CONTROL OF BACTERIAL GROWTH

FIELD OF THE INVENTION

This invention relates to the discovery that sulfonylurea compounds can prevent the growth of certain bacterial pathogens.

All documents cited in this text ("herein cited documents") and all documents cited or referenced in herein cited documents are incorporated by reference in their entirety for all purposes. There is no admission that any of the various documents etc. cited in this text are prior art as to the present invention.

BACKGROUND

A gram-negative rod-shaped bacteria, Bwkholderia pseudomallei is present in muddy soil throughout Southeast Asia and Northern Australia and can readily be isolated from over 50% of the rice paddies in northeastern Thailand. Human or animal infection can occur after exposure to contaminated soil or water via direct contact, inhalation, or even ingestion which was thought to be the route on infection in some cases of near-drowning victims after the 2004 Indian Ocean tsunami.

Infection can result in a range of clinical symptoms such as skin abscesses or pneumonia with the most severe outcome being sepsis leading to death in sometimes as little as 2 - 3 days. In northeastern Thailand, a largely agricultural and rural part of the country, melioidosis accounts for 20% of all community acquired cases of septicemia; at the Royal Darwin Hospital in Northern Australia, melioidosis is the most common cause of fatal community acquired bacteraemic pneumonia. Case fatality rates from primary disease range from approximately 20% in Northern Australia to nearly 50% in Thailand.

There is currently no approved vaccine. Current antibiotic treatment regiments are both difficult to complete and expensive as they entail intravenous therapy for 10 - 1 days followed by oral administration for an additional 3 -6 months. Recurrence often occurs due to failure to complete the oral component of the regiment. Novel approaches are clearly needed both to help limit the number of cases of melioidosis as well as to treat infected patients.

It has been over forty years since a new class of compounds was approved for treating infections by Gram-negative pathogens. With the rising prevalence of antibiotic resistance, once easily managed infections caused by Pseudomonas aeruginosa and Acinetobacter baumannii are now being treated with polymyxins, compounds previously abandoned due to concerns about toxicity. Antibiotic resistance genes are widely found in both the human microflora and the soil microbiota, with one specific example being B. pseudomallei, which is endemic in wet soils and stagnant water in Southeast Asia and Northern Australia. Another example, the recent clinical emergence of the New Delhi metallo^-lactamase gene, also further highlights the ongoing need to develop new classes of antibiotics effective against established and emerging human pathogens.

Antibiotic resistance in Gram-negative bacteria is an ever-increasing clinical problem yet few new antimicrobial compounds are under development. Targeting microbial amino acid biosynthetic pathways may represent a promising therapeutic approach as mammalian species often lack these enzymes and enzyme-deficient pathogen mutants are often attenuated in vivo.

Due to their low application rates and low toxicity towards animals, crop-selective sulfonylurea herbicides have been developed for weed control for a wide variety of crops including wheat, corn, barley, and rice. Sulfonylurea herbicides work by inhibiting the activity of acetohydroxy acid synthase (AHAS; also known as acetolactate synthase, ALS), an enzyme required for the biosynthesis of the branched-chain amino acids (BCAA) valine, leucine, and isoleucine. B. pseudomallei, Burkholderia mallei. Mycobacterium tuberculosis, and Actinobacillus pleuropneumoniae mutants deficient in this pathway are attenuated in vivo, suggesting a requirement for active BCAA biosynthesis in infected hosts.

In bacteria, there are three known variants of AHAS, type I, II. and III, of which type II and type III can be inhibited by sulfonylurea herbicides but not type I. Although enteric bacteria, gut-resident microbes such as E. coli, often contain two different variants of AHAS one of which is often a type I AHAS, most non-enteric bacteria appear to contain a single AHAS which typically resemble type III. This is certainly the case of Bp as well as for closely related but avirulent bacteria, Burkholderia thailandensis (Bt). Animals (along with plants) lack this enzyme which explains these compounds low toxicity.

It is known in the agricultural community that sulfonylurea herbicides can inhibit the growth of soil-resident bacteria. To date, the most effective sulfonylurea compounds at preventing both Burkholderia pseudomallei and Pseudomonas aerugonosa growth are bensulfuron methyl (trade name Londax from United Phosphorus), metsulfuron methyl (trade name Escort XP Herbicide from DuPont), chlorimuron ethyl (Classic Herbicide also from DuPont), and rimsulfuron. Although these compounds have all been patented and licensed for agricultural use. the potential to control bacterial pathogens has never been described.

Here, it is shown that SHs can potently inhibit the growth of three clinically important Gram- negative pathogens: Pseudomonas aeruginosa and Acinetobacter baumannii, both major sources of hospital acquired infections; and B. pseudomallei, an emerging infectious disease and a potential bioterror agent. A phylogenetic analysis revealed that many other pathogenic bacteria encode AHAS isozymes which may be sensitive to these inexpensive and non-toxic compounds. Both mutational and structural evidence demonstrate that SHs directly inhibit AHAS in these pathogens. Treatment of infected mice with SHs at doses well below reported levels of toxicity provided significant protection against otherwise acute, lethal challenges of B. pseudomallei or P. aeruginosa. These findings suggest that SHs are potentially a new class of antibiotics, while highlighting essential amino acid biosynthetic pathways as a promising target for anti-microbial compound development.

US5998420 discloses a method for treating tuberculosis in a mammal which comprises administering to the mammal a therapeutically effective amount of an inhibitor compound that inhibits an enzyme in the branched chain amino acid biosynthetic pathway in

Mycobacterium tuberculosis. Also, mice infected with Mycobacterium tuberculosis strain ATCC35801 were injected with sulfometuron methyl, a herbicidal compound that inhibits AHAs, which resulted in a significant reduction in growth of Mycobacterium tuberculosis in the lungs.

US7666404 discloses a treatment for Burkholderia pseudomallei infection such as melioidosis. The treatment of which involves inhibiting the expression of one or more transcriptional regulatory proteins and/or synthase enzymes. Inhibitors may include agents or drugs which either bind or sequester synthase substrate(s) or cofactor(s), or inhibit the synthase itself or which inhibit binding of the signal produced by the synthase to the synthase transcriptional regulator, agents which inhibit binding of the transcriptional regulator itself. Inhibitors of synthase and/or synthase enzyme transcriptional regulator may be used in the treatment or amelioration of glanders disease or melioidosis, and diseases associated with Burkholderia infection.

Atkins et al.. Infection and immunity, Vol. 70, No.9: 5290-5294 discloses that Burkholderia pseudomallei auxotrophic in the branched chain amino acid biosynthetic pathway are unable to grow without the supply of branched amino acids. The transposon was shown to have interrupted the ilvl gene encoding the large subunit of the acetolactate synthase enzyme. Compared to the wild type, this mutant was significantly attenuated in a murine model of disease. Mice inoculated intraperitoneal ly with the auxotrophic mutant, 35 days prior to challenge, were protected against a challenge dose of 6,000 median lethal doses of wild-type B. pseudomallei.

SUMMARY

Although it is well known in the agricultural community that many commonly used sulfonylurea herbicides inhibit the growth of soil-dwelling microbes along with unwanted plant species (i.e. weeds), this finding has attracted little interest in the infectious disease community. The inventors have found that some of these same compounds can inhibit the growth of the soil-dwelling human pathogen Burkholderia pseudomallei (Bp), the causative agent of the often fatal disease melioidosis and a possible bioterror agent, in laboratory studies and wish to explore the possible significance of this finding both in an agricultural setting, a common source of melioidosis infections, and as a possible new therapeutic agent for bacterial infections e.g. by B. Pseudomallei (e.g. melioidosis) and other human pathogens. As well as envisaging the use of SHs in a clinical context, the use of SHs in an environmental context is also envisaged, such as to treat infected fields so that fewer farmers will get infections by B. pseudomallei and Pseudomonas aeruginosa. SHs could be used to '"treat" B. pseudomallei containing rice paddies so as to hopefully reduce the levels of Bp in the soil and subsequent number of infections in the local population.

GLOSSARY OF TERMS

This section is intended to provide guidance on the interpretation of the words and phrases set forth below (and where appropriate grammatical variants thereof). Further guidance on the interpretation of certain words and phrases as used herein (and where appropriate grammatical variants thereof) may additionally be found in other sections of this specification.

The words "a" or "an" are employed to describe elements and components of the invention. This is done merely for convenience and to give a general sense of the invention. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.

As used herein, the term "about" as used in relation to a numerical value means, for example, +50% of the numerical value, preferably ±20%, more preferably + 10%, more preferably still ±5%, and most preferably +1%. Where necessary, the word "about' ^' may be omitted from the definition of the invention.

As used herein, the term "comprising' ^' means "including". Thus, for example, a composition "comprising" X may consist exclusively of X or may include one or more additional components.

As used herein the term "treatment" is to be construed broadly and includes both therapeutic treatment and prophylactic or preventative measures, and includes all uses which remedy a disease state or one or more symptoms thereof, prevent the establishment of disease, or otherwise prevent, hinder, retard, stabilize, reduce or reverse the onset or progression of disease or other undesirable symptoms in any way whatsoever.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs. BRIEF DESCRIPTION OF DRAWINGS

Figure 1. AHAS from Bp is sensitive to inhibition by bensulfuron methyl

Figure 2. Metsulfuron methyl inhibits the growth of Bt

Figure. 3. Type III AHAS from B. pseudomallei is sensitive to inhibition by sulfonylurea herbicides. (A) High-resolution growth curves of parental E. coli or 18G 12 treated with DMSO or SHs were generated with OD600 readings collected every 5 minutes. Valine (75 μg/ml each) was included in as indicated. Leucine (75 μg/ml) was always present as the parental E. coli strain is a leucine auxotroph. (B) A phylogenetic tree of the catalytic subunit of AHAS was constructed using maximal likelihood methods. Bootstrap analysis was performed on 100 trees and nodes appearing more than 75 times are indicated with a circle. The clusters formed by each isozyme are highlighted. (C) Structural models of chlorimuron ethyl bound to the catalytic subunit of AHAS from B. pseudomallei and P. aeruginosa.

Carbon atoms from AHAS are shown in grey whereas chlorimuron ethyl carbon atoms are shown in green. For both AHAS and chlorimuron ethyl, nitrogen atoms are shown in blue, oxygen in red, and sulfur in yellow. The chlorine in the heterocyclic ring of chlorimuron ethyl is also colored green. Hydrogen atoms are not shown. Black dashed lines indicate potential hydrogen bonds.

Figure 4. Sulfonylurea herbicides inhibit growth of pathogenic bacteria. (A) B. thailandensis ATCC 700388 was exposed to increasing doses of DMSO or SHs and high-resolution growth curves obtained with OD600 readings collected even' 10 minutes. (B) Two reference strains, AYE and 5377, and two local clinical isolates, KAb 1 and KAb 2, of A. baumannii were treated with various SHs and high-resolution growth curves obtained with OD600 readings collected every 12 minutes. (C) CFUs were determined from the microdilution well corresponding to the MIC for B. thailandensis ATCC 700388 and P. aeruginosa PAOl .

Alongside chlorimuron ethyl (Ce) and bensulfuron methyl (Bm), the bacteriostatic antibiotic chloramphenicol (Cm) and the bacteriocidal antibiotic ceftazidime (Cef) were included as controls.

Figure 5. Sulfonylurea herbicides inhibit the growth of P. aeruginosa by targeting AHAS. (A) Highresolution growth curves of wild-type P. aeruginosa PAOl along with chlorimuron ethylresistant strains containing the following ilvl variants: P104S, P104L, or R559C. (B)

Highresultuion growth curves of P. aeruginosa PAOl transformed with pUCP28T expressing either wild-type ilvl or ilvl containing the mutations found in the chlorimuron ethyl-resistant strains. In both of experiments, cultures were exposed to DMSO ( 1%) or various doses of chlorimuron ethyl and OD600 readings were collected every 10 minutes. Figure 6. Sulfonylurea herbicides slow intracellular replication of B. thailandensis and increase mouse survival in response to an otherwise lethal challenge by B. pseudomallei or P. aeruginosa. (A) Intracellular replication of B. thailandensis ATCC 700388 in A549 human respiratory epithelial cells treated with DMSO ( 1%) or chiorimuron ethyl (2 mM) starting two hours after infection (marked with an arrow). (B) Phase contrast images (100x) of the infected cultures. Scale bars, 50 μπι. (C) The survival curves of BALB/c mice (n = 6 per group) inoculated through the intranasal route with 1 ^x 102 CFUs of B. pseudomallei strain 22 and orally treated twice daily with either PBS or metsulfuron methyl (50 mg/kg). (D) Survival curves or (E) CFUs present in the lungs of FVB/N mice (n = 12 per group in both

experiments) inoculated intratracheally with P. aeruginosa strain PAO 1 and given a single tail-vein injection with PBS or metsulfuron methyl (200 mg/kg).

Figure 7. (A) Biosynthetic pathway for branched-chain amino acids. (B) The generic chemical structure of SHs is shown along with the actual structures of some of the compounds used in this study. Trade names are shown in parenthesis.

Figure 8. Various controls along with additional sulfonylurea herbicides that can also dampen the growth of B. thailandensis. B. thailandensis ATCC 700388 was exposed to increasing doses of either chloramphenicol, imazapyr, or SHs and high-resolution growth curves obtained as described earlier with measurements taken every 10 minutes.

Figure 9. Chiorimuron ethyl and metsulfuron methyl slow intracellular replication of B. thailandensis in A549 human respiratory epithelial cells while preserving cellular integrity. (A) Intracellular CFUs after B. thailandensis-m ^' fected A549 cultures were treated with either DMSO or various AHAS inhibitors two hours after infection (multiplicity of infection approximately 3). Note that the chiorimuron ethyl (red) and metsulfuron methyl (blue) data points are nearly identical therefore largely obscuring the chiorimuron ethyl values. (B) Phase contrast images (100 ^x ) of infected A549 cells from the indicated time points. Scale bar, 50 μπι.

Figure 10. The survival curves of BALB/c mice (n = 6 per group) inoculated through the intranasal route with 10 times the LD50 of B. pseudomallei strain 22 and orally treated twice daily with with PBS or metsulfuron methyl (50 mg/kg). The increased survival was statistically significant (p <0.01 ; Log-rank test, Yates corrected).

DETAILED DESCRIPTION

The inventors have found that sulfonylurea herbicides, compounds currently used in agricultural settings to control growth of unwanted species (i.e. weeds), are capable of inhibiting the growth of two human bacterial pathogens, Burkholderia pseudomallei and Pseudomonas aeruginosa. These findings raise two interesting possibilities. One, as

Burkholderia pseudomallei normally lives in the soil and often affects rice farmers, one possible use could be to "treat" infected fields with some of these herbicides so that less people will get sick from the bacteria in the infected fields. The second possible application is the use of these compounds (or derivatives of these compounds) as a therapeutic agent for infection by the above-mentioned pathogens as well as other pathogens.

With regard to the use of sulfonylurea herbicides on bacteria other than Burkholderia pseudomallei and Pseudomonas aeruginosa. MICs (minimum inhibitory concentrations) of a number of these compounds on Pseudomonas aeruginosa, an opportunistic pathogen which often infects burn victims, have been determined and it has been found that many of the values are similar to Bt and Bp which is striking given that Burkholderia' s and Pseudomonas are only distantly related.

As discussed below, it is envisaged that the invention will find utility in controlling the growth of a range of bacteria, including both gram negative and gram positive bacteria. Preferably, the bacteria do not possess a type I enzyme acetohydroxy acid synthase.

Accordingly, a first aspect of the invention provides the use of a sulfonylamide compound or a derivative thereof in the preparation of a medicament for treating and preventing infections caused by bacteria, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase and bacteria having a type III enzyme acetohydroxy acid synthase.

A second aspect of the invention relates to a sulfonylamide compound or a derivative thereof for treating and preventing infections caused by bacteria, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase.

Unless the context dictates otherwise, derivatives of sulfonylamide compounds may be encompassed within the term "sulfonylamide compound ^" as used herein. The term

"derivative thereof in connection with a sulfonylamide compound of the invention includes any compound structurally related to said sulfonylamide compound (and optionally formed from said sulfonylamide compound) but still retaining the biological activity of said sulfonylamide compound in all essential respects.

Included within the scope of the term "derivative" are chemically or biologically modified versions of the compound that are structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound. As examples of derivatives, mention can be made of functional derivatives containing one or more additional functional groups such as OH, NH2 and the like, or with one or more of the functional groups being removed from or displaced within the compound.

Derivatization (i.e., modification) may involve substitution of one or more moieties within the molecule (e.g., a change in functional group). For example, a hydrogen may be substituted with a halogen, such as fluorine or chlorine, or a hydroxyl group (- OH) may be replaced with a carboxylic acid moiety (-COOH).

The term "derivative" also includes conjugates, and prodrugs of a parent compound (i.e., chemically modified derivatives which can be converted into the original compound under physiological conditions). For example, the prodrug may be an inactive form of an active agent. Under physiological conditions, the prodrug may be converted into the active form of the compound. Prodrugs may be formed, for example, by replacing one or two hydrogen atoms on nitrogen atoms by an acyl group (acyl prodrugs) or a carbamate group (carbamate prodrugs).

The term "derivative" is also used to describe all solvates, for example hydrates or adducts (e.g., adducts with alcohols), active metabolites, and salts (e.g. pharmaceutically acceptable salts) of the parent compound.

Pharmaceutically acceptable salts are well known in the art and refer to the relatively nontoxic, inorganic and organic acid addition salts of the compounds used in the present invention. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977) and see also Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991 ).

Salts which may be conveniently used in the present invention include physiologically acceptable base salts, for example, derived from an appropriate base, such as an alkali metal (eg sodium), alkaline earth metal (eg magnesium) salts, and ammonium. Physiologically acceptable acid salts include hydrochloride, sulphate, mesylate, besylate, phosphate and glutamate. Salts according to the invention may be prepared in conventional manner, for example by reaction of the parent compound with an appropriate base to form the corresponding base salt, or with an appropriate acid to form the corresponding acid salt.

The treating or preventing by a sulfonylamide compound or a derivative thereof may be carried out in vivo or in vitro. An example of the latter type of use would be the treatment of water (e.g. stagnant water) and fields (e.g. rice paddies). In such embodiments, the sulfonylamide compound (or derivative thereof) may be contacted with the water or field, such as by adding the sulfonylamide compound (or derivative thereof) to the field or water to be treated. A further example of where the invention may find utility in an in vitro context would be the use of a sulfonylamide compound or a derivative thereof in a cleaning or disinfecting composition. Such a cleaning or disinfecting composition may, for example, take the form of a rinse, spray, cream or the like and may, for example, find utility in cleaning or disinfecting objects, surfaces or equipment. It is envisaged that such a cleaning or disinfecting agent may find utility in the domestic environment, in hospitals (e.g. in cleaning hospitals or hospital equipment such as medical devices (e.g. catheters)), in industry and in agricultural settings.

The sulfonylamide compound (or derivative thereof) may be contacted with the object / surface / equipment etc. to be treated. The term "contacting" is to be construed broadly and includes, but is not limited to, soaking, rinsing, flushing, submerging, and washing. It will be appreciated that the contacting should be performed for sufficient time for the sulfonylamide compound (or derivative thereof) to exert a useful cleaning or disinfecting effect. In one specific embodiment, the contacting is for at least 10 seconds, 20 seconds, 40 seconds, 1 minute, 3 minutes, 5 minutes. 8 minutes, 10 minutes, 20 minutes. 30 minutes. 45 minutes, 60 minutes or 120 minutes. The concentration of active components used may vary as desired or necessary to decrease the amount of time the amount of contact time required. Persons skilled in the art easily determine these variations in the concentrations of active components and suitable contact times.

A third aspect of the invention relates to a method of treating and preventing infections caused by bacteria comprising administering a sulfonylamide compound or a derivative thereof to a patent in need thereof, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase.

In at least some embodiments of the first, second, third, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the invention the bacteria are selected from bacteria having: (i) a type II enzyme acetohydroxy acid synthase and not a type I enzyme acetohydroxy acid synthase; or (ii) a type III enzyme acetohydroxy acid synthase and not a type I enzyme acetohydroxy acid synthase.

In at least some embodiments of the first, second, third, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the invention the bacteria are gram negative bacteria. Examples of gram negative bacteria include Escherichia coli. Salmonella species, other

Enterobacteriaceae, Pseudomonas species, Haemophilus species, Burkholderi species,

Moraxella species, Helicobacter species, Stenotrophomonas species, Bdellovibrio species, acetic acid bacteria, and Legionella species. In other embodiments of the first, second, third, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the invention the bacteria are gram positive bacteria. Examples of gram positive bacteria include: Bacillus species, Listeria species, Staphylococcus species,

Streptococcus species, Enterococcus species, and Clostridium species.

In at least some embodiments of the first, second, third, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the invention the bacteria are pathogenic bacteria, for example human pathogenic bacteria. As used herein, the term "pathogenic" refers to bacterial cells capable of infecting and causing disease in a host, as well as producing infection-related symptoms in the infected host, such as fever. The term "disease" as used herein is intended to be synonymous with the terms "disorder" and "condition" in that these terms all reflect an abnormal condition of the human or animal body (or a part thereof) that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.

In at least some embodiments of the first, second, third, seventh, eighth, ninth, tenth, eleventh and twelfth aspects of the invention the bacteria may optionally be selected from the group consisting of Burkholderia pseudomallei, Pseudomonas aeruginosa, Haemophilus influenza, Neisseria ghonorrhea, Bordetella pertussis, Campylobacter jejuni, Bacillus anthracis, Listeria monocytogenes, Staphylococcus epidermidis, Streptococcus pneumoniae.

Staphylococcus aureus. Neisseria meningitidis, Acinetobacter baumannii, Burkholderia cepacia, Burkholderia cenocepacia and Vibrio cholera. Preferably, the bacteria are not

Mycobacterium, and more preferably the bacteria are not M. tuberculosis.

It is envisaged that some bacteria, and the diseases that they cause, which may usefully be controlled by the present invention include: Haemophilus influenza (respiratory tract infections), Neisseria ghonorrhea, Bordetella pertussis (whooping cough), Campylobacter jejuni (food poisoning), Bacillus anthracis (anthrax). Listeria monocytogenes (food poisoning), and Staphylococcus epidermidis (hospital acquired infection). Preferably, the bacterium and disease is not Mycobacterium tuberculosis and a disease caused thereby.

It is envisaged that the various aspects of the present invention may be useful in treating or preventing infections by multidrug resistant bacteria.

For the avoidance of doubt, the terms '"control" and "prevent' ^" as used herein in relation to bacteria / bacterial growth / disease etc. are to be interpreted broadly and include, not onl substantially stopping bacterial growth (e.g. by a bactericidal or bacteriostatic action), but also any reduction or retardation in bacterial growth (e.g. by a bactericidal or bacteriostatic action). Thus, the various aspects of the invention may find utility in reducing the rate of increase in the numbers of a population of a particular bacterium, stopping the growth of the population, reducing the number of bacteria in the population, or eliminating the bacteria in the population.

A fourth aspect of the invention relates to the use of a sulfonylamide compound or a derivative thereof in the preparation of a medicament for treating and preventing melioidosis or skin abscesses, bacteraemic pneumonia or sepsis associated with infection with the bacteria B. pseudomallei.

A fifth aspect of the invention relates to a sulfonylamide compound or a derivative thereof for treating and preventing melioidosis or skin abscesses, bacteraemic pneumonia or sepsis associated with infection with the bacteria B. pseudomallei.

A sixth aspect of the invention relates to a method of treating and preventing melioidosis or skin abscesses, bacteraemic pneumonia or sepsis associated with infection with the bacteria B. pseudomallei comprising administering a sulfonylamide compound or a derivative thereof to a patent in need thereof.

B. Pseudomallei is an opportunistic pathogen which can cause disease in susceptible individuals such as those that are immunocompromised or suffer from diabetes mellitus.

Accordingly, in some embodiments it is envisaged that the patient is immunocompromised or is suffering from diabetes mellitus.

The treating and preventing in the first to sixth aspects of the invention may comprise administering a sulfonylamide compound or a derivative thereof to a patient in need thereof by one or more various routes of administration. The route of administration may, for example, be topical, oral, intravenous, cutaneous, subcutaneous, transdermal, transmucosal. intramuscular, intraperitoneal routes. An example of a topical administration would be a disinfecting hand wash or lotion etc. which may, for example, be useful in a domestic environment and hospital environments to prevent the spread of potentially disease causing bacteria. The sulfonylamide compound or a derivative thereof may be administered topically, systematical!)', or locally (e.g. as an implant).

The formulation of medicaments comprising a sulfonylamide compound or a derivative thereof will be within the skill of persons skilled in the art and a wide variety of formulations are contemplated. The compounds can be formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations etc. Such medicaments may comprise, in addition to the sulfonylamide compound (or derivative thereof), a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient(s). Some examples of the kinds of materials that can serve as pharmaceutically acceptable substances include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatine, talc, oils, glycols, esters, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer solutions, coloring agents, releasing agents, coating agents, sweetening and flavoring agents, preservatives and antioxidants. The precise nature of the carrier or other material may depend on the route of administration and will be within the ability and judgement of the skilled person.

Medicaments for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Medicaments for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if des!ired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone. agar, and alginic acid or a salt thereof, such as sodium alginate.

Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e.. dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate. and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution. Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyi cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The medicaments employed in the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping, or iyophilizing processes.

The medicament is to be administered in a therapeutically effective amount (either as a single dose or as part of a series of doses). By a "therapeutically effective amount ^" is meant the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient such that beneficial or desired results are achieved.

Preferably, the medicament comprises from at least about 1% to about 50% (and more preferably from at least about 0.5% to about 25%) of the sulfonylamide compound (or derivative thereof) by weight based upon the total weight of the composition of the invention being employed. The sulfonylamide compound (or derivative thereof) may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time.

The precise effective amount for a patient will depend upon the severity of the disease state, general health of the patient, age, weight, whether the treatment is prophylactic or to treat an existing infection, the gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.

The effective dose for a given situation can be determined by routine experimentation and is within the judgement of the skilled person. For example, in order to formulate a range of dosage values, cell culture assays and animal studies can be used. In one embodiment, the dosage range is between 0.1 to 50mg/kg.

The term "patient' ^" refers to patients of human or other mammal and includes any individual it is desired to treat (including prophylatically) using the methods of the invention. However, it will be understood that '"patient" does not imply that symptoms are present. "Mammal" refers to any animal classified as a mammal, including humans, primates, domestic and farm animals (eg. sheep, cows, horses, donkeys, pigs), and zoo, sports, and pet companion animals, laboratory test animals (eg. rabbits, mice, rats, guinea pigs, hamsters) and captive wild animals (eg. foxes, deer, dingoes). Preferred companion animals are dogs and cats. In one embodiment, the patient is an immunocompromised patient (e.g. an AIDS patient, a chemotherapy patient, a malnourished individual, an elderly person, or the very young) or agricultural worker (e.g. a farmer, such as a rice paddy farmer).

Embodiments of the various aspects of the invention are envisaged whereby more than one (e.g. 2, 3, 4 or more) sulfonylamide compounds (or derivatives thereof) are employed. The more than one sulfonylamide compounds (or derivatives thereof) may be provided in the same formulation / composition etc. or separately (e.g. in the form of a kit of parts). It is envisaged that the more than one sulfonylamide compounds (or derivatives thereof) may be administered or employed simultaneously, separately or sequentially.

Also within the scope of the various aspects of the invention are embodiments where the one or more sulfonylamide compounds (or derivatives thereof) are used in conjunction with one or more further active agents. The one or more sulfonylamide compounds (or derivatives thereof) and the one or more further active agents may be provided in the same formulation / composition etc. or separately (e.g. in the form of a kit of parts) so that they may be administered or employed simultaneously, separately or sequentially. By a "further active agent' ^" we include compounds which are useful in achieving one or more of the following: (i) treating and preventing infections caused by bacteria, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase and bacteria having a type III enzyme acetohydroxy acid synthase; (ii) treating and preventing melioidosis or skiri abscesses, bacteraemic pneumonia or sepsis associated with infection with the bacteria B. pseudomallei: (iii) reducing or substantially preventing the growth of soil bacteria in a crop-planting site; and (iv) reducing or substantially preventing the growth of bacteria in a water supply.

The one or more further active agent may act synergistically or additively with the one or more sulfonylamide compounds (or derivatives thereof). Examples of potential "further active agents" may include anti-bacterial agents such as natural and synthetic penicillins and cephalosporins, sulphonamides, erythromycin, kanomycin, tetracycline, chloramphenicol, rifampicin and including gentamicin. ampicillin, benzypenicillin, benethamine penicillin, benzathine penicillin, phenethicillin, phenoxy-methyl penicillin, procaine penicillin, cloxacillin, flucloxacillin, methicillin sodium, amoxicillin, bacampicillin hydrochloride, ciclacillin, mezlocillin, pivampicillin, talampicillin hydrochloride, carfecillin sodium, piperacillin, ticarcillin, mecilUnam, pirmeciilinan, cefaclor, cefadroxil, cefotaxime, cefoxitin, cefsulodin sodium, ceftazidime, cettizoxime, cefuroxime, cephalexin, cephalothin, cephamandole, cephazolin, cephradine, latamoxef disodium, aztreonam, chlortetracyciine hydrochloride, clomocycmie sodium, demeclocydine hydrochloride, doxycycline, lymecychne, minocycline, oxytetracycline, amikacin, ftamycetin sulphate, neomycin sulphate, netilmicin, tobramycin, cohstin, sodium fusidate, polymyxin B sulphate., spectinomycin, vancomycin, calcium sulphaloxate. sulfametopyrazine, sulphadiazine, sulphadimidine, sulphaguanidine, sulphaurea, capreomycin, metronidazole, tinidazole, cinoxacin, ciprofloxacin, nitrofurantoin, hexamine, streptomycin, carbeniciHin, colistimethate, polymyxin B, furazolidone, nalidixic acid, trimethoprim-sulfaniethoxazole, clindamycin, lincomycin, cycloserine, isoniazid, ethambutol, ethionamide, pyrazinamide and the like.

In alternative embodiments of the invention, the one or more sulfonylamide compounds (or derivatives thereof) is / are the sole active agent(s) in terms of achieving the following: (i) treating and preventing infections caused by bacteria, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase and bacteria having a type III enzyme acetohydroxy acid synthase; (ii) treating and preventing melioidosis or skin abscesses, bacteraemic pneumonia or sepsis associated with infection with the bacteria B. pseudomallei; (iii) reducing or substantially preventing the growth of soil bacteria in a crop-planting site; or (iv) reducing or substantially preventing the growth of bacteria in a water supply.

The sulfonylamide compounds or pharmaceutically acceptable derivatives used in the first to sixth aspects of the invention can be packaged as articles of manufacture comprising:

packaging material (e.g. blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes and bottles), the one or more sulfonylamide compounds (or derivative thereof), and optionally instructions for use.

In addition to the use of sulfonylamide compounds (including derivatives thereof) in a clinical or therapeutic context, it is envisaged that the invention may find utility in treating soil or water. In this way, the transmission of bacterial pathogens, and consequent infections, may be reduced. In one particular embodiment, it is envisaged that sulfonylamide compounds

(including derivatives thereof) may be used directly on rice paddies containing Bp with the aim of reducing incidence of melioidosis in endemic regions.

Accordingly, a seventh aspect of the invention provides a sulfonylamide compound or a derivative thereof for preparing a composition for reducing or substantially preventing the growth of soil bacteria in a crop-planting site, in which the soil bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase, to thereby reduce the bacterial contamination of said crop-planting site.

An eighth aspect of the invention relates to sulfonylamide compound or a derivative thereof for reducing or substantially preventing the growth of soil bacteria in a crop-planting site, in which the soil bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase, to thereby reduce the bacterial contamination of said crop-planting site.

A ninth aspect of the invention relates to a method of reducing or substantially preventing the growth of soil bacteria in a crop-planting site comprising the step of providing a

sulfonylamide compound or a derivative thereof to said crop-planting site, in which the soil bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase.

In at least some embodiments of the seventh, eighth and ninth aspects of the invention, the crop-planting site is selected from the group consisting of: a paddy field, a field, an orchard, a glass house, a vineyard, forest, or a tea plantation. The crop may. for example, be an ornamental, vegetable, field, cereal, or fruit crop.

The sulfonylamide compound (or derivative thereof) as used in the seventh, eighth and ninth aspects of the invention may be provided in a composition which comprises one or more additional components selected from the group consisting of the following: a fungicide, an insecticide, a pesticide, an acaricides, a nematocide, a herbicide, a plant growth regulator, soil improvement agent and a fertilizer. Other moieties which may usefully be incorporated into a composition used in the seventh, eighth or ninth aspect of the invention includes carriers, surfactants, solid diluents and liquid diluents, and the preparation of suitable compositions will be within the abilities of the skilled person.

The sulfonylamide compound (or derivative thereof) may be provided in a form suitable for application to the crop-planting site. The sulfonylamide compound (or derivative thereof) may conveniently be provided in the form of a liquid (e.g. as a ready-to-use solution, emulsion or suspension) or solid (e.g. in the form of a powder, wettable powder, soluble powder or granules).

Various means of applying the sulfonylamide compound (or derivative thereof) to the crop- planting site are contemplated by the present invention. For example, the composition comprising the one or more sulfonylamide compounds (or derivatives thereof) may be sprayed on (by hand or by means of a spray race or arch or vehicle- or aircraft-mounted apparatus), applied by granule scattering, administered via watering etc. The amount applied may be varied within a wide range depending on the desired effect, the location of application, the time of application, the interval between treatments, weather and climate and the like.

The sulfonylamide compound (or derivative thereof) may be prepared either as a formulation ready for use on the crop planting site, or as a formulation requiring dilution prior to application, but both types of formulation comprise the sulfonylamide compound (or derivative thereof) in admixture with one or more carriers or diluents. The carriers may be liquid, solid or gaseous or comprise mixtures of such substances, and the sulfonylamide compound (or derivative thereof) may be present in a suitable concentration depending upon whether the formulation requires further dilution.

A tenth aspect of the invention relates to the use of a sulfonylamide compound or a derivative thereof for preparing a composition for reducing or substantially preventing the growth of bacteria in a water supply, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase, to thereby reduce the bacterial contamination of said water supply.

An eleventh aspect of the invention relates to a sulfonylamide compound or a derivative thereof for reducing or substantially preventing the growth of bacteria in a water supply, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase, to thereby reduce the bacterial contamination of said water supply.

A twelfth aspect of the invention relates to a method of reducing or substantially preventing the growth of bacteria in a water supply comprising the step of providing a sulfonylamide compound or a derivative thereof to said water supply, in which the bacteria are selected from the group consisting of bacteria having a type II enzyme acetohydroxy acid synthase or bacteria having a type III enzyme acetohydroxy acid synthase.

Preferably, in the seventh to twelfth aspects of the invention the concentration of the sulfonylamide compound is from about 5 to about 100 g / ha, preferably from about 5 to about 20 g /ha, about 10 to about 90 g / ha, about 20 to about 80 g/ha, about 30 to about 70 g / ha, about 40 to about 60 g/ha.. about 15 to about 90 g /ha, about 25 to about 85 g /ha. or about 35 to about 80 g /ha.

In one embodiment of the seventh to twelfth aspects of the invention the sulfonylamide compound is bensulfuron methyl and the concentration is from about 50 to about 85g / ha, preferably from about 60 to about 75 g/ha. In another embodiment, the sulfonylamide compound is metsulfuron methyl and the concentration is from about 5 to about 7g / ha.

In yet another embodiment, the sulfonylamide compound is chlorsulfuron and the concentration of is from about 15 to about 25g / ha, preferably from about 10 to about 18 g/ha.

As will be appreciated from the foregoing, the invention envisages various uses of sulfonylamide compounds (including their derivatives). In one embodiment, the

sulfonylamide compound is a sulfonylurea compound.

Optionally, the sulfonylurea compound is a compound of general formula 1 :

wherein

R ^! is H or CH _: -NHR ^a , where R ^a is an acyl radical,

R ² is Cl-C4-alkoxy, CO-(C,-C ₄ -alkoxy), S0 ₂ -(C C ₄ -alkyl), or a halide selected from F.

Br, CI. I or At,

R ³ is H or Ci-C ₄ -alkyl,

R ⁴ and R ^" independently of one another are identical or different and are selected from the group consisting of Cj-Ce-alkyl, Q-Cg-alkoxy and C]-C ₄ -alkylthio, each radical being optionally substituted by one or more groups selected from halogen, (_VC ₄ -alkoxy, Q-C ₄ - alkylthio, C ₃ -C ₆ -cycloalkyl. C2-C ₆ -alkenyl, C ₂ -C6-alkynyl, C ₆ -C ₆ -alkenyloxy or C ₆ -C ₆ - alkynyloxy,

R ⁶ is CH or N _. - R' is C]-C ₄ -alkyl. and

n is 0 or 1. The term "acyl," as used herein includes a carbonyl attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, or any other moiety where the atom attached to the carbonyl is carbon. Examples of acyl groups include formyl, alkanoyl (e.g. acetyl), aroyl (e.g. benzoyl), heteroaroyl, propanoyl, 2-methylpropanoyl, butanoyi, propenoyl, and caproyl. The term "alkoxy" includes alkoxy moieties having varying numbers of carbon atoms, with examples including methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy. and the like. C1-C4 alkoxy means an alkoxy moiety containing 1, 2, 3 or 4 carbon atoms. C I -C6 alkoxy means an alkoxy moiety containing 1, 2, 3, 4, 5 or 6 carbon atoms.

The term "alkylthio" as used herein refers to those alkyl groups attached to the remainder of the molecule via a sulfur atom. C]-C ₄ -alkylthio means an alkylthio moiety containing 1, 2, 3 or 4 carbon atoms.

The terms "halogen" or "halo" as used herein indicate fluorine, chlorine, bromine, astatine, or iodine.

The phrase "alkyl" as used herein includes linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof. Alkyl moieties, as used herein, may be saturated, or unsaturated, i.e., the moiety may comprise one, two, three or more independently selected double bonds or triple bonds. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl. butyl, iso-. sec- and tert-butyl, pentyl. hexyl, heptyl, 3-ethylbutyl, and the like. The number of carbon atoms in an alkyl group or moiety can vary. CI -C4 alkyl means an alkyl moiety containing 1, 2, 3 or 4 carbon atoms and likewise C1 -C6 alkyl means an alkyl moiety containing 1 , 2, 3, 4, 5 or 6 carbon atoms.

The phrase "alkenyl" as used herein includes a moiety' that comprises linked normal, secondary, tertiary or cyclic carbon atoms, i.e., linear, branched, cyclic or any combination thereof, that comprises one or more double bonds, e.g.. 1, 2. 3. 4. 5, 6 or more, typically 1 or 2. The number of carbon atoms in an alkenyl group or moiety can vary. C2-C6 alkenyl means an alkenyl moiety containing 2, 3, 4. 5 or 6 carbon atoms.

The term "cycloalkyl" includes cycloalkyl moieties having varying numbers of carbon atoms, with examples including cyclopropyl, cyclobutyl. cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptanyl. cycloheptenyl, phenyl, indanyl, indenyl, benzocyclobutanyl, dihydronaphthyl, tetrahydronaphthyl, naphthyl, decahydronaphthyl, benzocycloheptanyl, benzocycloheptenyl, 2-methylcyclopropyl, cyclobutyl, cyclopentyl, 2-methylcyclopentyl, 3- methylcyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and adamantyl. C ₃ -C ₆ -cycloalkyl means a cycloalkyl moiety containing 3. 4, 5 or 6 carbon atoms. C?-C ₆ -alkynyl means an alkynyl moiety containing 2, 3, 4, 5 or 6 carbon atoms.

CrC ₆ -alkenyloxy means an alkenyloxy moiety containing 3, 4, 5 or 6 carbon atoms. Similarly, C ₃ -C ₆ -aIkynyloxy means an alkynyloxy moiety containing 3, 4, 5 or 6 carbon atoms.

The phrase "optionally substituted" is used interchangeably with the phrase

"substituted or unsubstituted". Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and each substituent is independent of the other.Optionally, the sulfonylurea compound is selected from the group consisting of bensulfuron methyl, metsulfuron methyl, chlorimuron ethyl, amidosulfuron, chlorsulfuron, cinosulfuron, ethametsulfuron methyl, flazasulfuron, halosulfuron, imazosulfuron, nicosulfuron, primisulfuron, pyrazosulfuron ethyl, sulfometuron methyl, thifensulfuron. triasulfuron, tribenuron, triflusulfuron, prosulfuron, azisulfuron, cyclosulfuron, ethoxysulfuron, flupyrsulfuron-methyl-sodium, oxasulfuron, sulfosulfuron. rimsulfuron, iodosulfuron-methyl and its sodium salt, mesosulfuron-methyl and its sodium salt and foramsulfuron and its sodium salt.

In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES

Example 1

Interest in AHAS was raised when it was found that expression of this enzyme from Bp in a lab strain of E. coli provided a fitness advantage when grown in minimal media lacking in BCAA but that this fitness advantage was eliminated when BCAA were provided in the media. Taking advantage of this experimental system, it was found that sulfonylurea herbicides can also eliminate this growth advantage (Figure 1 ) but again, only in the absence of BCAA. These finding show that the type III variant of AHAS present in Bp can be inhibited by sulfonylurea herbicides.

It was next tested whether or not these compounds could affect growth of Bp and Bt in the laboratory. First, nine different sulfonylurea herbicides were added into minimal media used to grow Bt and it was found that seven of the nine had some inhibiton' effect on the growth of Bt (Figure 2) whereas four other herbicides, two that also target AHAS and two that target other pathways, did not show any effects. Inhibitory activity of various compounds was also tested using a standard laboratory protocol to measure the minimum inhibitory concentration (MIC) against either Bp or Bt. Of the nine, four of the compounds were often very effective at preventing growth showing activity down to 62.5 μΜ (approximately 25 μg/ml). In both assays, when BCAA was added to the media, sulfonylurea herbicides had no effect on growth. Of the four most effective sulfonylurea herbicides, two, metsulfuron methyl (trade name Escort) and chlorimuron ethyl (trade name Classic), have been tested for use on rice paddies in India and one, bensulfuron methyl (trade name Londax) is a commonly used herbicide on rice paddies in the US, China, and many other parts of the world.

Although these compounds are only effective in these laboratory studies when BCAA are absent, these culture conditions might approximately model nutrient conditions encountered outside the lab. Given that these compounds can successfully prevent the growth of plants and bacteria in soil, this suggests that BCAA are not widely available. In addition, during infections of animals, BCAA might also be scarce as a Bp mutant lacking AHAS grows much more slowly that wild-type in mouse infection studies. Were BCAA to be plentiful, one would expect that an AHAS mutant would grow without difficulty but this is not the case. These findings suggest that sulfonylurea herbicides might be effective at slowing or even preventing the growth of Bp both in the soil and during human infection

Example 2

Interest in the anti-microbial effects of SHa was raised as the result of a screen to identify genes in B. pseudomallei capable of conferring a growth advantage to E. coli in nutrient- limiting conditions, as previous studies have suggested that pathogens may encounter such conditions in vivo. A fosmid library in E. coli containing genomic DNA from 39 B.

pseudomallei clinical isolates was generated. End-sequencing and array-based comparative genomic hybridization (array-CGH) showed that the approximately 12,000-member library demonstrated extensive chromosomal coverage, lacking only regions associated with reference strain-specific genomic islands. Functional expression of B. pseudomallei genes in E. coli was confirmed, both at the level of single genes (metalloprotease production) and multi-gene clusters (O-antigen synthesis). To identify B. pseudomallei genes capable of conferring a growth advantage under nutrient-limiting conditions, the pooled fosmid library was grown in either rich or minimal media and enrichment assessed by array-CGH and endsequencing. Array-CGH repeatedly identified a region enriched after growth in minimal media centered at ilvl. End-sequencing of individually recovered fosmids confirmed this finding and revealed that these fosmids always encoded three genes, ilvIHC.

The ilvIHC (BPSL1 196 - 1198) cluster encodes the first two enzymes in the BCAA biosynthetic pathway (Fig. 7A). The catalytic and regulatory subunits of type III AHAS are encoded by //v/ and ilvH, respectively, and acetohydroxvacid isomeroreductase (EC 1.1.1.86) is encoded by ilvC. To explain the increased prevalence of //v/HC-containing fosmids from the previous experiment, the growth profiles of individual fosmids containing either ilvIHC (fosmid 18G 12) or a highly similar fosmid albeit one that does not include ilvIHC (fosmid 1 IB 19) were compared. High-resolution growth curves revealed that in minimal, but not rich media, /7v/HC-positive 18G12 always grew more as determined by maximum optical density and colony counts (data not shown). To determine which genes were necessary for this phenotype, 18G12 was subjected to transposon mutagenesis. Of the approximately 40 mutagenized fosmids tested, only transposon insertions in ilvl completely eliminated the growth advantage. An insertion in z ^' vHyielded an intermediate phenotype whereas an insertion in z ^' vC and all other loci yielded 18G12-like growth profiles. Consistent with expression of B. pseudomallei AHAS providing a growth advantage due to nutritional benefits, enrichment of z ^' z HC-containing fosmids was not observed when the pooled library was grown in minimal media supplemented with BCAAs (data not shown).

In the agricultural community, it is known that soil-dwelling bacteria are inhibited by SHs. As B. pseudomallei also inhabits damp soil, it was tested if SHs (Fig. 7B) could inhibit AHAS activity from B. pseudomallei. E. coli containing the z ^' /WHC-positive fosmid 18G12 was grown in minimal media and exposed to four different SHs individually. It is important to note that although wild-type strains of E. coli contain both type I and type III AHAS isozymes, most laboratory strains only contain the SH-resistant type I AHAS. In the presence of SHs, growth of 18G 12 in minimal media was dampened to levels now comparable to the parental E. coli strain (Fig. 3 A top panel). The effects of SHs were also evident when using minimal media containing valine, a potent inhibitor of type I but not type III AHAS thereby suppressing the endogenous E. coli type I AHAS. Under these conditions, only 18G12 was capable of any growth which could then be completely blocked by the addition of SHs (Fig. 3A, bottom panel). High levels of growth by both the parental E. coli strain and 18G12 were seen upon the inclusion of BCAAs regardless of the presence of SHs (data not shown). These results show that when BCAAs are limiting, type III AHAS in B. pseudomallei is inhibited by SHs.

It was then examined whether the inhibition of AHAS seen in B. pseudomallei might be extended to other human pathogens. Although many pathogenic bacteria contain genes annotated as either catalytic or regulatory subunits of AHAS, these annotations are often in conflict. For example, the genome of M. tuberculosis strain H37Rv contains a type I catalytic subunit, ilvBl (GenelD 887286), adjacent to a type III regulator)' subunit, z ^' /vH (GenelD

887226), yet M. tuberculosis is sensitive to SHs arguing that the AHAS encoded is a type III. To revisit this issue and determine the prevalence of AHAS isozymes across a range of clinically relevant bacteria (Table SI), a phylogenetic analysis using the AHAS catalytic subunit was performed which also contains the SH binding site. It was found that type I AHAS was exclusively present in Enter obacteriacae, which additionally always encoded a type II and / or type III isozyme as well, consistent with previous, albeit limited, findings. More importantly, it was found that almost all the other bacteria in the analysis, including important human pathogens such as P. aeruginosa, A. baumannii, Mycobacterium

tuberculosis, Streptococcus pneumoniae, and Staphylococcus aureus, only encoded a type III AHAS (Fig. 3B). To explore the potential interactions of these type III AHASs with SHs, computational structural studies were performed. Using the co-crystal structure of the SH chlorimuron ethyl bound to the catalytic subunit of AHAS from A. thaliana as a starting point, structural models of chlorimuron ethyl binding to the catalytic subunits of AHAS from B. pseudomallei or P. aeruginosa were generated (Fig. 3C). In both models, chlorimuron ethyl made numerous contacts with conserved residues and sites of known resistance mutations in other species (Table S2). Just as in the co-crystal structure, these contacts occured in a channel leading to the enzymatic active site. Overall, these findings suggest that SHs may exert potentially broad anti-microbial effects against a wide range of pathogens.

It was next tested whether SHs could inhibit growth in vitro of various human pathogens. When tested on Burkholderia thailandensis, a species closely related to B. pseudomallei, growth was completely inhibited for three days by the SHs chlorimuron ethyl or metsulfuron methyl (Fig. 4A). Bensulfuron methyl (Fig. 4A) and rimsulfuron (Fig. 8) prevented growth for nearly two days while other SHs demonstrated modest effects (Fig. 8). In contrast to SHs, imazapyr, a structurally unrelated herbicide that also inhibits AHAS from plants, did not show any inhibitory effects (Fig. 8). Using the same experimental design, four strains of

A.baumannii were also tested: two sequenced reference strains, AYE (Fournier PE, et al.

(2006) PLoS Genet 2(l ):e7.35) and 5377 (Smith MG. et al. (2007) Genes Dev 21(5):60I-614), and two local clinical isolates, KAb 1 and KAb 2. Both AYE and Ab2 are multidrug- resistant strains. Growth of all four strains was sensitive to chlorimuron ethyl and metsulfuron methyl (Fig. 4B) with metsulfuron methyl consistently being more potent. As before, addition of BCAAs to the media restored rapid growth (data not shown) suggesting that the effect of SHs is due to specific inhibition of AHAS.

Table SI. Sequences used in the construction of the AHAS phylogeny.

Onanism (Strain) NCBI-GenelD Isorvme

Acinewbaaer baumarinii ( ^' 5? ^" 7 i 49 Π404 m

Acinewbaaer baumannii (AYE t 60033S6 ΠΙ

Actinobaci!ius pieitropneumoniae (L20 ) 4S48528 Π

Actinobacilius pleiiropneumoniae (L20 ) 4848759 ΠΙ

Arabidopsis thaliana (Columbia ) S2401 N/A

Bacillus anthracis (Ames.) 10863S3 ΠΙ

Bad !i us siibriiis (168 > - 936~92 ΠΙ

Bordeteila pertussis (Toliama I) 26640S1 ΠΙ

Brucella suis ( 1330 ) 1167071 in

Burkholderia cenocepacia (AU1054) 4093524 m

Burkholderi cenocepacia (J2 15) 6953~55 III

Burkholderia mutnvoran (ATCC 17616; 5765S3" ΠΙ

Bwkholderia pseudomallei (K9624 ! 309394" ΠΙ

Burkholderia thai!andensis (ATCC 700388 ; 3847786 ΠΙ

Campylobaaer jejuni (NCTC 11168 i 904899 III

Cauiobacter crcscentus (CB 15) 943022 ΠΙ

Emerobacter ( 638 5110480 Π

Emerobacter ( 6 81 5113032 I

Emerobacter (6381 5113663 m

Escherichia co!i (K-12 MG1655 ) 948182 I

Escherichia con (K-12 MG1655 j 948 93 III

Haemophilus influenzae (Rd K 0 i 950449 m

Klebsiella pneumoniae (MGH 78578) 5339104 m

Klebsiella pneumoniae (MGK 78578 ) n

Klebsiella pneumoniae (MGK 78 78 ) 5341318 I

Listeria innocua (Clipl l262 1130811 ΠΙ

Listeria monocytogenes (EGD-e) 984805 III

Mycobacterium bovis (BCG Pastern' 1 1 ~3P2 ) 4698974 in

Mycobacterium marinum (M i 622596 ^" m

Mycobacterium tuberculosis (H3"R SS~28D ΠΙ

Neisseria gononhoeae (FA 10901 32S2600 m

Neisseria laciamica ( 020-06 i 1000~32S in

Neisseria meningitidis (22491 ) 90765S ni

Pseudomonas aeruginosa (PA 4 > 4384635 ΠΙ

Pseudomonas aeruginosa (PAOl ) 881496 m

Ralstonia solanacearum (GMIIOOO) ^" 122091 $ in

Salmonella enterica (Typhmiurium LT2 i 1251634 ΠΙ

Salmonella enterica (Typ urium LT2 ) 125 320 I

Salmonella enterica (Typlumurium LT2.i 125542 ^" Π

Staphylococcus aureus (Μιι50ι 1122066 in

Staphylococcus epidenmdis (ATCC 12228) 105"224 in

Stenotrophomonas maitophilia (Κ2~9α · 6394810 n

Snvptococcus pneumoniae (TIGR4) 930383 ΠΙ

Vibrio cholerae (Ol El Tor Nl 6961 ) 2613025 in

Vibrio cholerae ⁽ Ol Ξ1 Tor Nl 6961 ) 2614464 n

Vibrio vulnificus fCMCP6 i 11 "596 ΠΙ

Vibrio vulnificus ⁽ CMCP6 ) 11 ^" 7966 n

Yersinia pestis ⁽ C0921 11 ^" 3384 m

Yersinia pestis (C092 /> 1 P5125 I

Yersinia pestis (C092 ) 11 ^" 6"3 n Table S2. Alignment of kev residues in three different AHAS catalytic subunits.

Location of known

thalimia B. Dsendoiiiciilei P.aemgitiosa resistance mutations ^{0 A} Conserved ^{0 B} 256 180 (K163 ) ^c Yes Ye ^D

R3 ^" 301 R286 No ^E Yes

W5 ^" 4 W5D0 W4S6 Yes Yes in type Π and type

ΠΙ bur Q present in type I

(F57S ⁾ (Ε504 · Q490 Yes No

(P652 ) Q572 (Ί5 8 ) No No f S653 i ! ^' A5~3 ^' i R.559 Yes No

(A) Data about the location of known resistance mutations is from Duggleby ex ah. Pianr Phvsiol Biochem 2008

(B I Conservation was determined by referring to our alignment of 50 sequences across 3S species used when performing the phylogenenc analysis.

(C) Residues in parenthesis are the equivalent, aligned residue but do not appear to make key strucraral contacts and are thus not depicted in the model.

I ^' D K conserved expect for A. pieuropneumoniae where there is a R present.

(E i Two residues where resistance mutations are found occur just before this residue.

To study the effects of SHs on B. pseudomallei in a BSL3-compliant manner, minimum inhibitory concentration (MIC) values were determined using a modified microdilution procedure in which rich media was substituted with minimal media. Similar experiments were also performed using B. thailandensis and P. aeruginosa. Of the numerous SHs tested against all three bacteria, chlorimuron ethyl and metsulfuron methyl again were the most effective with MIC values ranging from 15.6 - 62.5 μΜ (Table 1). Chlorimuron ethyl and metsulfuron methyl were further tested against 29 local, clinical P. aeuruginosa isolates, including multidrug resistant strains, resulting in MIC50 values for both SHs of 62.5 μΜ, with no differences seen between the multidrug resistant and sensitive strains (data not shown). As it is difficult to compare these MIC values against standard values, the Burkholderia species was also used to test sensitivity to chloramphenicol, a clinically effective antibiotic, under the modified conditions and comparable levels of inhibitory activity were found (Table 1 ). In these as in the other experiments, addition of BCAAs to the minimal media rendered SHs ineffective (data not shown). Table 1. MICs (μΜ) of Selected AHAS Inhibitors and Chloramphenicol in Minimal Media

Burkhotderia Burk olderia Burkholderia Pseudomonas Pseudomonas pseudomallei pseudomallei thailandensis aeruginosa aeruginosa K 6423 strain 22 ATCC 700388 PAOl PA14

Growth inhibition by SHs could be due to bacteriostatic or bacteriocidal effects. To address this, microdilution MIC assays were again performed after which the number of colony forming units (CFUs) present in the well corresponding to the MIC were assessed. For both B. thailandensis and P. aeruginosa, MIC levels of ceftazidime resulted in a 50- to 1000-fold decrease in CFUs relative to input indicating bacteriocidal effects. In contrast, MIC levels of chloramphenicol or SHs resulted in a slight increase in CFUs relative to input (Fig. 4C). Even at SH concentrations up to 8-times MIC, there was only a minimal reduction in CFUs (data not shown) suggesting that SHs are bacteriostatic.

To directly confirm the mechanism of SH-induced growth inhibition, mutational studies in P. aeruginosa were conducted. Spontaneous resistance to chlorimuron ethyl was selected for and five mutants were identified. Sequencing ilvIH from the five mutants revealed that each one contained a single, nonsynonymous mutation in ilvl resulting in three different variants of AHAS : P 104S, P 104L, or R559C (Fig. 5 A). In A. thaliana, mutations at this proline (P 197) also confer resistance to sulfonylurea herbicides (Duggleby RG, McCourt JA, & Guddat LW (2008) Plant Physiol Biochem 46(3):309-324.). Structural models of the proline mutants found that these changes relieved stress on a nearby loop region allowing the formation of a small alphahelix resulting in a partial collapse of the channel leading to the SH binding site (data not shown).

The structural models also showed that mutations at R559 might lead to resistance due to the loss of multiple H-bonds between this residue and chlorimuron ethyl (Fig. 3C). To

demonstrate that these mutations alone were sufficient to confer resistance, plasmids were generated expressing FLAG epitope tagged versions of either wild-type z7v/or the three different mutants. Upon expression in P. aeruginosa, only the mutant versions of ilvl conferred resistant to chlorimuron ethyl (Fig. 5B) with both the wild-type and mutant versions expressed at similar levels (data not shown). These results demonstrate that in P. aeruginosa, SHs prevent growth by directly targeting AHAS.

It was then investigated if SHs could inhibit bacterial growth outside of chemically denned in vitro conditions. Both B. pseudomallei and B. thailandensis can invade eukaryotic cells and replicate within the cytoplasm, a niche that might protect the bacteria from the inhibitor} effects of SHs. To test for this, intracellular replication assays incorporating SHs were performed using A549 human respiratory epithelial cells infected with B. thailandensis. In these experiments, BCAAs were readily present in the tissue culture media. Nevertheless, treatment with either chlorimuron ethyl or metsulfuron methyl greatly slowed intracellular replication as measured by both intracellular CFUs (Fig. 6A and Fig. 9A) and the prolonged survival of the A549 cells (Fig. 6B and Fig. 9B).

To test SHs for therapeutic potential, two different mouse infection studies were performed. In the first, mice were infected intranasally with a highly virulent strain of B. pseudomallei and A549 cells (Fig. 6B and Fig. 9B). To test SHs for therapeutic potential, two different mouse infection studies were performed. In the first, mice were infected intranasally with a highly virulent strain of B. pseudomallei and orally treated twice daily with either phosphate- buffered saline (PBS) or metsulfuron methyl (50 mg/kg). After eight days, all of the PBS- treated mice had died whereas all of the metsulfuron methyl -treated mice were still alive (Fig. 6C) leading to a statistically significant increase in survival over the course of the entire experiment (p < 0.01; Log-rank test, Yates corrected). Significant levels of protection were also seen when mice were exposed to a higher inoculums (Fig. 10). In a second set of experiments, mice were infected with P. aeruginosa intratracheally and then given a single tail-vein injection with either PBS or metsulfuron methyl (200 mg/kg) (Fig. 6D-E). Even with just this single dose regimen, the group treated with metsulfuron methyl showed a much higher rate of survival than the PBS-treated group (p < 0.01 ; Log-rank test. Yates corrected). In addition, treatment with metsulfuron methyl resulted in nearly a 100-fold reduction in CFUs present in the lungs 24 hours after infection (p < 0.01; Mann-Whitney rank-sum test). No observable toxicities were seen in either of these regimens which is consistent with the treatment doses used being well below reported levels of acute, chronic or reproductive toxicity (DuPont Escort XP Technical Bulletin, K-14796, 2007).

Discussion Based on these findings, it is proposed that SHs constitute a new class of antibiotics with potentially broad applications. A wide variety of low cost, non-toxic SHs are readily available for testing against additional Gram-negative and Gram-positive pathogens. The structural models suggest potential starting points for chemical modifications to increase potency or specificity.

It is clear that SHs can inhibit the growth of various antibiotic-resistant Gram-negative pathogens when BCAAs are absent in vitro. Notably, evidence suggests that BCAA-limiting conditions are likely to occur in vivo as well. For example, BCAA auxotrophs of certain pathogens are attenuated when inoculated into the blood, lungs, or peritoneum (Atkins T, et al. (2002) Infect Immun 70(9):5290-5294: Cuccui J. et al. (2007) Infect Immun 75(3): 1 186- 1 195; Ulrich RL, Amemiya K, Waag DM, Roy CJ, & DeShazer D (2005) Vaccine

23(16): 1986-1992 ; Hondalus MK, et al. (2000) Infect Immun 68(5):2888-2898;

Subashchandrabose S, et al. (2009) Infect Immun 77(1 1 ):4925-4933). SHs then might be effective at combating human pathogens lacking a type I AHAS during infections of particular anatomical sites. As the lungs appear to be one of these sites, increasingly common cases of ventilator-associated pneumonia by P. aeruginosa and A. baumannii might be candidates for treatment. When treating immunocompromised patients, bacteriostatic agents might actually be the preferred course of treatment so to avoid overwhelming a poorly functioning immune system with bacterial debris such as endotoxin (Pankey GA & Sabath LD (2004) Clin Infect Dis 38(6):864-870.).

Outside of therapeutic applications, one sulfonylurea in particular, bensulfuron methyl, might have an immediate application outside of the clinic. Bensulfuron methyl is marketed as Londax to control broadleaf weed growth in rice crops. As rates of melioidosis infection are known to correlate with the levels of B. pseudomallei present in the soil, treating

B. pseudomallei-contaminated rice paddies with bensulfuron methyl might reduce the burden of melioidosis among rice farmers. As these compounds work as herbicides, it is clear that BCAAs are not plentiful in the soil. Perhaps then small, carefully controlled pilot studies could begin to address this potentially exciting application. To verify such a use, these compounds could be used in the lab to see if they can block the growth of Bp under conditions designed to mimic the nutrient conditions in soil. It could then be determined whether treating a rice paddy known to contain Bp with e.g. bensulfuron methyl (Londax), a sulfonylurea herbicide used to prevent the growth of weeds in rice paddies all across the world, would reduce the levels of Bp in the soil. If treating Bp containing rice paddies with bensulfuron methyl or similar compounds results in decreased levels of Bp, then a larger and longer study could then be carried to see if treatment of rice paddies decreases the actual number of melioidosis cases. Materials and Methods

High-Resolution Growth Curves

High-resolution growth curves were obtained using a Bioscreen C MBR (Oy Growth Curves Ab Ltd) integrated incubator and micro-plate reader. All cultures (250 μΐ) were grown aerobically at 37 °C with SHs included as indicated and optical density readings at 600 nm (OD600) collected at regular intervals. Preparation of input inoculums varied depending on the experiment as described in SI Materials and Methods.

Phyiogenic Analysis

Protein sequences corresponding to fifty catalytic AHAS subunits from thirty-eight species (Table S 1 ) were aligned (Clustal W2), gaps stripped (BioEdit), and a phylogenetic tree derived using maximal likelihood methods (PHYLIP). Further details are provided in SI Materials and Methods.

Structural Models

The catalytic subunits of AHAS from B. pseudomallei K96423 (BPSL1196) and

P. aeruginosa PAOl (PA4696) were modeled using the co-crystal structure of chlorimuron ethyl bound to the catalytic subunit of AHAS from A. thaliana, 1 YBH, as a template. Detailed procedures are provided in SI Materials and Methods.

Determination of Minimum Inhibitory Concentrations

Minimum inhibitory concentrations (MICs) were determined in M9 minimal medium based off the two-fold microdilution broth technique described in the Clinical and Laboratory Standards Institute guidelines (M7-A6) and described in further detail in SI Materials and Methods.

Chlorimuron Ethyl-Resistant AHAS from P. aeruginosa

Spontaneous P. aeruginosa PAOl resistance mutants were selected for on M9 plates containing chlorimuron ethyl. Sequencing ilvIH from the five mutants identified three separate, nonsynonymous changes in ilvl. Expression plasmids containing either wild-type or mutant versions of ilvl were constructed and used to transform parental P. aeruginosa PAOl as described SI Materials and Methods.

Intracellular Replication

Human respiratory epithelial cells (A549) were exposed to B. thailandensis ATCC

700388 for two hour after which the cells were washed and cultured in media containing gentamicin (250 μg/ml) and either DMSO (1%) or chlorimuron ethyl (2 mM). At indicated time points, the cells were again washed and intracellular CFUs determined as is detailed in SI Materials and Methods.

Mouse Infections

The protocols describing B. psendomallei and P. aeruginosa mouse infections are described in SI Materials and Methods.

Example 3 - Supporting Information

Construction and Validation of the B. pseudomallei Fosmid Librar

The fosmid library was created using the CopyControl HTP Fosmid Library Production Kit (Epicentre Biotechnologies). Input DNA consisted of genomic DNA from 39 clinical isolates and the library is composed of approximately 12.000 colonies. To prepare a pooled version of the fosmid library, all colonies were robotically transferred from the 384-well master plates on to Q-trays (Genetix) containing LB agar and chloramphenicol (12.5 μ°/ιη1). After overnight growth at 37 °C, the colonies were harvested in LB and, after the addition of glycerol (40% final concentration), small, single-use aliquots of the pooled library were stored at -80 °C.

To assess the coverage and integrity of the library, both end-sequencing of individual fosmids and array-CGH of the pooled library were performed. For end-sequencing, cultures of randomly chosen fosmids were grown in LB containing streptomycin (100 μg/ml), trimethoprim (50 μg mI). chloramphenicol (12.5 μg/ml) and fosmid induction solution (Epicentre) aerobically at 37 °C overnight. Fosmid DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen). For each fosmid DNA sample, two sequencing reactions were performed using primers that bound to and sequenced away from the fosmid backbone (Fl 5 ^' - AGGCGATTAAGTTGGGTAAC-3 ' and Rl 5'-AGCGGATAACAATTTCACAC-3 '). The resulting sequencing data was mapped back to the B. pseudomallei K96423 genome.

For array-CGH, fosmid DNA was purified using the FosmidMAX DNA Purification Kit (Epicentre) from a culture of the pooled library grown in LB containing streptomycin ( 100 μg/ml), trimethoprim (50 μg ml), chloramphenicol (12.5 μg/ml) and induction solution (Epicentre) aerobically at 37 °C overnight. The resulting fosmid library DNA or reference genomic DNA from B. pseudomallei K96423 was hybridized to a custom designed tiling array (Nimblegen Systems) in which 50 bp probes were tiled approximately every 35 bp across a single strand of the B. pseudomallei K96423 genome except for highly repetitive regions resulting in an array with 97% genomic coverage. Fosmid DNA was sonicated to fragments approximately 100 to 500 bp in length, fiuorescently labeled (Kreatech BAC Array Labeling Kit), and hybridized to the arrays for 16 h at 60 °C. The arrays were then washed, dried, and scanned using an Axon Genepix 4000B scanner (Molecular Devices) at 5 μηι resolutions. The fluorescent images were converted to digital signal intensities using

NimbleScan Version 2.4 (Nimblegen).

To identify regions absent from the fosmid library using the collection of single-color hybridizations, the entire data set (two library and twelve B. pseudomallei K96423 reference hybridizations) was quantile normalized and then transformed as described by Skvortsov and colleagues (Skvortsov D, Abdueva D, Stitzer ME, Finkel SE, & Tavare S (2007), BMC Bioinformatics 8:203). Using this approach, it is important that any regions absent must only be missing in a small fraction of the overall data set which is why so many reference samples were included. After normalization and transformation. CGH Scan (Anderson BD, et al. (2006). BMC Genomics 7:91) was used to identify missing regions. This program uses a random walk method to identify missing regions in a high-density data set and assigns each of these regions a separate statistical significance score while also controlling for the overall statistical significance of the analysis. In addition, this program allows the user to specify both an intensity cut-off value below which probes are considered absent and a Bonferroni corrected significance cut-off for entire analysis. For the analysis, an intensity cut-off of mean - 2 x standard deviations and a significance cut-off at 0.001 were used. Other values were deemed sub-optimal either due to the absence of identified gaps in the library (as occurred when the intensity cut-off was set at mean - 3 x standard deviations) or because too many, small, low pvalue gaps were found in the reference data sets (as occurred when lower significance thresholds such as 0.05 or 0.01 were used).

Growth Enrichment in Rich or Minimal Media

An aliquot of the pooled fosmid library was used to inoculate a 50 ml culture of LB or M9 minimal media and grown to early-stationary phase (typically 8 - 12 h for LB, 24 - 30 h for M9). In addition to M9 salts (Raleigh EA, Elbing K. & Brent R (2002) Selected topics from classical bacterial genetics.Cw/r Protoc Mo! Biol Chapter 1 :Unit 1 4.). the M9 media also included glucose (0.2%), thiamine (0.5 μg/ml), and leucine (75 μg/mL). Leucine is included as the parental strain of E. coli for the fosmid library, EPI300 (Epicentre), is derived from a leucine auxotroph. Both the LB and M9 cultures also contained streptomycin (100 μg ml), trimethoprim (50 μg ml), chloramphenicol (12.5 μg/ml) and induction solution (Epicentre).

To assess enrichment by array-CGH. fosmid DNA was purified from both cultures using the FosmidMAX DNA Purification Kit (Epicentre). In these experiments, a custom-made tiling array (Nimblegen) was again used but these differed from the ones used earlier as the probes on these arrays were tiled across both strands of the B. pseudomallei K96423 genome. In addition, these experiments were performed as two-color hybridizations in which fosmid DNA was sonicated into fragments approximately 100 to 300 bp in length, fluorescently labeled with either Cy3 or Cy5 (Kreatech Diagnostics aRNA Labeling Kit), and hybridized to the arrays for 17h at 65 °C. Processing and scanning of the arrays was performed as described previously. To determine which regions of the genome were enriched, the ratio of signal from the M9 culture to the LB culture was calculated, log2 transformed, and smoothed using a 1 kb sliding window.

To assess enrichment by end-sequencing, early-stationary phase cultures were streaked out to LB plates and colonies randomly selected for end-sequencing. DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen) and end-sequencing was performed as described earlier. After mapping the sequencing results back to the B. pseudomallei K96423 genome, the resulting fosmid locations were then plotted across the genome such that the height of any peak corresponded to the number of fosmids which contained that part of the genome.

In addition to the LB and M9 cultures described above, similar experiments were also performed with M9 containing valine and isoleucine (75 μg/ml each) along with leucine (75 g/mL). When the fosmid library was cultured in M9 contained all three BCAAs, there was no evidence for enrichment of any loci either by array-CGH or end-sequencing.

High-Resolution Growth Curves

For experiments using members of the fosmid library, glycerol stocks were streaked out to LB agar plates containing streptomycin (100 μg/ml). trimethoprim (50 μg/ml), and

chloramphenicol (12.5 με/πιΐ) and incubated overnight at 37 °C. The next day, a single colony was picked and diluted into M9 salts (250 μΐ) of which 5 μΐ was used to inoculate LB or M9 medium (250 μΐ) containing the same antibiotics as above along with induction solution (Epicentre). For these experiment, the M9 medium contained glucose (0.2%), thiamine (0.5 μg/ml) and leucine (75 μg/ml). For cultures of the parental E. coli strain, EPI300 (Epicentre), chloramphenicol was omitted.

For the B. thailandensis experiments, the following changes were made: streptomycin, trimethoprim, and chloramphenicol were omitted; the 5 μΐ inoculum was prepared by diluting one colony into 100 μΐ of M9 salts: and the M9 medium contained glucose (0.2%) and thiamine (0.5 μg/ml).

For the A. baumannii experiments, an LB agar plate containing single colonies was again prepared and used to inoculate a starter culture of 3 ml of M9 medium containing acetate (0.2%) and thiamine (0.5 μg/ml). After 4 h of aerobic growth at 37 °C, the starter culture was then diluted to an OD600 of 0.01 in M9 salts and 5 μΐ used to inoculate M9 medium (250 μΐ) as described above again without any antibiotics. The experimental setup for P. aeruginosa experiments was similar to that for B. thailandensis except that thiamine was omitted and trimethoprim (200 μ§/ιη1) was included in experiments involving pUCP28T.

The reported OD600 readings were the average value from three different wells in one experiment and each experiment shown is representative of at least two others.

Transposon Mutagenesis and Screening

Fosmid DNA for was purified using the FosmidMAX DNA Purification Kit (Epicentre) and subjected to transposon mutagenesis using the EZ-Tn5 <KAN-2> Insertion Kit (Epicentre). Approximately 40 kanamycin resistant (50 g/ml colonies were then subjected to

endsequencing and growth screening in both LB and M9 medium. High-resolution growth curves were obtained as described previously using M9 medium containing glucose (0.2%). thiamine (0.5 μg/ml). leucine (75 μg/ml). streptomycin ( 100 μg ml), trimethoprim (50 μ§/ηι1), chloramphenicol (12.5 ugjm\), kanamycin (50 μg/ml) and induction solution (Epicentre).

For end-sequencing, fosmid DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen) from overnight cultures grown in LB containing streptomycin (100 μg/ml), trimethoprim (50 μg/ml), chloramphenicol ( 12.5 μg ml), kanamycin (50 μg ml) and induction solution

(Epicentre). For each fosmid. two sequencing reactions were performed using primers that bound near the ends of and sequenced away from the transposon (Kan FP- 1 5'- ACCTACAAC AAAGCTCTCATCAACC-3 ' and Kan RP-1 5 ^' - GCAATGTAACATCAGAGATTTTGAG-3 both from Epicentre). The subsequent sequencing data was mapped back to the B. pseudomallei K96423 genome.

Phylogenetic Analysis

Catalytic subunits of AHAS were identified by searching through the genomes of select organisms, mostly pathogenic bacteria, using the enzyme commission number 2.2.1.6 (Durfee T, et al. (2008), J Bacteriol 190(7):2597-2606; and Duggleby RG, McCourt JA. & Guddat LW (2008), Plant Physiol Biochem 46(3):309-324). In most genomes, multiple genes have been annotated as AHAS catalytic subunits. To ensure that only actual catalytic subunits were analyzed, only those genes adjacent to a regulatory subunit were included. Nucleotide sequences of fifty AHAS catalytic subunits from thirty-eight species were translated into protein sequences, aligned (ClustalW2), and gaps stripped from the alignment (BioEdit). A phylogenetic tree was derived using maximum likelihood methods and subjected to bootstrap analysis 100 times (PHYLIP). Other phylogenetic approaches such as distance-matrix methods or parsimony analysis (PHYLIP) yielded similar trees.

Structural Models The catalytic subunits of AHAS from B. pseudomallei K96423 (BPSL1 196) and

P. aeruginosa PAO l (PA4696) were modeled as dimers using Modeler 9.7 (Sali A &

Blundell TL (1993) J Mol Biol 234(3):779-815) with 1YBH (McCourt JA, Pang SS, King- Scott J, Guddat LW, & Duggleby RG (2006) Proc Natl Acad Sci USA 103(3):569-573), the co-crystal structure of chlorimuron ethyl bound to the catalytic subunit of AHAS from

A.thaliana, as the template. Docking chlorimuron ethyl to the modeled dimers was carried out using a protocol that imparted flexibility both to the ligands and the protein. Since the protein is too large to be included as a flexible unit, only residues within a 15 A sphere across the dimeric interface (which consists of the active site) were included to be flexible. The

CHARMm force field (Brooks BR et al. (2009), J Comput Chem 30(10): 1545-1614) was used to generate the conformers of the protein. A grid with resolution of 0.5 A (default) was set on this sphere, and the ligand accessible grid was defined such that the minimum distance between a grid point and the protein was 2.0 A for hydrogen atoms and 2.5 A for heavy atoms.

The docking procedure was started with the random generation of ligand conformations. For each conformation, fitting of the ligand into the active site was carried out by comparing the shape of the ligand to the shape of the active site and, if deemed acceptable, a dock energy was computed between the protein and the ligand. Annealing from 300 K to 700 K was simulated next and the top ten (i.e. the best docking scores) poses of the ligand were rescored. The two poses with the best scores for each molecule were retained and subject to molecular dynamic (MD) simulations.

For the simulations, the N- and C-termini were capped with acetyl (ACE) and N-methyl (NME) moieties respectively to keep them neutral. MD simulations were performed with the SANDER module of the AMBER9 package (Case DA. et al. (2006) AMBER 9, University of California. San Francisco. AMBER 9, University of California, San Francisco) employing the all-atom Cornell force field (Cornell WD, et al. (1995), J Am Che Soc 1 17(19):5179-5197). Chlorimuron ethyl parameters were generated using antechamber (Wang J, Wolf RM, Caldwell JW, Kollman PA, & Case DA (2004) J Comput Chem 25: 1 157-1174; and Wang J, Wang W. Kollman PA, & Case DA (2006) J Mol Graph Model 25:247-6077). Each system was solvated with a T1P3P water box (Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, & Klein ML (1983) J Chem Phys 79:926-935) whose sides were at least 8 A from any protein atom. Particle Mesh Ewald method (PME) (Darden T & York DP, L. (1993) J Chem Phys 98: 10089-10092) was used for treating the long range electrostatics. All bonds involving hydrogen were constrained by SHAKE (van Gunsteren WF & Berendsen HJC (1977) Mol Phys 34: 131 1-1327) to enable an integration time step of 2 fs. Initially, the whole system was minimized for 3.000 steps, to remove any unfavorable interactions. Subsequently, the systems were each heated to 300 K for 75 ps under NPT conditions followed by 10 ns simulations at constant temperature (300 K) and pressure ( 1 atm) with structures saved every 1 ps. The free energy of binding (AGbind) of the chlorimuron ethyl to AHAS was computed using the MM-GBSA (molecular mechanics/Generalized Born surface area) method (Bashford D & Case DA (2000) Annu Rev Phys Chem 51 : 129- 152: and Tsui V & Case DA (2000) J Am Chem Soc 122:2489-2498) using the GB module (Jayaram B, Sprous D, & Beveridge DL (1998) J Phys Chem B 102:9571-9576) in Amber while the non- polar component was estimated from the solvent accessible surface area using MOLSURF(Connolly ML (1983) Science 221 (4612):709-713) using: AGsolv.np = 0.00542*SASA + 0.92 (Sanner MF. Olson AJ, & Spehner JC (1996) Biopoly ers 38(3):305- 320). Each energy term was averaged over frames taken ever}' 50 ps from the simulations. The representative structural models used here were based on the best AGbind (data not shown) values of the respective poses sampled.

To examine the structural effects of the chlorimuron ethyl-resistant mutations (P104L. P104S, and R559C), the model of the wild-type P. aeruginosa catalytic subunit was mutated and subjected to MD simulations using AMBER for 10 ns each.

Determination of Minimum Inhibitory Concentrations and Bacteriostatic Effects

Minimum inhibitory concentrations (MICs) were determined in M9 minimal medium based off the two-fold microdilution broth technique described in the Clinical and Laboratory Standards Institute guidelines (M7-A6). In brief, starter cultures in LB broth were grown aerobically at 37 °C for 3 - 4 h. The exponential-phase cultures were then washed twice with M9 salts before resuspending in M9 medium and diluted such that each well of a 96-well microtiter plate was inoculated with approximately 5 ^x 104 CFUs. The M9 medium used in these experiments contained glucose (0.2%) and thiamine {0.5 μg/ml). Nine different concentrations of sulfonylurea herbicides were typically tested in each experiment with concentrations ranging from 500 μΜ to 1.95 μΜ. For B. pseudomallei and B. thailandensis, growth was assessed by eye after 20 - 24 h; for P. aeruginosa, growth was assessed after 16 - 20 h. The values in Table 1 were based on 3 - 5 independent experiments.

To determine if compounds had bacteriostatic or bacteriocidal effects, MIC assays were performed as described above and the well corresponding to the MIC serially diluted in M9 salts before plating on LB agar to determine the number of CFUs present in the MIC well. The "input" well was prepared along with the rest of the microdilution assay plate but before incubating the plate at 37 °C overnight, the well was immediately harvested, serially diluted and plated to determine "input" CFUs.

Chlorimuron Ethyl-Resistant AHAS from P. aeruginosa To select for spontaneous resistance to chlorimuron ethyl, overnight cultures of

P. aeruginosa PAOl in LB were washed twice in M9 salts and then plated on to M9 plates containing glucose (0.2%) and chlorimuron ethyl (400 uM). Two days later, five chlorimuron ethyl-resistant colonies were isolated from which genomic DNA was purified and ilvIH sequenced.

The E. coli-P. aeruginosa shuttle vector, pUCP28T (West SE, Schweizer HP, Dall C, Sample AK, & Runyen-Janecky LJ (1994) Gene 148(1 ): 81-86), was used to express wild-type or mutated versions of ilvl in P. aeruginosa PAOl . Wild-type ilvl from P. aeruginosa PAOl was PCR amplified to include a C-terminal glycine spacer (5x) and FLAG epitope along with the necessary restriction sites for cloning into the MCS of pUCP28T. Mutant versions of ilvl were generated using the QuikChange II XL Site Directed Mutagenesis Kit (Agilent). Prior to usage, the entire ilvl gene from each of the pUCP28T-based constructs was sequenced.

Electrocompetent P. aeruginosa PAOl was generated as previously described (Choi KH, Kumar A, & Schweizer HP (2006) J Microbiol Methods 64(3):391 -397) and the presence of pUCP28T was selected for using trimethoprim (200 μg/ml).

Intracellular Replication Assays

A stationary phase culture of B. thailandensis ATCC 700388 was washed twice with phosphate-buffered saline (PBS) before resuspending in DMEM containing heat-inactivated FBS (10%: Invitrogen) and then used to infect A549 human respiratory epithelial cells at a multiplicity of infection of approximately 10. To promote contact between the adherent cells and bacteria in suspension, the tissue-culture plates were spun at 200 χ g for 10 min at room temperature prior to incubation at 37 °C. After two hours, the A549 cells were washed three times with PBS and then cultured in DMEM containing heat-inactivated FBS (10%), gentamicin (250 μg/ml) to kill the extracellular bacteria, and DMSO (1 %) or AHAS inhibitors (2 mM) as indicated. At various time points, the A549 cells were washed three times with PBS and lysed by exposure to Triton-X (0.1%) at 37 °C for 20 min. Lysates were harvested by scraping, serially diluted in M9 salts and plated on LB agar to determine the CFUs present in the lysate.

Mouse Infections

Survival curves were generated using B. pseudomallei strain 22. also known as KHW, a highly virulent, clinical isolate from a fatal case of melioidosis in Singapore (Tan GY, et al. (2008) J Med Microbiol 57(Pt 4):508-515). Bacterial colonies grown on tryptone soya agar were resuspended in PBS to achieve an OD600 of 1.9 (equivalent to 109 CFU/mL) and then diluted to 5 χ 103 CFU/mL. Bacterial suspensions were plated in duplicate for retrospective determination of the actual number of viable bacteria delivered to the mice. Female 6-week-old BALB/c mice were intranasally challenged with 1 x l02 CFUs, five-times the established LD50 (Tan GY, et al. (2008) J Med Microbiol 57(Pt 4):508-515). For intranasal inoculation of bacteria, the mice were lightly anaesthetized with 3% isoflurane in oxygen. The desired dose of bacteria in a total volume of 20 μΐ PBS was then delivered through one nostril with a pipette tip. Solutions of metsulfuron methyl were prepared fresh each day by dissolving the powder in DMSO (500 mg/ml) and then diluting into PBS pH 8.0 (5 mg/ml). Groups of 6 mice were fed 200 μΐ of either PBS pH 8.0 (also containing 1% DMSO) or metsulfuron methyl twice daily using a gavage tube. The treatment was initiated 24 hours before challenge and continued for 10 days after infection. Body weights were measured everyday and routine observations were performed twice daily. All experimental procedures were carried out in a BSL3 laboratory and in accordance with the Animal Care and Use Committee ^' s Guidelines, DSO National Laboratories. Singapore, using protocols reviewed and approved by the DSO National Laboratories Biosafety Committee.

For mouse infections using P. aeruginosa strain PAO 1 , 5 ml of an overnight culture was added to 50 ml of LB and grown for 3 h to OD 0.8 - 1.0. Bacteria were washed twice with PBS then resuspended in PBS. Intratracheal instillation was carried out using a modification of previously described method (Guilbault C, et al. (2005) Lab Anim 39(3):336-352). To establish pulmonary infection, 8-10 week old FVB/N mice were anesthetized with a mixture of Hypnorm/Dormicum after which the trachea was exposed though an anterior midline incision and the P. aeruginosa inoculum (3 x 105 CFU in 50 μΐ of PBS for survival curves; 2 x 105 CFU in 50 μΐ of PBS for lung CFUs) was delivered just beneath the cricoid cartilage. The incision was sealed by sterile surgical clips (Braintree Scientific). Metsulfuron methyl (5 mg/ml) was injected through the tail vein immediately after intra-tracheal infection. To determine CFUs present in the lungs, mice were killed by inhalation of carbon dioxide after which the lungs were removed aseptically, suspended PBS (2 ml), and homogenized using a Wheaton overhead stirrer (Wheaton Instruments.). Dilutions of the homogenate were spread on LB agar plates and incubated at 37 °C overnight. Animal protocols were based on guidelines from the National Advisory Committee for Laboratory Animal Research and were approved by the Institutional Animal Care and Use Committee, Singapore.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.