceutical metabolite may be one or more metabolites. In an embodiment, the method comprises assaying a sample for a plurality of metabolites.

[0020] An aspect of the invention provides a method to reduce or prevent the development of antibiotic resistance, the method comprises assaying a sample from the environment for the presence or absence of one or more antibiotic or pharmaceutical metabolites. When the assay indicates the presence of the metabolite in the sample, the method may further comprise a step of removing or degrading the metabolite from the source environment. Notably, a positive result provides an indication that an effective capture system is needed to avoid entry of the metabolite into the environment.

[0021] An “environmental sample” is material taken from one or more of air, water, soil, biological material and wastes. Water includes water from the environment, sludge from wastewater treatment plants, wastewater, rivers, streams, sewage, and groundwater. Waste includes, agricultural waste, including slurry, sewage, hospital waste, pharmaceutical waste and industrial waste. This includes sewage plants, farmhouse urine or faeces collection tanks. Sewage may comprise waste material from residences, health care systems (e.g. hospitals, community homes), institutions, and commercial and industrial establishments. Raw sewage may include household waste liquid from toilets, showers, kitchen etc. Sewage may comprise also liquid waste from industry or commerce, or it may comprise surface waters (e.g. stormwater).

[0022] In an embodiment, the environment may be any environment in which an antibiotic or antibiotic metabolite may be present.

[0023] The method of the current invention may also be used for agricultural products, such as dairy products. The dairy product may be any known dairy product, such as milk, cheese and yoghurt. The milk also comprises infant formula.

[0024] The method of the current invention may also be used for any food or beverage product. This can be an agricultural product as defined above. The food product may be a meat or fish product.

[0025] Thus, an aspect of the invention provides a method to detect or monitor an antibiotic metabolite and / or pharmaceutical metabolite in an agricultural product, the method comprising assaying a sample from an agricultural product for the presence or absence of the metabolite. When the assay indicates the presence of the metabolite in the sample, the method may further comprise a step of removing or degrading, the metabolite from the sample source or the environment of the sample source. In this context, the sample source is the source the product originated from, such as an animal., e.g. a cow. Notably, a positive result provides an indication that an effective capture system is needed to avoid entry of the metabolite into the agricultural product.

[0026] In an embodiment of any aspect of the invention the method comprises assaying a sample for a plurality of metabolites. It is considered by the current inventors that two or more metabolites could work synergistically to enhance the development of resistance to the parent antibiotic.

[0027] In any aspect of the invention, the antibiotic metabolite may be from any known antibiotic. The antibiotic metabolite may be any antibiotic metabolite. This includes but is not limited to metabolites of antibiotics of fluoroquinolone (FQ), p-lactam, and sulfonamide classes. The antibiotic may be selected from ciprofloxacin, amoxicillin and sulfamethoxazole. Examples of metabolites of ciprofloxacin is N-formylciprofloxacin, and oxo ciprofloxacin. Later generation of fluoroquinolones are also included, such as levofloxacin and moxifloxacin.

[0028] Preferably, in any aspect of the invention the antibiotic or pharmaceutical metabolite is a human metabolite. In an embodiment, the metabolite is a veterinary metabolite. In an embodiment, the metabolite is an environmental metabolite. The method may be used to detect a combination of human, veterinary and environmental metabolites in a sample. Such metabolites are known in the art.

[0029] The means to assay or detect the metabolite may be any known method in the art. The method may be quantitative or qualitative. Examples include but are not limited to liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), ultra performance liquid chromatography (uPLC) - detected with LIV / DAD (diode array detector), tandem LC-MS / MS analysis, gas chromatography (GC)-MS, tandem GC-MS / MS, and enzyme linked immunosorbent assay (ELISA) (Vumazonke et al., Int. J. Environ. Res, Public Health 2020, 17(11), 4067).

[0030] In any aspect of the current invention, the method may comprise one or more treatment steps. The treatment may comprise physical, chemical, and biological processes to remove or degrade the contaminant(s) from the sample source or source environment. This will prevent the further spread of metabolites in the environment.

[0031] Methods to remove antibiotics from the environment are known in the art. It will be appreciated that the same methods, or modified methods, can be used to remove metabolites of these antibiotics. These include but are not limited to antibiotic absorbents. It is known that carbonbased materials have good adsorption capacity for antibiotics and heavy metals.

[0032] The reduction or avoidance of development of antibiotic resistance is in one or more bacterial families, species or strains. In an embodiment, the species may be one or more of Pseudomonas aeruginosa and Staphylococcus, aureus.

[0033] In an embodiment, the method of the invention reduced antibiotic resistance or avoidance of development of resistance in Gram negative species, e.g. Pseudomonas aeruginosa and / or Gram-positive species, e.g. S. aureus against one or more fluoroquinolones, for example ciprofloxacin. Definitions and General Preferences

[0034] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0035] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0036] As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method / process steps.

[0037] An “environmental sample” is material taken from one or more of air, water, soil, biological material and wastes.

[0038] The term “antibiotic” is an agent that is used to treat or prevent bacterial infection.

[0039] The term “antibiotic metabolite” when used herein is a breakdown product of an antibiotic structure. Typically, it is structurally similar to the parent antibiotic compound. In some cases, the antibiotic metabolite has antibacterial activity. Typically, breakdown occurs in the human or animal body and the metabolites are excreted from the body. It may occur from exposure to the environment and such products are called environmental transformation products (ETPs) For example, the breakdown may be caused by sunlight (photolytic breakdown product), or by the presence of metal ions, especially heavy metals (cadmium, chromium, copper, lead, etc.). Breakdown by the presence of metal ions could be exacerbated by sunlight due to a synergistic effect. Soil bacteria can also degrade / metabolise chemical structures in the environment. Antibiotic metabolites are known in the art. Examples are disclosed in Sima, M., et al., Factors Affecting the Metabolic Conversion of Ciprofloxacin and Exposure to Its Main Active Metabolites in Critically III Patients: Population Pharmacokinetic Analysis of Desethylene Ciprofloxacin. Pharmaceutics 2022, 14 (8), 1627; Hanaya, K. et al., Synthesis of Linezolid Metabolites PNU-142300 and PNU-142586 toward the Exploration of Metabolite-Related Events. Chem. Pharm. Bull. 2017, 65 (2), 194-199; Haginaka, J.; et al., Liquid Chromatographic Determination of Amoxicillin and Its Metabolites in Human Urine by Postcolumn Degradation with Sodium Hypochlorite. J. Chromatogr. B: Biomed. Sci. Appt. 1987, 413, 219-226. DOI: 10.1016 / 0378-4347(87)80229-3; and Zeiler, H et al., Antibacterial Activity of the Metabolites of Ciprofloxacin and Its Significance in the Bioassay. Arzneim. Forsch. 1987, 37 (2), 131-134.

[0040] The term “pharmaceutical metabolite” when used herein is a breakdown product of the pharmaceutical structure. Typically, it is structurally similar to the parent compound. In some cases, the metabolite has the same activity as the parent pharmaceutical compound. Typically, breakdown occurs in the human or animal body and the metabolites are excreted from the body. It may occur from exposure to the environment. For example, the breakdown may be caused by sunlight (photolytic breakdown product), or by the presence of metal ions, especially heavy metals (cadmium, chromium, copper, lead, etc.). Breakdown by the presence of metal ions could be exacerbated by sunlight due to a synergistic effect. Soil bacteria can also degrade / metabolise chemical structures in the environment.

[0041] The term “wastewater” when used herein is a type of water that is produced by people. It can include greywater, which is generally household wastewater from sinks, bathtubs, showers, dishwashers, clothes washers. It can include blackwater, which is the water used to flush toilets, combined with the human waste that it flushes away, soaps, detergent and toilet paper.

[0042] “Fluoroquinolones” is a class of antibiotics that inhibits the activity of DNA gyrase and topoisomerase, enzyme that are essential for bacterial DNA replication. The class includes ciprofloxacin, norfloxacin, ofloxacin, delafloxacin, Gemifloxacin, levofloxacin, and moxifloxacin. Fluoroquinolones contain one or more fluorine atoms in their chemical structure. Ciprofloxacin is used to treat a wide variety of infections and it is on the WHO’s list of essential medicines (EML). Its chemical structure is illustrated in Figure 5.

[0043] “P-lactam” antibiotics are antibiotics that contain a p-lactam ring in their chemical structure. This class includes penicillin derivatives or penicillins, e.g. amoxicillin, cephalosporins, cephamycins, monobactams, carbapenems and carbacephems. These antibiotics work by inhibiting cell wall biosynthesis by binding to and inhibiting penicillin binding protein (PBP), a group of D-alanyl-D-alanine transpeptidases, and are the most widely used group of antibiotics. The first p-lactam discovered was penicillin. Amoxicillin belongs to the aminopenicillin class of penicillin family. Its chemical structure is illustrated in Figure 5.

[0044] “Sulfonamides” is a large class of antibiotics that have multiple clinical uses. The sulfonamides were the first effective antibiotics to be introduced into clinical medicine and have been in use continuously since around 1930. They are considered bacteriostatic and act by inhibition of bacterial biosynthesis of folic acid, which is needed for cell growth. They are competitive inhibitors of the enzyme dihydropteroate synthase (DHPS), which is involved in folate synthesis. Examples include sulfamethoxazole, sulfasalazine, sulfisoxazole, acetazolamide and zonisamide. The chemical structure of sulfamethoxazole is in Figure 3.

[0045] The term “assaying” should be understood to mean quantitative or qualitative detection of one or more antibiotic or pharmaceutical metabolites in the sample. Typically, it involves determining the presence or absence of the one or more metabolite in a sample. Suitable methods will be known to a person skilled in the art. Examples include liquid chromatographymass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), ultra performance liquid chromatography (uPLC) - detected with LIV / DAD (diode array detector), tandem LC-MS / MS analysis, gas chromatography (GC)-MS, tandem GC-MS / MS, and enzyme linked immunosorbent assay (ELISA).

[0046] The “presence” of the metabolite is when the metabolite is determined to be above a specified threshold or value. This threshold may zero. The threshold may be a predetermined value.

[0047] Brief Description of the Figures

[0048] The current invention will now be described with reference to the following Figures in which;

[0049] Figure 1 : Synthesis of three of the main human metabolites of ciprofloxacin, / V-formyl ciprofloxacin (4), oxo ciprofloxacin (5), and desethylene ciprofloxacin (6).

[0050] Figure 2: Synthesis of the main human metabolites of amoxicillin, amoxicilloic acid (7) and amoxicillin diketopiperazine (8).

[0051] Figure 3: Synthesis of one of the main human metabolites of sulfamethoxazole, / V-acetyl sulfamethoxazole (9) and proposed synthesis of sulfamethoxazole N -glucuronide. Figure 4: Assessment of the effect of exposure to 0.03125 pg.mL-1 / V-formyl ciprofloxacin for a continuous 30 day period on biofilm formation, over 24 h.

[0052] Figure 5: Structures of ciprofloxacin (left) and amoxicillin (right) with main areas of metabolism in humans highlighted.

[0053] Figure 6: Yields in milligram of ciprofloxacin metabolites obtained for microbiological assessment.

[0054] Figure ?: Yields in milligram of amoxicillin metabolites obtained for microbiological assessment.

[0055] Figure 8: Yields in milligram of n-acetyl sulfamethoxazole obtained for microbiological assessment.

[0056] Figure 9: 24-hour Biofilm formation by P. aeruginosa (PAO1) using the crystal violet assay showing statistically significant enhanced biofilm formation from strains cultured for 30 days day in the presence of / V-formyl (left A) and oxo (right B) ciprofloxacin, compared to relevant controls.

[0057] Figure 10: Minimum Inhibitory Concentration analysis of S. aureus following 30-day exposure and repeated culture in the presence of / V-formyl ciprofloxacin metabolite showing a 2-fold increase in the concentration of the parent compound (ciprofloxacin) needed to kill these strains over relevant controls.

[0058] Figure 11 : Minimum Inhibitory Concentration analysis of S. aureus following 30-day exposure and repeated culture in the presence of oxo ciprofloxacin metabolite showing a 2-fold increase in the concentration of the parent compound (ciprofloxacin) needed to kill these strains over relevant controls.

[0059] Detailed Description of the Invention

[0060] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full. The inventors have surprisingly discovered that human metabolites of known antibiotics can cause antibiotic resistance to the parent antibiotic The invention detects the presence of an antibiotic metabolite in the environment. The invention further comprises steps to remove the metabolite from the environment and / or steps to stop further entry of the metabolite into the environment. In this way, the method of the invention reduces the spread of antibiotic resistance and / or the development of antibiotic resistance to the parent antibiotic.

[0061] During the COVID-19 pandemic may non-antimicrobial pharmaceuticals were tested for their ability to “kill” the virus. In parallel, the pharmaceuticals were tested for antibacterial activity. It was shown by groups that different types of pharmaceuticals are also antibacterial, e.g. antidepressants (Caldara, M.; et al., Antimicrobial Properties of Antidepressants and Antipsychotics — Possibilities and Implications. Pharmaceuticals 2021 , 14, 915; Nocentini, A.; et al., Carbonic Anhydrase Inhibitors as Novel Antibacterials in the Era of Antibiotic Resistance: Where Are We Now? Antibiotics 2023, 12, 142; Antimicrobial Properties on NonAntibiotic Drugs in the Era of Increased Bacterial Resistance: Antibiotics 2020, 9, 107). Therefore, many of these pharmaceutical metabolites may also contribute to antibiotic resistance. Therefore, the method of the current invention also aims to detect or monitor pharmaceutical metabolites in the environment.

[0062] The method of the invention detects the presence of an antibiotic or pharmaceutical metabolite in the environment. This will positively influence the proper use of antibiotics and pharmaceuticals, e.g. more mindful and careful use of the levels of antibiotic being used per treatment.

[0063] The method of the invention will also influence the proper disposal and waste treatment of all antibiotic and pharmaceutical residues. For example, if a metabolite is found in a sample, such as an environmental sample or a food or beverage product sample, this will indicate contamination in that environment or the product source, which will allow proper systems to be implemented to avoid further contamination by the metabolite in that environment. Notably, the system is a capture system to capture or remove metabolites or residues at source before they enter the environment. The system may be a destruction system, where the metabolite is decomposed or degraded into harmless by products through biological processes or chemical / electrochemical oxidation. Both systems may be present in the one unit or system. This source could be a hospital source, a household source, or an agricultural source. Any suitable system for metabolites may be used. The system may be for the most common, and / or potent metabolites. “Common” includes inactive metabolites. It will be appreciated that the system is one for any metabolite. Additional steps that can be taken after detection of one or more metabolites in the environmental sample includes antibiotic stewardship, where levels of antimicrobial / antibiotic use are continuously reduced. Results show that consumption levels in Europe are reducing (EFSA Journal. 2024) but in low and middle-income countries (LMIC) in Africa and Asia, this is not the case (Mohammed Kananm et al., Antibiotics, 2023, 12(10), 1504).

[0064] In an embodiment, repeated use of the method is intended. This can be used to continually monitor an environment or source for metabolite contamination.

[0065] Ciprofloxacin and amoxicillin are commonly prescribed antibiotics belonging to the fluoroquinolone and p-lactam classes, respectively. Both antibiotics are included in the World Health Organization’s EML.

[0066] The structures of ciprofloxacin (left) and amoxicillin (right), with main areas of human metabolism highlighted are show in Figure 5.

[0067] Ciprofloxacin is 1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-ylquinoline-3-carboxylic acid. In an embodiment of the method the metabolites of ciprofloxacin are one or more of:

[0068] • Oxo Ciprofloxacin: 1-Cyclopropyl-6-fluoro-4-oxo-7-(3-oxopiperazin-1-yl)-1 ,4- dihydroquinoline-3-carboxylic acid.

[0069] • / V-Formyl Ciprofloxacin: 1-Cyclopropyl-6-fluoro-4-oxo-7-(4-formylpiperazin-1-yl)-1 ,4- dihydroquinoline-3-carboxylic acid.

[0070] • Desethylene Ciprofloxacin: 7-(2-aminoethylamino)-1-cyclopropyl-6-fluoro-4- oxoquinoline-3-carboxylic acid.

[0071] • Sulfo Ciprofloxacin / Ciprofloxacin piperazinyl- / V4-sulfate: 1-cyclopropyl-6-fluoro-4-oxo- 7-(4-sulfopiperazin-1-yl)quinoline-3-carboxylic acid.

[0072] Ciprofloxacin metabolites are described in Zeiler, H.; et al., Antibacterial Activity of the Metabolites of Ciprofloxacin and Its Significance in the Bioassay. Arzneim. Forsch. 1987, 37 (2), 131-134 and Al-Omar, M.A. Ciprofloxacin: Drug Metabolism and Pharmacokinetic Profile. Profiles of Drug Substances, Excipients and related Methodology, 2004, 31 , 209.

[0073] Example metabolites of ciprofloxacin are shown in in Figure 6. Amoxicillin is (2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7- oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid. In an embodiment of the method the metabolites of amoxicillin are one or more of:

[0074] • Amoxicilloic Acid.

[0075] • Amoxicillin diketopiperazine.

[0076] Example metabolites of amoxicillin are shown in in Figure 7.

[0077] Example metabolites of amoxicillin are described in Nagele, E. et al.’, Structure Elucidation of Degradation Products of the Antibiotic Amoxicillin with Ion Trap MSn and Accurate Mass Determination by ESI TOF. J Am Soc Mass Spectrom 2005, 16, 1670.

[0078] Sulfamethoxazole is 4-Amino-N-(5-methylisoxazol-3-yl)-benzenesulfonamide. In an embodiment of the method the metabolites of sulfamethoxazole are one or more of:

[0079] • N-acetyl sulfamethoxazole: N-(4-(N-(5-Methylisoxazol-3- yl)sulfamoyl)phenyl)acetamide.

[0080] • Sulfamethoxazole N -glucuronide: 6-[(4-aminophenyl)sulfonyl-(5-methyl-1,2-oxazol- 3-yl)amino]-3,4,5-trihydroxyoxane-2-carboxylic acid.

[0081] Example metabolites of sulfamethoxazole are shown in in Figure 8.

[0082] Example metabolites of sulfamethoxazole are described in Garcia-Galan, M.J. et al. Identification and determination of metabolites and degradation products of sulfonamide antibiotics. Trends Analytical Chem 2008, 27 (11), 1008.

[0083] AMR may occur in any bacterial species which are commonly treated by antibiotics. For example, bacterial pathogens responsible for community-acquired pneumonias, bronchitis, urinary tract infections, and gastroenteritis. It may occur in Gram-negative bacteria (such as Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Moraxella catarrhalis, Proteus mirabilis, and Pseudomonas aeruginosa). It may occur in Gram-positive bacteria (such as methicillin-sensitive Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis).\t will be appreciated that it may be any species, especially part of the ESKAPE pathogens, which stands for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterspp. A person skilled in the art would be aware of these species (WHO bacterial priority pathogen list, 2024 bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance.

[0084] Geneva: World Health Organization; 2024)

[0085] An aspect of the invention provides a method to detect or monitor an antibiotic and / or pharmaceutical metabolite in an agricultural product.

[0086] An aspect of the invention provides a method to validate an antibiotic or pharmaceutical metabolite capture or destruction system, the method comprising assaying a sample for the presence or absence of the metabolite. Notably, the sample is an environmental sample. For instance, in an embodiment the sample is a waste sample. In this method, the presence of the metabolite in the sample indicates that the system is not working to prevent entry or release of the metabolite into the environment, or destruction of the metabolite. The absence of the metabolite indicates that the method is working to prevent entry of the metabolite into the environment.

[0087] An aspect of the invention provides a method to trace the source of an antibiotic or pharmaceutical metabolite in the environment, the method comprising assaying a sample from the source for the presence or absence of the metabolite as described herein. The source may be a hospital or a farm for example.

[0088] A method to track an antibiotic or pharmaceutical metabolite in the environment, the method comprising assaying a sample for the presence or absence of the metabolite as described herein.

[0089] In any aspect of the invention, multiple samples may be tested from one or more environmental samples.

[0090] In an aspect the method is one that comprises a step of assaying a sample for the amount or level of metabolite in an environmental sample. The amount may be compared with a reference. The reference is a known or predetermined amount. It may be the amount determined from the same environment at a different day or time. This comparison may be used to determine if there is an increase or a decrease in a metabolite in an environment. Assays are known in the art.

[0091] An aspect of the invention provides an assay to detect an antibiotic or pharmaceutical metabolite. A kit is also provided comprising the assay of the invention. The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0092] EXAMPLES

[0093] Synthesis and characterization of the main human metabolites of antibiotics from the fluoroquinolone (FQ), p-lactam, and sulfonamide classes.

[0094] Methods used are those known in the art. The Gould Gould-Jacobs synthesis method is described in Gould, R. Gordon., et al., The Synthesis of Certain Substituted Quinolines and 5,6-Benzoquinolines. J. Am. Chem. Soc. 1939, 61 (10), 2890-2895. The Bayer method is described in Lie, J. et al., Contemporary Drug Synthesis, 1st ed.; Wiley - VCH, 2004.

[0095] As an example for ciprofloxacin, the synthesis and characterization of / V-formyl ciprofloxacin is as follows:

[0096] 38

[0097] Microwave Assisted Synthesis (MAS)

[0098] 1 ,4-Diazabicyclo[2.2.2]octane (DABCO) (0.17 g, 1.51 mmol) was added to a 40 mL microwave synthesis flask containing a suspension of 1-cyclopropyl-6,7-difluoro-1 ,4-dihydroquinoline-3- carboxylic acid (0.20 g, 0.75 mmol) and N-formyl piperazine (0.09 mL, 0.90 mmol) in DMSO (6 mL). The flask was heated in the microwave synthesiser for 20 mins at 170 °C. The reaction mixture was a white suspension prior to heating with the progress of the reaction being monitored by TLC. The flask was allowed to cool to rt after heating and the mixture was partitioned between water / ethyl acetate (30 mL, 1 :1). A yellow solid was observed at the interface of the layers, which was filtered, washed with water (5 x 10 mL) and ethyl acetate (5 x 10 mL). The crude solid was purified by solubilising in the minimum volume of methanol / chloroform (9:1). Following cooling overnight in the -20 °C freezer, a yellow crystalline solid was obtained which was filtered and dried overnight at 55 °C in a vacuum oven. Molecular Formula: C18H18FN3O4; Yield: 0.08 g, 30%; M.P. (°C): decomposed at 282 - 284 ; Rf: 0.31 , chloroform / methanol (90:10);1H NMR (DMSO-d6, b / ppm): 15.13 (s, 1 H), 8.67 (s, 1 H), 8.12 (s, 1 H), 7.94 (d, 1 H, J = 13.1 Hz), 7.60 (d, 1 H, J = 7.4 Hz), 3.82 (tt, 1 H, J = 7.3, 4.0 Hz), 3.66 - 3.57 (m, 4H), 3.39 - 3.33 (m, 2H), 3.34 - 3.27 (m, 2H), 1.38 - 1.27 (m, 2H), 1.23 - 1.13 (m, 2H);13C NMR (DMSO-d6, b / ppm): 176.8 (d, J = 2.8 Hz, C=Oketone), 166.2, (C=Oacid), 161.5 (C=OfOrmamide), 153.5 (d, C-F, J = 249.2 Hz), 148.6, 145.4 (d, J = 10.0 Hz), 139.6, 119.6, 111.5 (d, J = 22.9 Hz), 107.5 (d, J = 3.1 Hz), 107.2, 50.7 (d, J = 4.5 Hz), 49.6 (d, J = 4.5 Hz), 44.9, 39.6, 36.3, 8.0;19F NMR (DMSO-d6, b / ppm): -121 .9 (dd, J = 13.3, 7.3 Hz); I.R. (KBr disc, cm’1): 3550 - 3250, 3099, 3051 , 2962, 2874, 2834, 1718, 1671 , 1628; Microanalysis Calcd. for Ci8Hi8FN3O4: C, 60.16, H, 5.05, N, 11.69, Found: C, 59.70, H, 4.92, N, 11.49; HRMS: ES+for Ci8Hi8FN3O4calculated [M+H]+(m / z) of 360.1360, observed [M+H]+(m / z) of 360.1346.

[0099] Thermal Synthesis

[0100] 1 ,4-Diazabicyclo[2.2.2]octane (DABCO) (0.17 g, 1.51 mmol) was added to a 100 mL RBF containing a suspension of 1-cyclopropyl-6,7-difluoro-1 ,4-dihydroquinoline-3-carboxylic acid (0.20 g, 0.75 mmol) and N-formyl piperazine (0.09 mL, 0.90 mmol) in DMSO (6 mL). The white suspension was stirred for 1 hr at 130 °C, became a clear yellow solution over 100 °C, with the progress of the reaction being monitored by TLC. The reaction was allowed to cool to rt and a solid was observed in solution. Ice cold water (15 mL) was added to the reaction vessel, and it was cooled at 4 °C in the fridge. The product was observed as a shiny white solid which was purified in a similar manner to MAS synthesis. Following cooling overnight in the -20 °C freezer, a white solid was obtained which was filtered and dried overnight at 55 °C in a vacuum oven. Molecular Formula: Ci8HisFN3O4; Yield: 0.01 g, 37%; M.P. (°C): decomposed at 280 - 282; Rt: 0.34, chloroform / methanol (90:10);1H,13C,19F, IR & HRMS analysis was consistent with data reported above for MAS method.

[0101] The synthesis of three of the main human metabolites of ciprofloxacin, / V-formyl ciprofloxacin (4), oxo ciprofloxacin (5), and desethylene ciprofloxacin (6) is illustrated in Figure 1.

[0102] For Figure 1 : (i) (COCI) , DMF, DCM, N atm, rt, 24 h; (ii) Ethyl 3-(diethylamino) acrylate, Et N, 2 2 3 toluene, N atm, 90 °C, 5 h; (iii) Cyclopropylamine, EtOH / Et O (1 :2), rt, 3 h; (iv) K CO , DMF, 2 2 2 3

[0103] 100 °C, 21 h; (v) Piperazine, Et N, ACN, reflux, 1 week; (vii) 2-Oxopiperazine, DABCO, DMSO, 3 170 °C / 130 °C, 15 min / 1.5 h; (viii) / V-Formylpiperazine, DABCO, DMSO, 170 °C / 130 °C, 20 min / 1 h; (ix) N-Boc ethylenediamine, pyridine, N atm, 120°C, 16 h: Boc removal: TFA: DCM

[0104] 2

[0105] (1 :1), 1h;

[0106] The synthesis of the main human metabolites of amoxicillin, amoxicilloic acid (7) and amoxicillin diketopiperazine (8) is shown in Figure 2. The synthesis of one of the main human metabolites of sulfamethoxazole, / V-acetyl sulfamethoxazole (9) and proposed synthesis of sulfamethoxazole N -glucuronide is shown in Figure 3.

[0107] Ciprofloxacin, amoxicillin, and sulfamethoxazole metabolites were tested. All three antibiotics on WHO’s List of Essential Medicines.

[0108] Table 1 : High resolution MS data (ES+, [M+H]+) for synthesized antibiotic metabolites.

[0109] Susceptibility and SIC studies using clinically relevant ESKAPE pathogens and assessment of resistance.

[0110] Ciprofloxacin, oxo ciprofloxacin, and / V-formyl ciprofloxacin against Pseudomonas aeruginosa strain PAO1.

[0111] Figure 4 illustrates biofilm formation over a 24-hour period for P. aeruginosa strain PA01 passaged for 30 days in the presence of N-formyl ciprofloxacin (Sub30M) compared to a passage culture not exposed to the test compound. A statistically significant increase in biofilm formation was observed in Sub 30M in comparison to the untreated control. Enhanced biofilm formation leads to a larger physical barrier for the antibiotic to overcome.

[0112] These results signify a development of AMR in P. aeruginosa to ciprofloxacin by exposure to subclinical levels of its metabolite N-formyl ciprofloxacin. Studies using clinically relevant ESKAPE pathogens, P. aeruginosa and S. aureus and assessment of resistance to ciprofloxacin

[0113] The Minimum Inhibitory Concentration (MIC) assay is a technique used to determine the lowest concentration of an antimicrobial agent that inhibits the visible growth of a microorganism. The assay typically involves diluting the antimicrobial agent in a series of concentrations and exposing the microorganism to these varying concentrations. After an incubation period, the growth of the microorganism is assessed, usually by visual inspection or with a spectrophotometer. The MIC is the lowest concentration that shows no visible growth, indicating effective inhibition of the microorganism. This method is crucial for determining the potency of antibiotics and guiding appropriate therapeutic dosages.

[0114] No shift in MIC was observed for passaged cultures of P. aeruginosa exposed to oxo and N- formyl ciprofloxacin for 30 days. However, 24-hour biofilm formation assays for passaged cultures showed statistically significant increases in biofilm formation following exposure to N- formyl ciprofloxacin (left Figure 9A) and oxo ciprofloxacin (right Figure 9B) for 30 days (n=3). These results are illustrated in Figure 9.

[0115] Biofilm Method

[0116] The crystal violet biofilm assay is a quantitative method used to assess biofilm formation by microorganisms. In this technique, biofilms are grown on the surface of microtiter plates, stained with crystal violet dye, and subsequently washed to remove excess dye. The bound dye, which stains the biofilm biomass, is then solubilised using acetic acid. The absorbance of the solubilised dye is measured spectrophotometrically at 600nm, providing a quantitative measure of the biofilm mass. This assay is widely used due to its simplicity, cost-effectiveness, and ability to analyse multiple samples simultaneously.

[0117] These results signify a development of AMR in P. aeruginosa to ciprofloxacin by exposure to subclinical levels of its metabolite N-formyl ciprofloxacin and oxo ciprofloxacin. S. aureus:

[0118] The Minimum Inhibitory Concentration (MIC) assay is a technique used to determine the lowest concentration of an antimicrobial agent that inhibits the visible growth of a microorganism. The assay typically involves diluting the antimicrobial agent in a series of concentrations and exposing the microorganism to these varying concentrations. After an incubation period, the growth of the microorganism is assessed, usually by visual inspection or with a spectrophotometer. The MIC is the lowest concentration that shows no visible growth, indicating effective inhibition of the microorganism. This method is crucial for determining the potency of antibiotics and guiding appropriate therapeutic dosages.

[0119] A 2-fold shift in MIC was observed for passaged cultures of S. aureus exposed to oxo and N- formyl ciprofloxacin for 30 days. The MIC of the two control strains (uncultured unexposed and cultured unexposed) was 0.25 p.g.ml’1. However, the 30-day cultured and exposed strain of S. aureus saw the MIC value for the organism rise to 1 .g.ml'1following exposure to N-formyl ciprofloxacin (10) and oxo ciprofloxacin (11) for 30 days (n=3). These results are illustrated in Figures 10 and 11.

Claims

Claims1 . A method to reduce, or avoid development of, antibiotic resistance in one or more bacterial species, the method comprising: assaying a sample from a food or beverage product, or a sample from the environment, for the presence of one or more antibiotic or pharmaceutical metabolites, wherein when the assay indicates presence of the one or more metabolites, the method further comprises one or more of the following steps: removing or degrading the metabolite from the environment or the product source or environment, implementing metabolite capture system, and preventing entry of the metabolite into the environment or the product.

2. The method of Claim 1 , wherein the environmental sample is a sample taken from air, water, soil, biological material or waste.

3. The method of Claim 1 , wherein the food or beverage product is one or more dairy products or meat products.

4. The method of any one of the preceding claims wherein the metabolite is an antibiotic metabolite.

5. The method of Claim 4, wherein the metabolite is a human metabolite.

6. The method of Claim 4, wherein the metabolite is an animal metabolite.

7. The method of Claim 4, wherein the metabolite is an environmental transformation product (ETP).

8. The method of any one of Claims 4 to 7 wherein the antibiotic is selected from the group comprising a fluoroquinolone (FQ), p-lactam, and sulfonamide antibiotic.

9. The method of Claim 8, wherein the antibiotic is a fluoroquinolone.

10. The method of Claim 9, wherein the metabolite is a ciprofloxacin metabolite.

11. The method of Claim 10, wherein the ciprofloxacin metabolite is one or more of N- formylciprofloxacin, oxo ciprofloxacin desethylene ciprofloxacin and sulfo ciprofloxacin.

12. The method of Claim 11 , wherein the ciprofloxacin metabolite is N-formylciprofloxacin and / or oxo ciprofloxacin.

13. The method of any one of the preceding claims, wherein the bacterial species is one or more ESKAPE pathogens.

14. The method of any one of the preceding claims, wherein the species is P. aeruginosa and / or S. aureus.

15. A method to validate an antibiotic or pharmaceutical metabolite capture system, the method comprising assaying a sample for the presence or absence of the metabolite, wherein the presence of the metabolite in the sample indicates that the capture system is not working effectively to prevent entry or release of the metabolite into the sample source and wherein the absence of the metabolite in the sample indicates that the method is working effectively.

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