RESISTANT BACTERIA

FIELD OF THE INVENTION

The present invention relates to bacteria with increased resistance to antibiotic compounds, specifically inhibitors of the type II topoisomerases, and methods of generating bacteria with such increased resistance. It also relates to methods of increasing the resistance of bacteria to antibiotic compounds, such as inhibitors of the type II topoisomerases. The invention further relates to uses of bacteria in developing antibiotic compounds, and uses of bacteria as biosensors. The invention also relates to methods of diagnosing a subject, methods of determining a suitable antibiotic treatment regimen, and methods of treatment. The invention relates further still to antibiotic compounds for use in the treatment of a bacterial infection of a subject.

BACKGROUND TO THE INVENTION

The bacterial type II topoisomerases, DNA gyrase and topoisomerase IV, are essential enzymes that catalyse changes in DNA topology. DNA gyrase comprises two protein subunits termed GyrA and GyrB. Topoisomerase IV is comprised of two subunits called GrIA and GrIB in the Gram-positive bacterium Staphylococcus aureus and ParC and ParE in the Gram- negative bacterium Escherichia coli. DNA gyrase and topoisomerase IV are important targets for antibiotic therapy. Two classes of antibiotics that act at this target have been approved for clinical use in the treatment of human infection. The quinolone class of antibiotics targets the GyrA and GrIA (ParC) subunits of topoisomerase IV (Current Topics in Medicinal Chemistry 9, 981-998). Examples of quinolone antibiotics are nalidixic acid, ciprofloxacin, levofloxacin, moxifloxacin and delafloxacin. The aminocoumarin class, represented by novobiocin, inhibits GyrB and to a lesser extent the GrIB (ParE) subunit (The EMBO Journal 15, 1412-1420; Antimicrobial Agents and Chemotherapy 40, 1060-1062).

Bacterial resistance to the quinolone antibiotics has become prevalent. Quinolone resistance can result from target-based mutations in one of the four topoisomerase gene products, the expression from extrachromosomal elements of proteins such as the Qnr proteins that disrupt quinolone-enzyme binding, or drug efflux via membrane-localised pumps (Biochemistry 53, 1565-1574). Target-mediated quinolone resistance is most frequently associated with mutations in the Ser84 and Glu88 residues in Staphylococcus aureus GyrA (Biochemistry 53, 1565-1574). These residues are important in forming hydrogen bonds with the water molecules of the water-Mg ²⁺ ion bridge that is chelated by the keto acid groups of the quinolone. Other amino acid residues that have been associated with quinolone resistance in Staphylococcus aureus include GyrA Gly82, GrIA Ser80, GrIA Glu84 and GrIA Ala1 16 {Journal of Antimicrobial Chemotherapy 60, 269-273; Antimicrobial Agents and Chemotherapy 55, 5512-5521). Novobiocin resistance in Staphylococcus aureus has been commonly associated with mutations in the Gly85, Asp89, Ile102, Arg144 and Thr173 residues in GyrB (Antimicrobial Agents and Chemotherapy 40, 1060-1062; Journal of Antimicrobial Chemotherapy 60, 269- 273; Antimicrobial Agents and Chemotherapy 57 ^' , 5977-5986).

Several other chemical classes of inhibitors of the type II topoisomerases have been discovered that seek to circumvent quinolone resistance via differential binding interactions with the target enzymes. None of these has yet been approved for therapeutic use although some are undergoing clinical development. Examples include the quinazolinediones (Journal of Medicinal Chemistry 49, 6435-6438), the isothiazoloquinolones (Antimicrobial Agents and Chemotherapy 55, 2860-2871), the 2-pyridones (Antimicrobial Agents and Chemotherapy 39, 964-970), the spiropyrimidinetrione (Antimicrobial Agents and Chemotherapy 59, 467-474) and the novel bacterial topoisomerase inhibitors (Nature 466, 935-940).

WO 2015/155549 discloses a series of novel small-molecule tricyclic inhibitors of the bacterial type II topoisomerases. Compounds disclosed in WO 2015/155549 retained antibiotic potency against quinolone-resistant strains of Staphylococcus aureus harbouring double mutations in the type II topoisomerase gene products such as GyrA Ser84Leu and GrIA Ser80Phe, or GyrA Ser84Leu and GrIA Glu84Gly.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a bacterium with increased resistance to inhibitors of the type II topoisomerases, characterised in that the bacterium comprises a gene encoding a GrIB protein having a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus.

In a second aspect, the invention provides a method of generating a bacterium with increased resistance to inhibitors of the type II topoisomerases, the method comprising modifying the bacterium to incorporate a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus.

In a third aspect, the invention provides a method of increasing resistance of a bacterium to inhibitors of the type II topoisomerases, the method comprising modifying the bacterium to incorporate a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus.

In a fourth aspect, the invention provides the use of a bacterium in accordance with the first aspect of the invention in a method of developing antibiotic compounds.

As explained in more detail below, for the purposes of the present invention, references to "developing" antibiotic compounds should be taken to encompass not only the development of known antibiotic compounds, but also the generation, identification, and optionally subsequent development, of new antibiotic compounds.

In a fifth aspect, the invention provides the use of a bacterium in accordance with the first aspect of the invention as a biosensor.

As discussed elsewhere in the present specification, a bacterium in accordance with the invention may suitably be used as a biosensor for antibiotic compounds.

In a sixth aspect, the invention provides a method of diagnosing a subject as having an infection with bacteria resistant to inhibitors of the type II topoisomerases, the method comprising assaying bacteria associated with the bacterial infection of the subject for the presence of a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus, wherein the presence of such a gene indicates that the subject has an infection with resistant bacteria.

In a seventh aspect, the invention provides a method of determining a suitable antibiotic treatment regimen, the method comprising:

• assaying bacteria associated with a bacterial infection of a subject for the presence of a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus, wherein

• the presence of such a gene indicates that a suitable treatment regimen comprises treatment with an antibiotic other than one selected from the group consisting of: Example Compound 1 ; Example Compound 2; Example Compound 3; Example Compound 4; Example Compound 5; Example Compound 6; Example Compound 7; Example Compound 8; and Example Compound 9.

• the absence of such a gene indicates that a suitable treatment regimen comprises treatment with an antibiotic inhibitor of the type II topoisomerases. In an eighth aspect, the invention provides an antibiotic for use in the treatment of a bacterial infection of a subject, characterised in that:

• the subject has been identified as infected with bacteria that do not possess a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus, and

• the antibiotic is an inhibitor of the type II topoisomerases.

In a ninth aspect, the invention provides an antibiotic for use in the treatment of a bacterial infection of a subject, characterised in that:

• the subject has been identified as infected with bacteria that possess a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus, and

• the antibiotic is other than one selected from the group consisting of: Example Compound 1 ; Example Compound 2; Example Compound 3; Example Compound 4; Example Compound 5; Example Compound 6; Example Compound 7; Example Compound 8; and Example Compound 9.

In a tenth aspect, the invention provides a method of treating a bacterial infection of a subject, the method comprising:

• assaying bacteria associated with the bacterial infection for the presence of a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus, and

• when the assay indicates the presence of such a gene, providing the subject with a therapeutically-effective amount of an antibiotic other than one selected from the group consisting of: Example Compound 1 ; Example Compound 2; Example Compound 3; Example Compound 4; Example Compound 5; Example Compound 6; Example Compound 7; Example Compound 8; and Example Compound 9;

• when the assay indicates the absence of such a gene, providing the subject with a therapeutically-effective amount of an antibiotic inhibitor of the type II topoisomerases.

In an eleventh aspect, the invention provides a method of treating a bacterial infection, the method comprising:

• selecting a subject as having an infection with bacteria that lack the presence of a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus, and • providing the subject with a therapeutically-effective amount of an antibiotic inhibitor of the type II topoisomerases.

In a twelfth aspect, the invention provides a method of treating a bacterial infection, the method comprising:

• selecting a subject as having an infection with bacteria that possess a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus, and

• providing the subject with a therapeutically-effective amount of an antibiotic other than one selected from the group consisting of: Example Compound 1 ; Example Compound 2; Example Compound 3; Example Compound 4; Example Compound 5; Example Compound 6; Example Compound 7; Example Compound 8; and Example Compound 9.

Suitable examples of inhibitors of the type II topoisomerases that may be used in accordance with the seventh, eighth, tenth, or eleventh aspect of the invention are discussed elsewhere in the present specification. The specification also provides further details of suitable embodiments that may be used when the seventh, ninth, tenth, or twelfth aspects of the invention indicate that the use of an antibiotic other than those selected from the group consisting of: Example Compound 1 ; Example Compound 2; Example Compound 3; Example Compound 4; Example Compound 5; Example Compound 6; Example Compound 7; Example Compound 8; and Example Compound 9 is desired.

DETAILED DESCRIPTION OF THE INVENTION

A number of the aspects and embodiments of the invention are based upon the inventors' finding that bacteria expressing mutated forms of GrIB protein, in which a mutation is present at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus, have increased resistance to selected antibiotic inhibitors of the type II topoisomerases. While the involvement of GrIB in antibiotic resistance is well known, the importance of this particular amino acid residue, and particularly its importance in resistance to inhibitors of the type II topoisomerases, such as those of WO 2015/155549, PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7, had not previously been recognised. The inventors' determination of the ability of this mutated form of GrIB to confer resistance to antibiotics such as selected inhibitors of the type II topoisomerases gives rise to a number of useful and inventive applications, as set out herein.

Various aspects and embodiments of the invention are described further in the paragraphs below. Certain terms used in the definitions provided herein are also explained further below, in order to facilitate understanding of the invention.

Bacteria of the invention

The first aspect of the invention provides bacteria that have increased resistance to inhibitors of the type II topoisomerases, and which comprise a gene encoding a GrIB protein having a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus.

Suitably, bacteria of the invention may be produced by the methods of the second or third aspects of the invention (respectively methods of generating bacteria with increased resistance to inhibitors of the type II topoisomerases, and methods for increasing resistance of bacteria to inhibitors of the type II topoisomerases). In light of this it will be appreciated that the following considerations are also applicable to methods of these second or third aspects of the invention, except for where the context requires otherwise.

A bacterium of the invention may be a genetically-modified bacterium comprising a gene encoding a GrIB protein having a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus.

A genetically-modified bacterium of the invention may be one that has been transformed with the gene encoding a mutated GrIB protein.

Examples of such genes that may be employed, for example in genetically-modified bacteria such as those set out above, are described elsewhere in the present specification.

A bacterium of the invention may be a Gram-positive bacterium.

In a suitable embodiment, a bacterium of the invention may be a Staphylococcus, such as selected from the group consisting of: Staphylococcus aureus; Staphylococcus auricularis; Staphylococcus capitis; Staphylococcus caprae; Staphylococcus cohnii; Staphylococcus epidermidis; Staphylococcus haemolyticus; Staphylococcus hominis; Staphylococcus lugdunensis; Staphylococcus pasteuri; Staphylococcus saccharolyticus; Staphylococcus saprophytics; Staphylococcus simulans; Staphylococcus warnerii; and Staphylococcus xylosis.

In a particularly suitable embodiment, a bacterium of the invention is a Staphylococcus aureus. For example, a bacterium of the invention may be derived from Staphylococcus aureus ATCC 29213. It will be appreciated that, in such embodiments, a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus will be a mutation of the arginine 453 residue itself.

Examples of such bacteria of the invention have been deposited by the inventors under the Budapest Treaty.

Accordingly, the invention provides a bacterium deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB), NCIMB Ltd, Aberdeen, UK, under the Budapest Treaty on 30 ^th October 2015, and given the Accession No. NCIMB 42475. This strain of Staphylococcus aureus is also referred to as SA4139-SP40 elsewhere within the present specification.

The bacteria of the invention may further comprise a mutation at amino acid residue 185 in a NagD-like phosphatase/HAD family hydrolase protein. The mutation may be a substitution of the amino acid residue proline with the amino acid residue serine. Strain SA4139-SP40 comprises this mutation.

The bacteria of the invention may further comprise a mutation at amino acid residue 79 in an autolysin/mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase protein. The mutation may be a substitution of the amino acid residue alanine with the amino acid residue valine. Strain SA4139-SP40 comprises this mutation.

The bacteria of the invention may further comprise a mutation at amino acid residue 70 in an organic hydroperoxide resistance transcriptional regulator/MarR family transcriptional regulator protein. The mutation may be a substitution of the amino acid residue serine with the amino acid residue leucine. Strain SA4139-SP40 comprises this mutation. These further mutations are characteristic of strain SA4139-SP40 and are present in the bacteria deposited under Accession No. NCIMB 42475. Accordingly, these further mutations may be used to characterise bacteria as being of strain SA4139-SP40.

The invention also provides a bacterium deposited at the NCIMB under the Budapest Treaty on 30 ^th October 2015, and given the Accession No. NCIMB 42476. This strain of Staphylococcus aureus is also referred to as SA6485-A2 elsewhere within the present specification.

These bacteria of the invention have been produced by the inventors, and both constitute examples of bacteria in accordance with the first aspect of the invention. Furthermore, these bacteria deposited at NCIMB under accession numbers NCIMB 42475 and NCIMB 42476 both represent examples of bacteria suitable for use according to the fourth aspect of the invention.

Resistance to inhibitors of the type II topoisomerases

For the purposes of the present invention, increased resistance of bacteria to inhibitors of the type II topoisomerases may be demonstrated when a bacterium has elevated resistance to such antibiotics as compared to a suitable control bacterium. By way of example, a suitable control bacterium may comprise a bacterium that does not express a GrIB protein including a mutation. For example, in the case of a bacterium of the invention based upon Staphylococcus aureus, but expressing a mutated GrIB protein, a suitable control may be a Staphylococcus aureus without the mutated GrIB protein. In the case of a bacterium of the invention based upon Staphylococcus aureus ATCC 29213, but expressing a mutated GrIB protein, a suitable control may be Staphylococcus aureus ATCC 29213 without the mutated GrIB protein, such as a Staphylococcus aureus ATCC 29213 with wild-type GrIB protein.

Suitably a bacterium of the invention may have a resistance to inhibitors of the type II topoisomerases that is at least four-fold higher than that of a suitable control. Indeed a bacterium according to the invention may have a resistance to inhibitors of the type II topoisomerase that is four-fold or more, eight-fold or more, 16-fold or more, 32 -fold or more, 64-fold or more, or 128-fold or more higher than that of a suitable control.

Suitably a bacterium of the invention may have a resistance to inhibitors of the type II topoisomerases that is at least four-fold higher than that of Staphylococcus aureus ATCC 29213. In certain embodiments a bacterium of the invention may have a resistance to inhibitors of the type II topoisomerases that is at least eight-fold higher or at least 16-fold higher than that of Staphylococcus aureus ATCC 29213.

Bacteria of the invention may, in particular, be resistant to the specific inhibitor compounds described elsewhere in the present specification (including Example Compounds 1 to 9, or the broader group of compounds disclosed in the applicant's co-pending international patent application published as WO 2015/155549 and in the applicant's copending applications PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7). Suitably a resistant bacterium may demonstrate a resistance to one (or more) of these compounds that is four-fold or more, eight-fold or more, 16-fold or more, 32 -fold or more, 64- fold or more, or 128-fold or more higher than the resistance of a suitable control bacterium.

Examples of inhibitors of the type II topoisomerases to which resistance may be demonstrated are considered elsewhere in the specification. Merely as examples, such inhibitors of the type II topoisomerases may include one or more compounds selected from the group consisting of: Example Compound 1 ; Example Compound 2; Example Compound 3; Example Compound 4; Example Compound 5; Example Compound 6; Example Compound 7; Example Compound 8; and Example Compound 9.

Mutated GrIB proteins

The present specification includes many references to "a GrIB protein having a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus". For the sake of brevity such GrIB proteins may simply be referred to as "mutated GrIB proteins", on the basis that these are understood to incorporate a mutation of the sort specified above.

In a suitable embodiment, the mutation of the GrIB protein is a substitution of the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus. The wild type amino acid sequence of GrIB of Staphylococcus aureus is set out in SEQ ID NO:4.

Merely by way of example, the substitution may be a substitution with a histidine residue. Substitution with histidine in accordance with this embodiment of the invention is found in the bacteria of the invention deposited under accession numbers NCIMB 42475 and NCIMB 42476. Suitably the GrIB protein may comprise the amino acid sequence of SEQ ID NO:2. Indeed, the GrIB protein may consist of, or essentially consist of, the amino acid sequence of SEQ ID NO:2.

Except for where the context requires otherwise, the considerations set out above are applicable in the context of bacteria of the invention and also methods of the invention.

Genes encoding mutated GrIB proteins

The first aspect of the invention provides a bacterium with increased resistance to inhibitors of the type II topoisomerases, characterised in that the bacterium comprises a gene encoding a GrIB protein having a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus. For the sake of brevity such genes may simply be referred to in the present specification as "mutated GrIB genes", or "mutated genes", on the understanding that such mutated genes must encode a GrIB protein with a mutation meeting the requirements set out herein.

The mutation may be any mutation that gives rise to a gene encoding a mutated form of a GrIB protein having a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus. For example, the mutation may be one that causes a change in the codon encoding the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus.

In a suitable embodiment, a mutated gene encoding a GrIB protein comprises a mutation at the nucleotide homologous to guanine 1358 of the grIB gene in Staphylococcus aureus. The polynucleotide sequence of grIB encoding the wild-type GrIB protein of Staphylococcus aureus is set out in SEQ ID NO:3.

Merely by way of example, the mutation may be a substitution of the nucleotide homologous to guanine 1358 of the grIB gene in Staphylococcus aureus. In particular, the substitution may be a substitution with an adenine residue.

Suitably the gene encoding a mutated GrIB protein may comprise a polynucleotide of SEQ ID NO: 1. Indeed, the portion of the gene that encodes the mutated GrIB protein may consist of, or essentially consist of, a polynucleotide of SEQ ID NO: 1. Except for where the context requires otherwise, the considerations set out above are applicable in relation to any references to mutated GrIB genes set out herein, whether in relation to bacteria of the invention or to methods of the invention.

Assaying a bacterium for the presence of a mutated gene or mutated protein

Various aspects of the invention make use of methods in which bacteria are assayed for the presence of a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the GrIB protein in Staphylococcus aureus. These methods may be practiced using any suitable method by which the presence of such a gene may be determined. The skilled person will be aware of many suitable methods that may be used in such assays.

Merely by way of example, a suitable method may involve analysis of nucleic acids of the bacterium. For example, a suitable technique may involve analysis of the DNA of the bacterial genome. Such an embodiment may involve analysis for the presence of a mutation at the codon encoding the amino acid residue homologous to arginine 453 of the GrIB protein in Staphylococcus aureus. The mutation may be any mutation that does not give rise to an arginine residue. For example, the mutation may be one that gives rise to a codon encoding a histidine residue.

Alternatively, a suitable method may involve analysis of RNA, such as mRNA, that is indicative of gene expression within the bacterium. In this case, the sequence of the mRNA is utilised to determine the presence in the bacterium of a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the GrIB protein in Staphylococcus aureus.

A still further suitable embodiment may involve analysis of proteins expressed by the bacterium. It will be appreciated that expression by the bacterium of a GrIB protein comprising a mutation of the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus will provide an indication that the bacterium possesses a gene encoding such a protein.

Methods in which an assay is based upon the analysis of nucleic acids, whether DNA or RNA, may utilise suitable techniques by which the nucleic acid may be sequenced. In the case of methods based upon analysis of mRNA, a suitable technique may involve the sequencing of mRNA, or the use of mRNA to generate cDNA that is sequenced. In methods in which an assay is based upon analysis of proteins may similarly use sequencing techniques to determine the sequence of the protein in question. Alternatively, suitable techniques may investigate the biological function of such proteins (for example techniques in which enzyme activity is used to determine whether or not a GrIB protein is a mutated protein of the sort described herein), or the conformation of such proteins (for example techniques using a suitable antibody able to specifically distinguish a form of GrIB comprising a mutation of the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus from other forms of this protein).

In the context of the present invention a method that makes use of analysis of DNA of the bacterial genome may be considered to be a "direct" assay for the presence of a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the GrIB protein in Staphylococcus aureus. Suitable embodiments of the invention may use direct assays of this sort.

On the other hand, a method that makes use of analysis of mRNA or protein associated with gene expression by a bacterium may be considered to be an "indirect" assay for the presence in the bacterium of a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the GrIB protein in Staphylococcus aureus. The use of such indirect assays is also considered in suitable embodiments of the invention.

Suitable "direct" or "indirect" techniques by which the presence of a mutated gene encoding GrIB may be assayed include those selected from the group consisting of: DNA sequencing; restriction enzyme analysis; immunoassays; and peptide sequencing. Of this group, the first two techniques provide examples of direct techniques, and the second two provide examples of indirect techniques. Suitably, peptide sequencing may be performed by MALDI-TOF mass spectrometry.

It will be appreciated that, if the presence of a wild-type gene encoding GrIB is identified in respect of any given bacterium, this may be taken as indicative of the absence of a mutated gene encoding GrIB in the bacterium in question.

Methods of the second or third aspects of the invention

The second and third aspects of the invention respectively provide methods of generating bacteria with increased resistance to inhibitors of the type II topoisomerases, and methods of increasing resistance of bacteria to such inhibitors. Both methods involve modifying the bacterium to incorporate a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus. It will be appreciated that the same considerations and techniques may be employed in many embodiments of these two different aspects.

There are many methods by which a suitable gene encoding a mutated GrIB protein may be introduced into a bacterium, and the gene thereby incorporated. The skilled person will be able to determine and select an appropriate technique in order to practice the invention.

Suitable examples may include well-established techniques, such as bacterial transformation to incorporate the mutated gene. Alternatively, techniques such as gene editing, such as by CRISPR-based approaches, may be employed.

In a suitable embodiment of these methods the bacterium is a Staphylococcus aureus. For example, the bacterium may be Staphylococcus aureus ATCC 29213.

Uses of the fourth aspect of the invention

The bacteria of the invention are suitable for use in a number of methods that employ their biological properties, and in particular their increased antibiotic resistance.

Bacteria of the invention will be generally useful in the discovery and development of antibiotic compounds, including novel antibiotic compounds, and particularly in the discovery and development of bacterial type II topoisomerase inhibitors. It will be appreciated that, in its common use in relation to "discovery and development", the word "discovery" encompasses not only the identification of previously existing compounds having antibiotic properties, but also the generation of novel compounds with antibiotic activity.

The fourth aspect of the present invention provides the use of a bacterium in accordance with the first aspect of the invention in a method of developing antibiotic compounds. As clarified above, for the purposes of the present invention, references to "developing" antibiotic compounds should be taken to encompass not only the development of known antibiotic compounds, but also the generation, identification, and optionally subsequent development, of new antibiotic compounds. Methods by which bacteria of the invention may be used in accordance with the fourth aspect of the invention may comprise incubating at least one antibiotic compound, or putative antibiotic compound, in the presence of a bacterium of the invention. The compound may be an inhibitor of the type II topoisomerases, or a putative inhibitor of the type II topoisomerases.

In a suitable embodiment, methods by which bacteria of the invention are used according to the fourth aspect of the invention may further comprise assessing the ability of the antibiotic compound, or putative antibiotic compound, to inhibit growth of bacteria of the invention. Compounds able to inhibit growth of the bacteria of the invention may be taken as exhibiting antibiotic properties. It will be appreciated that in the case of compounds previously only putatively having antibiotic activity, use of the bacteria of the invention in this way may be useful in the discovery of novel antibiotic compounds. In the case of compounds that are based upon known antibiotics, the use of the bacteria of the invention may be useful in developing new antibiotic compounds, for example through processes in which the impact of modifications of the structure of such compounds is assessed with reference to the ability of such modified compounds to inhibit bacterial growth.

Suitably uses in accordance with the fourth aspect of the invention may comprise incubating an antibiotic in the presence of bacteria including a bacterium of the invention and another bacterium (not of the invention) that has different antibiotic resistance characteristics to those of the bacterium of the invention.

Uses of bacteria of the invention may comprise incubating different populations of bacteria of the invention (and optionally bacteria not of the invention) with combinations of antibiotic or putative antibiotic compounds.

Uses of bacteria of the invention may include a range of studies used in the discovery or development of new antibiotic compounds. These include mechanism-of-action studies, and studies investigating the activities of combinations of antibiotic compounds.

Bacteria of the invention may be used for the generation and development of new antibiotic compounds, including, but not limited to, new non-quinolone-based type II topoisomerase inhibitors.

Bacteria of the invention may, for example, be used in in vitro susceptibility testing of antibiotics. Suitably such a method may comprise the use of bacteria of the invention in the determination of the minimum inhibitory concentration (MIC) with respect to an antibiotic. The MIC may be determined using the broth or agar dilution method, by the disc diffusion assay, by Etest ^® , the M.I.C. Evaluator™, or by another suitable method. In a suitable example of the broth dilution protocol, the bacteria of the invention are inoculated into samples of an appropriate growth medium containing serial dilutions of the antibiotic. Following incubation for an appropriate duration in the relevant atmospheric conditions and temperature, the MIC is determined as the lowest concentration of the antibiotic that is able to inhibit visible growth of bacteria of the invention (and optionally other bacteria). Bacteria of the invention are particularly useful in this method because they may be derived from the well-characterised Clinical and Laboratory Standards Institute (CLSI) reference strain Staphylococcus aureus ATCC 29213 for which established susceptibility data are available. Bacteria of the invention are also useful because they retain similar growth rates, yields and morphologies to the reference strains.

Bacteria of the invention may be used in antibiotic interaction studies of antibiotics. For example, bacteria of the invention may be used in in vitro checkerboard combination studies with two or more antibiotics. In this assay the MIC of an antibiotic is determined as described above but in the presence of varying concentrations of a second antibiotic to evaluate the potential for synergistic or antagonistic interactions between the antibiotics.

Bacteria of the invention may be used in methods to determine the bactericidal activity of antibiotics. For example, bacteria of the invention may be used in the measurement of the minimum bactericidal concentration (MBC) using a dilution method. In this assay, aliquots of the test cultures from the standard broth dilution MIC method described above are removed at the end of the MIC assay for serial dilution in antibiotic-free buffer and plating on to antibiotic- free agar for the enumeration of colony forming units per millilitre (CFU/mL). The MBC is defined as the concentration of antibiotic that achieves a ≥3 logio decrease in CFU/mL following incubation of the agar plates for an appropriate duration in the relevant atmospheric conditions and temperature.

In a suitable embodiment of the fourth aspect of the invention, bacteria of the invention may be used to determine bactericidal activity of antibiotics using the time-kill assay. In this assay a culture of the bacteria of the invention is exposed to concentrations of antibiotic and the number of viable bacterial cells in the culture are monitored over time by enumerating CFU/mL on antibiotic-free agar plates. A≥3 logio decrease in the total number of CFU/mL indicates a bactericidal effect. Bacteria of the invention may be used in the in vivo biological evaluation of antibiotics. In particular, bacteria of the invention may be used in suitable animal models of bacterial infection, for example in sepsis, pneumonia or thigh infection models. In the sepsis model, for example, a vertebrate organism such as the mouse may be infected with a lethal dose of bacteria of the invention by intraperitoneal inoculation. Antibiotic is administered to the animal at an appropriate dosing frequency and regimen by intravenous, oral, subcutaneous or another method of administration. Survival is monitored over a time course in order to give an indication of the doses of antibiotic that provide a protective effect. Bacteria of the invention are of particular benefit in such uses because they may be derived from the well-characterised CLSI reference strain Staphylococcus aureus ATCC 29213 for which a large amount of in vivo model data are available.

Bacteria of the invention may be used in mechanism-of-action studies of antibiotics. An important part of the process of the discovery of new antibiotics is the elucidation of the biological mechanism by which the putative antibiotic compound exerts its antibiotic effect. This is especially relevant when the putative antibiotic compound(s) has been discovered via whole-cell phenotypic screening as opposed to a target-based approach. An altered phenotypic response in a relevant assay for assessment of antibiotic resistance, for example the broth dilution MIC method described above, provides an indication of the putative target or pathway. Bacteria of the invention will prove particularly advantageous in elucidating the mechanism-of-action of antibiotics that target the mutated GrIB gene product found in strains SA4139-SP40 and SA6485-A2.

Bacteria of the invention may be used for the in silico design of new antibiotics. The bacteria of the invention may be of benefit for computer models of bacterial proteins for the purposes of the de novo structure-based design of antibiotics, for ligand-protein modelling studies, for the development of pharmacophore models or for virtual ligand docking and screening.

Bacteria of the invention may be used in the production of bacterial protein, specifically in the production of mutated GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus. Whole-cell samples of bacteria of the invention, or purified samples of the genomic DNA thereof, may be used as templates for the production of synthetic copies, for example by the process of polymerase chain reaction, of the mutated coding sequences. The resulting DNA amplicons may be cloned into a suitable plasmid vector for expression and purification of the corresponding gene products. Mutated GrIB protein, whether purified in native form from the bacteria of the invention or produced using DNA recombinant technology as described above, will be generally useful in the discovery and development of antibiotic compounds, and particularly in the discovery and development of bacterial type II topoisomerase inhibitors. Accordingly, the invention provides, as a further aspect, the use of a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus in the discovery or development of antibiotic compounds.

In a suitable embodiment of this aspect of the invention, the mutated GrIB protein may be present in situ within a bacterium of the invention. However, it may be preferred that the mutated GrIB protein is an isolated and purified protein.

Suitably, a mutated GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus is used in a technique selected from the group consisting of: in vitro binding assays; in vitro functional biochemical assays; and crystallography studies. Other techniques that may be used in embodiments of such uses will also be recognised by those skilled in the art.

Merely by way of example, the ability of a known or putative antibiotic compound to bind to a mutated GrIB protein may indicate that the compound in question will be of benefit in the treatment of bacterial infections associated with bacteria that possess a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus. This indication may be even stronger in the case of known or putative antibiotic compounds that are demonstrated to be able to inhibit such mutated GrIB proteins.

Uses of the fifth aspect of the invention

The fifth aspect of the invention provides the use of a bacterium of the invention as a biosensor. In particular, a bacterium in accordance with the invention may be used as a biosensor for antibiotic compounds.

Bacteria of the invention may be used in accordance with the fifth aspect of the invention as biosensors of antibiotic compounds in a range of samples, including environmental, clinical, or other samples. Bacteria of the invention may be useful as biosensors in the detection of bacterial type II topoisomerase inhibitor antibiotics. Bacteria of the invention may be particularly useful as biosensors in the detection of non-quinolone and non-coumarin type II topoisomerase inhibitors, such as those of WO 2015/155549,PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7.

Merely by way of example, a suitable use in accordance with the fifth aspect of the invention may comprise the use of a bacterium of the invention (such as the strain Staphylococcus aureus SA4139-SP40 disclosed herein) and its reference progenitor strain (such as Staphylococcus aureus ATCC 29213). A bacterium of the invention and reference strain may each be independently cultured in samples where it is wished to detect the presence of an antibiotic compound. In such an embodiment, growth of a bacterium of the invention (such as Staphylococcus aureus SA4139-SP40) but not of the reference strain (such as Staphylococcus aureus ATCC 29213) may indicate the presence of an antibiotic compound, and particularly a type II topoisomerase inhibitor, in the sample.

Bacterial infections

A number of aspects and embodiments of the present invention relate to the treatment of bacterial infections. Such bacterial infections may be infections with Gram-positive bacteria.

In a suitable embodiment, the bacterial infection may be a Staphylococcus infection. For example, the infection may be with a bacterium selected from the group consisting of: Staphylococcus aureus; Staphylococcus auricularis; Staphylococcus capitis; Staphylococcus caprae; Staphylococcus cohnii; Staphylococcus epidermidis; Staphylococcus haemolyticus; Staphylococcus hominis; Staphylococcus lugdunensis; Staphylococcus pasteuri; Staphylococcus saccharolyticus; Staphylococcus saprophytics; Staphylococcus simulans; Staphylococcus warnerii; and Staphylococcus xylosis.

In a particularly suitable embodiment, the bacterial infection is a Staphylococcus aureus infection. Suitably the bacterial infection is an infection with methicillin-resistant Staphylococcus aureus.

Merely by way of example, the bacterial infection may be selected from the group consisting of: an infection of the skin; a soft tissue infection; an infection of the gastrointestinal tract; a blood infection; an infection of the respiratory tract; an infection of the throat; an infection of the oral cavity; a dental infection; a nasal infection; an ear infection; a bone infection; and an infection of the genitourinary tract.

Diagnostic methods of the sixth aspect of the invention

The sixth aspect of the invention provides a method by which a subject may be diagnosed as having an infection with bacteria resistant to inhibitors of the type II topoisomerases. Such methods allow a determination to be made as to which antibiotics are most suitable for use in treatment of a subject's infection, and allow appropriate prescription choices to be made.

The subject may be one having a Staphylococcus aureus infection, or believed to have a Staphylococcus aureus infection.

In a suitable embodiment of a method of the sixth aspect of the invention, the subject is a human subject. However, it will be appreciated that the methods of this aspect of the invention are also applicable to non-human animals.

Samples of bacteria associated with the bacterial infection of the subject, which can then be assayed as required, may be collected by any suitable means. Merely by way of example, these may be collected from swabs, from wound dressings or wound aspirates, or from body fluid or tissue samples.

The methods of the sixth aspect of the invention are based upon assays for the presence of a mutated grIB gene (whether direct or indirect assays), rather than the culturing of bacteria associated with the infection in the presence of various antibiotics (including inhibitors of the type II topoisomerases), and observing the impact of the test compounds on bacterial growth. As a result, these methods of the invention allow much faster diagnosis to be performed. This provides significant advantages in terms of the reduced time elapsing prior to beginning appropriate antibiotic treatment.

Selection of treatment regimens in accordance with the invention

The identification of the relevance of the presence of mutated forms of GrIB to resistance to inhibitors of the type II topoisomerases also gives rise to the methods of the seventh aspect of the invention. These methods allow the selection of an appropriate antibiotic treatment regimen, based upon the presence or absence of genes encoding mutated GrIB in bacteria associated with an infection. Since the inventors have found that the presence of a gene encoding mutated GrIB in bacteria confers increased resistance to antibiotic inhibitors of the type II topoisomerases, the investigation of bacteria infecting a subject to identify the presence of such a gene allows selection of appropriate treatment to control the infection. If the bacteria possess genes encoding mutated GrIB then it is to be expected that the bacteria will be less susceptible to treatment with inhibitors of the type II topoisomerases (such as Example Compound 1 , Example Compound 2, Example Compound 3, Example Compound 4, Example Compound 5, Example Compound 6, Example Compound 7, Example Compound 8, and Example Compound 9, or other compounds disclosed in WO 2015/155549, PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7), and thus appropriate treatment should employ alternative antibiotics.

However, if such genes encoding mutated GrIB are not present, then antibiotic inhibitors of the type II topoisomerases, including inhibitors such as Example Compound 1 , Example Compound 2, Example Compound 3, Example Compound 4, Example Compound 5, Example Compound 6, Example Compound 7, Example Compound 8, and Example Compound 9, or other compounds disclosed in WO 2015/155549, PCT/GB2016/052899,

PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7, represent a suitable treatment that may be employed in dealing with the infection.

Alternative antibiotics other than Example Compounds 1 to 9 (or indeed the other compounds disclosed in WO 2015/155549, PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7) that may be used in the case that genes encoding mutated GrIB are identified among the infecting bacteria, will be apparent to those skilled in the art. Merely by way of example, these may include one or more of the group consisting of: ampicillin; ciprofloxacin; delafloxacin; erythromycin; gentamicin; levofloxacin; linezolid; novobiocin; penicillin G; rifampicin; tetracycline; trimethoprim; and vancomycin. The choice of alternative antibiotic compound to use will of course depend upon a number of additional factors, such as the presence of resistance determinants to any of the alternative antibiotics.

In a suitable embodiment, a method of the seventh aspect of the invention may further comprise providing an antibiotic to implement the treatment regimen that has been determined to be suitable. For example, in the event that the presence of a gene encoding a mutated GrIB protein has been identified among bacteria associated with the infection, the method may further comprise providing an antibiotic other than any of Example Compounds 1 to 9 (or indeed, as contemplated below, other than any of the compounds disclosed in WO 2015/155549, PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7). On the other hand, if the assay has indicated that a gene encoding a mutated GrIB protein is not present among bacteria associated with the infection, then the method may further comprise providing any antibiotic inhibitor of the type II topoisomerases (including Example Compounds 1 to 9 or any other compound disclosed in WO 2015/155549, PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7) as a suitable therapeutic agent.

Indeed, so useful are such embodiments that they lead to the methods of treatment of the tenth aspect of the invention. These provide methods of treating a bacterial infection of a subject, which involve assaying bacteria associated with the bacterial infection for the presence of a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus. In the event that the assay indicates the presence of such a gene, the subject is then provided with a therapeutically-effective amount of an antibiotic other than any of Example Compounds 1 to 9 (or other antibiotic compounds as discussed further below). Alternatively, in the event that the assay indicates the absence of such a gene, the subject is provided with a therapeutically- effective amount of antibiotic inhibitor of the type II topoisomerases, which may include any of Example Compounds 1 to 9 (or any other compound disclosed in WO 2015/155549, PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7).

Unless the context requires otherwise, the same considerations also apply to the methods of the eleventh and twelfth aspects of the invention.

Medical uses in accordance with the invention

The eighth and ninth aspects of the invention provides new medical uses of antibiotics for use in the treatment of a bacterial infection of a subject. These new uses are characterised with reference to the subject to be treated, and particularly with reference to the nature of the bacteria associated with the subject's bacterial infection.

In the eighth aspect of the invention, the subject is one who has been identified as infected with bacteria that do not possess a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus.

The absence of such a gene encoding a mutated GrIB indicates that the bacteria associated with the infection will respond to treatment with antibiotic inhibitors of the type II topoisomerases, including the compounds disclosed herein as Example Compound 1 , Example Compound 2, Example Compound 3, Example Compound 4, Example Compound 5, Example Compound 6, Example Compound 7, Example Compound 8 or Example Compound 9. Indeed, the infection may respond to treatment with any of the compounds disclosed in WO 2015/155549, PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7.

In the ninth aspect of the invention, the subject is one who has been identified as infected with bacteria that do possess a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus.

The presence of such a gene encoding a mutated GrIB indicates that the bacteria associated with the infection will not respond to treatment with the compounds disclosed herein as Example Compound 1 , Example Compound 2, Example Compound 3, Example Compound 4, Example Compound 5, Example Compound 6, Example Compound 7, Example Compound 8 or Example Compound 9 . Indeed, the infection may not respond to treatment with any of the compounds disclosed in WO 2015/155549, PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7.

Accordingly, the medical uses of the ninth aspect of the invention are limited to antibiotic compounds other than Example Compounds 1 to 9, as discussed in more detail below. In suitable embodiments, the medical uses of the ninth aspect of the invention may be limited to exclude one or more, or indeed all, of the other compounds disclosed in WO 2015/155549, PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7.

Antibiotic inhibitors of the type II topoisomerases suitable for use in the methods or medical uses of the invention

The seventh, eighth, tenth, and eleventh aspects of the invention each refer to circumstances in which it may be desired to use an inhibitor of the type II topoisomerases as an antibiotic. In suitable embodiments of these methods or uses of the invention the inhibitor of type II topoisomerase may be used in the treatment of a bacterial infection.

Generally, the contexts in which it is desirable to use such compounds as antibiotics are those in which it has been identified that a bacterium (such as a bacterium associated with an infection of a subject) does not comprise a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus.

As set out further herein, the inventors' findings have identified that bacteria from which such genes are absent retain susceptibility to inhibitors of the type II topoisomerases such as Example Compound 1 , Example Compound 2, Example Compound 3, Example Compound 4, Example Compound 5, Example Compound 6, Example Compound 7, Example Compound 8 and Example Compound 9 described herein. The inventors believe that the susceptibility of such bacteria to antibiotic inhibitors of type II topoisomerase is broadly applicable across all compounds exhibiting this biological activity, but that Example Compounds 1 to 9 represent particularly suitable examples of compounds that may be used in embodiments of those aspects of the invention where an inhibitor of the type II topoisomerases is to be used as an antibiotic.

Without wishing to detract from the above, further suitable examples of type II topoisomerase inhibitors that may be used in accordance with such embodiments of the invention are those set out in the applicant's co-pending International Patent Application number PCT/GB2015/051107 (published as WO 2015/155549), PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7. The contents of this application are incorporated by reference, in particular in respect of the disclosure of various antibiotic type II topoisomerase inhibitors.

Accordingly, in a suitable embodiment, an inhibitor of type II topoisomerase that may be used as an antibiotic in accordance with the invention may include a compound of formula (I), or a pharmaceutically acceptable salt thereof:

wherein X ¹ is independently selected from: N and CR ⁵

X ² is independently selected from: N and CR ² ;

=A is independently selected from: =0, =S, =NR ⁶ and Y ¹ and Y ² are each independently selected from C and N; Z ¹ , Z ² and Z ³ are each independently selected from O, S, S(O), NR ^{1 1} , CR ¹² and C=W; wherein W is selected from O, S or NR ⁶ ; with the proviso that if none of Z ¹ , Z ² and Z ³ is C=W, then the ring formed by Z ¹ , Z ² , Z ³ , Y ¹ and Y ² contains two endocyclic double bonds and, if one of Z ¹ , Z ² and Z ³ is C=W, then the ring formed by Z ¹ , Z ² , Z ³ , Y ¹ and Y ² contains a single endocyclic double bond; and with the further proviso that at least one of Z ¹ , Z ² , Z ³ , Y ¹ and Y ² is O, S, N or N R ^{1 1} ;

R ¹ is independently selected from: H, F, NR ⁶ R ⁷ , NR ⁶ NR ⁶ R ⁷ and Ci-C ₄ -alkyl;

R ² is independently selected from: H and F;

R ³ is independently selected from: -(CR ⁸ R ⁸ ) _n -3-io heterocycloalkyl, -(CR ⁸ R ⁸ ) _n -aryl, -(CR ⁸ R ⁸ ) _n - heteroaryl, and -(CR ⁸ R ⁸ ) _n -C3-Cio cycloalkyl; wherein the aryl, heteroaryl, heterocycloalkyl or cycloalkyl group is optionally substituted with 1 , 2 or 3 R ¹⁵ groups; wherein R ¹⁵ is independently at each occurrence selected from: oxo, =NR ⁶ , =NOR ⁶ , 3-5-heterocycloalkyl, halo, nitro, cyano, NR ⁶ R ⁷ , N R ⁶ S(0) ₂ R ⁶ , N R ⁶ CONR ⁶ R ⁶ , N R ⁶ C0 ₂ R ⁶ , OR ⁶ ; SR ⁶ , SOR ⁶ , SO3R ⁶ , SO2R ⁶ , S0 ₂ NR ⁶ R ⁶ C0 ₂ R ⁶ C(0)R ⁶ , CONR ⁶ R ⁶ , C(0)NR ⁶ CR ⁶ R ⁶ C(0)OR ⁶ , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, Ci-C ₄ haloalkyl, CR ⁶ R ⁶ OR ⁶ , CR ⁶ R ⁶ NR ⁷ R ⁶ , and =CR ⁶ CR ⁶ R ⁶ NR ⁷ R ⁶ ;

R ⁴ is independently selected from: Ci-Cs alkyl, C ₂ -Cs alkenyl, C ₂ -Cs alkynyl, Ci-Cs haloalkyl, - (CR ⁸ R ⁸ ) _n -C ₃ -C ₆ cycloalkyl, -(CR ⁸ R ⁸ ) _n -C ₃ -C ₆ heterocycloalkyl, -(CR ⁸ R ⁸ ) _n -C ₃ -C ₆ halocycloalkyl, - (CR ⁸ R ⁸ ) _n -phenyl, and -(CR ⁸ R ⁸ ) _n - heteroaryl;

R ⁵ is independently selected from: H, O-d-Cs alkyl, halo, Ci-Cs alkyl, C ₂ -Cs alkenyl, C ₂ -Cs alkynyl, Ci-Cs haloalkyl, O-d-Cs haloalkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 halocycloalkyl; or R ⁴ and R ⁵ together form an alkylene or heteroalkylene chain of the form - (CR ⁸ R ⁸ )rW ¹ -(CR ⁸ R ⁸ )s-W ² -(CR ⁸ R ⁸ )t- and which is attached at its respective ends to the substitution point for R ⁴ and R ⁵ respectively; wherein W ¹ and W ² are each independently selected from: a bond, O, S and NR ⁹ ; wherein r, s, and t are each independently an integer selected from 0, 1 and 2 and wherein definitions of r, s, t, W ¹ and W ² are chosen such that the total length of the alkylene or heteroalkylene chain is 2, 3 or 4 atoms;

R ⁶ , R ⁹ , R ¹⁰ and R ¹³ are independently at each occurrence selected from: H, Ci-C ₄ alkyl, and Ci-C ₄ haloalkyl;

R ⁷ and R ¹⁴ are each independently at each occurrence selected from: H, Ci-C ₄ alkyl, Ci-C ₄ haloalkyl, S(0) ₂ -Ci-C ₄ alkyl, C(0)-Ci-C ₄ alkyl, C(0)-0-Ci-C ₄ alkyl and CH ₂ -phenyl;

R ⁸ is independently at each occurrence selected from: H, Me, CF3 and F;

where the nitrogen to which R ¹¹ is attached has a formal double bond to one of its neighbouring atoms in the heteroaromatic ring, R ^{1 1} is absent; or, where the nitrogen to which R ^{1 1} is attached is attached via formal single bonds to both of its neighbouring atoms in the heteroaromatic ring, R ^{1 1} is independently selected from: H , Ci-C ₄ alkyl, and Ci-C ₄ haloalkyl;

R ¹² may be independently at each occurrence selected from: H, halo, nitro, cyano, NR ¹³ R ¹⁴ ,

NR ¹³ S(0) ₂ R ¹³ , NR ¹³ CONR ¹³ R ¹³ , NR ¹³ C0 ₂ R ¹³ , OR ¹³ ; SR ¹³ , SOR ¹³ , SO3R ¹³ , S0 ₂ R ¹³ , S0 ₂ NR ¹³ R ¹³ CO2R ¹³ C(0)R ¹³ , CONR ¹³ R ¹³ , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, Ci-C ₄ haloalkyi, CR ¹³ R ¹³ OR ¹³ , CR ¹³ R ¹³ OC(0)R ¹³ ; and CR ¹³ R ¹³ NR ¹³ R ¹⁴ ;and

n is an integer independently selected at each occurrence from 0, 1 , 2 and 3; and

wherein each of the aforementioned aryl, heteroaryl, C3-C10 heterocycloalkyi or C3-C10 cycloalkyi groups is monocyclic or bicyclic; and

where the groups R ¹ , R ⁴ , R ⁶ , R ⁷ , R ⁹ , R ¹⁰ and R ¹⁵ groups is an alkyl, alkenyl, alkynyl, haloalkyi, cycloalkyi, halocycloalkyi, heterocycloalkyi, aryl (e.g. phenyl) or heteroaryl groups , that group is optionally substituted, where chemically possible, by 1 to 5 substituents which are each independently at each occurrence selected from: oxo, =NR ^a , =NOR ^a , halo, nitro, cyano, NR ^a R ^a , NR ^a S(0) ₂ R ^a , NR ^a CONR ^a R ^a , NR ^a C0 ₂ R ^a , OR ^a ; SR ^a , S(0)R ^a , S(0) ₂ OR ^a , S(0) ₂ R ^a , S(0) ₂ NR ^a R ^a , C0 ₂ R ^a C(0)R ^a , CONR ^a R ^a , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, Ci-C ₄ haloalkyi, CR ^b R ^b OR ^a , CR ^b R ^b NR ^a R ^a , and =CR ^b CR ^b R ^b NR ^a R ^a ; wherein R ^a is independently at each occurrence selected from: H, Ci-C ₄ alkyl and Ci-C ₄ haloalkyi; and R ^b is independently at each occurrence selected from: H, halogen, Ci-C ₄ alkyl and Ci-C ₄ haloalkyi.

Suitably both Y ¹ and Y ² are C and Z ¹ , Z ² and Z ³ are selected from CR ¹² , O, S and N; wherein a single one of Z ¹ , Z ² and Z ³ is N and that N must form part of a C=N endocyclic double bond; and wherein a single one of Z ¹ , Z ² and Z ³ is CR ¹² .

In a suitable embodiment the compound of formula (I) is a compound of formula (VII):

(VII), wherein Z ¹ is selected from O and S.

For example, in a suitable embodiment of such a compound, wherein Z ¹ may be O.

In a suitable embodiment of such a compound R ¹² is independently at each occurrence selected from: H. Ci-C ₄ -alkyl, CR ¹³ R ¹³ OR ¹³ , CR ¹³ R ¹³ OC(0)R ¹³ and CR ¹³ R ¹³ NR ¹³ R ¹⁴ .

Merely by way of example, in any such compound A may be O.

By the same token, in any such compound R ¹ may be H. Alternately or additionally, in any such compound X ² may be CR ² . Merely by way of example, in any such compound X ¹ may be CR ⁵ .

In a suitable example of such a compound R ⁵ is independently selected from: CI, O-C1-C4 alkyl, and C1-C4 alkyl.

In an alternative example of such a compound R ⁴ and R ⁵ may together form an alkylene or heteroalkylene chain of the form -0-(CR ⁸ R ⁸ )2- and which is attached at its respective ends to the substitution point for R ⁴ and R ⁵ respectively.

Suitably R ⁴ may be independently selected from C1-C4 alkyl, C1-C4 haloalkyl, cyclopropyl and halocyclopropyl.

Alternatively, R ³ may be selected from phenyl and 6- or 9-membered heteroaryl comprising at least one nitrogen.

In a suitable embodiment, of such a compound R ³ is ; wherein R ¹⁶ is R ¹⁵ ; or wherein two R ¹⁶ groups together with the carbon or carbons to which they are attached form a 3-6 membered cycloalkyi, a 3-6 membered heterocycloalkyi ring or a 6-membered aryl or heteroaryl ring; wherein where two R ¹⁶ groups form a heterocycloalkyi ring, that ring will comprise 1 or 2 heteroatoms selected from N, O and S in the ring system; wherein where two R ¹⁶ groups form a cycloalkyi or heterocycloalkyi ring, that ring is optionally substituted with one or two R ¹⁵ groups; wherein R ¹⁵ is independently selected from oxo, =NOR ⁶ , NR ⁶ R ⁷ , OR ⁶ , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, CR ⁶ R ⁶ NR ⁶ R ⁷ and =CR ⁶ CR ⁶ R ⁶ NR ⁶ R ⁷ ; and m is an integer independently selected from 0, 1 , 2, 3 and 4.

Accordingly, in a suitable embodiment, an inhibitor of type II topoisomerase that may be used as an antibiotic in accordance with the invention may include a compound of formula (II), or a pharmaceutically acceptable salt thereof:

wherein X ^1a is independently selected from: N and CR ^5a ;

X ^2a is independently selected from: N and CR ^6a ;

=A ^a is independently selected from: =0, =S, (-F) ₂ , =NR ^7a and =NOR ^7a ;

Y ^1a and Y ^2a are each independently selected from C and N; Z ^1a , Z ^2a and Z ^3a are each independently selected from O, S, S(0) ₂ , S(O), NR ^8a , CR ^9a and C=W ^a ; wherein W ^a is selected from O, S or NR ^7a ; with the proviso that if none of Z ^1a , Z ^2a and Z ^3a is C=W ^a , then the ring formed by Z ^1a , Z ^2a , Z ^3a , Y ^1a and Y ^2a contains two endocyclic double bonds and, if one of Z ^1a , Z ^2a and Z ^3a is C=W ^a , then the ring formed by Z ^1a , Z ^2a , Z ^3a , Y ^1a and Y ^2a contains a single endocyclic double bond; with the further provisos that at least one of Z ^1a , Z ^2a , Z ^3a , Y ^1a and Y ^2a is O, S, N or NR ^8a and that no more than one of Z ^1a , Z ^2a and Z ^3a is C=W; and with the yet further proviso that at least one of Z ^1a , Z ^2a and Z ^3a is NR ^8a or CR ^9a ;

R ^1a is independently selected from: H, F, NR ^7a R ^10a , NR ^7a NR ^7a R ^10a and Ci-C ₄ -alkyl;

R ^2a is Co-C ₃ -alkylene-R ^11a ;

R ^3a is -W ^1a -Co-C3-alkylene-R ^12a ; wherein W ^1a is selected from a bond, Ci-C3-alkylene, acetylene, -0-, -S(0)y- (wherein y is an integer selected from 0, 1 and 2), -NR ^7a -, -NR ^7a S(0) ₂ - , -S(0) ₂ NR ^7a -, -C(0)NR ^7a , -NR ^7a C(0)-, -OC(O)-, -C(0)0-, -OC(0)NR ^7a -, -NR ^7a C(0)0 and - NR ^7a C(0)NR ^7a - ; and wherein R ^12a is independently selected from phenyl, monocyclic heteroaryl, monocyclic 3-10 heterocycloalkyl, monocyclic C3-Cio-cycloalkyl and a bicyclic group comprising two fused rings each independently selected from phenyl, heteroaryl, 3-7- heterocycloalkyl and C3-C7-cycloalkyl; wherein R ^12a is optionally substituted with 1 , 2 or 3 R ^13a groups; wherein R ^13a is independently at each occurrence selected from: oxo, =NR ^7a , =NOR ^7a , 3-5-heterocycloalkyl, halo, nitro, cyano, NR ^7a R ^10a , NR ^7a S(0) ₂ R ^7a , NR ^7a CONR ^7a R ^7a , NR ^7a C0 ₂ R ^7a , OR ^7a , SR ^7a , SOR ^7a , S0 ₃ R ^7a , S0 ₂ R ^7a , S0 ₂ NR ^7a R ^7a , C0 ₂ R ^7a , C(0)R ^7a , CONR ^7a R ^7a , C(0)NR ^7a CR ^7a R ^7a C(0)OR ^7a , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, Ci-C ₄ -haloalkyl, Ci-C ₄ - alkylene-OR ^7a , Ci-C ₄ -alkylene-NR ^7a R ^10a , and =CR ^7b CR ^7a R ^7a NR ^7a R ^10a ;

R ^4a is independently selected from: Ci-Cs-alkyl, C ₂ -Cs-alkenyl, C ₂ -Cs-alkynyl, Ci-Cs-haloalkyI and Co-C3-alkylene-R ^14a ; wherein R ^14a is selected from C3-C6-cycloalkyl, 3-6-heterocycloalkyl, C3-C6-halocycloalkyl, -phenyl and -heteroaryl;

R ^5a is independently selected from: H, O-d-Cs-alkyl, halo, Ci-Cs-alkyl, C ₂ -Cs-alkenyl, C ₂ -Cs- alkynyl, Ci-Cs-haloalkyl, O-d-Cs-haloalkyl, C3-C6-cycloalkyl, 3-6-heterocycloalkyl, C3-C6- halocycloalkyl; or R ^4a and R ^5a together form an alkylene or heteroalkylene chain of the form - (CR ^7a R ^7a ) _ra -W ^2a -(CR ^7a R ^7a )sa-W ^3a -(CR ^7a R ^7a )ta- and which is attached at its respective ends to the substitution point for R ^4a and R ^5a respectively; wherein W ²³ and W ³³ are each independently selected from: a bond, O, S and NR ^15a ; wherein ra, sa, and ta are each independently an integer selected from 0, 1 and 2 and wherein definitions of r, s, t, W ²³ and W ³³ are chosen such that the total length of the alkylene or heteroalkylene chain is 2, 3 or 4 atoms;

or R ^3a and R ^5a , together with the carbons to which they are attached, form a 5-7-heterocycloalkyl ring which is optionally substituted with a single R ^11a group and/or from 1 to 5 R ^13a groups; R ^6a is independently selected from: H, Ci-C4-alkyl and halo;

or R ^3a and R ^6a , together with the carbons to which they are attached, form a 5-7heterocycloalkyl ring which is optionally substituted with a single R ^11a group and/or from 1 to 5 R ^13a groups; R ^7a , R ^15a , R ^16a and R ^23a are independently at each occurrence selected from: H and C1-C4- alkyl;

R ^7b is independently selected from: H, halogen and Ci-C4-alkyl;

where the nitrogen to which R ^8a is attached has a formal double bond to one of its neighbouring atoms in the ring comprising Z ^1a and Z ^2a , R ^8a is absent; or, where the nitrogen to which R ^8a is attached is attached via formal single bonds to both of its neighbouring atoms in the ring comprising Z ^1a and Z ^2a , R ^8a is independently selected from: R ^2a , H, Ci-C4-alkyl, and C1-C4- haloalkyl;

R ^9a is independently at each occurrence selected from: R ^2a , H, halo, nitro, cyano, Ci-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl and Ci-C4-haloalkyl;

providing that Z ^1a , Z ^2a , Z ^3a , Y ^1a , Y ^2a , R ^8a and R ^9a are selected such that the compound comprises either no R ^2a group or a single R ^2a group;

R ^10a is independently at each occurrence selected from: H, Ci-C4-alkyl, Ci-C4-haloalkyl, S(0)2- Ci-C ₄ -alkyl, C(0)-Ci-C ₄ -alkyl, C(0)-0-Ci-C ₄ -alkyl and CH ₂ -phenyl;

R ^11a is independently selected from: aryl, C3-C7-cycloalkyl, heteroaryl, 3-12-heterocycloalkyl, CN, NR ^16a R ^17a , OR ^18a , SR ^18a , C(0)R ^19a , S(0) _y R ^19a (wherein y is 1 or 2) and S(0)(NR ^7a )R ^19a ; R ^17a is independently selected from: H, Ci-C4-alkyl, aryl, heteroaryl, 3-7-heterocycloalkyl, C3- C ₇ -cycloalkyl, C(0)R ^19a , C(S)R ^19a , C(NR ^6a )R ^19a ; S(0) ₂ R ^19a ; Ci-C ₃ -alkylene-R ^20a ; or R ^16a and R ^17a , togetherwith the nitrogen to which they are attached togetherform a 5-12-heterocycloalkyl group or a heteroaromatic group;

R ^18a is independently selected from Ci-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, aryl, heteroaryl, 3-7-heterocycloalkyl, C ₃ -C ₇ -cycloalkyl, C(0)R ^21a and Ci-C ₄ -alkylene-R ^20a ;

R ^19a is independently selected from Ci-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, heteroaryl, 3-12- heterocycloalkyl, C ₃ -C ₇ -cycloalkyl, aryl, NR ^22a R ^23a , OR ^22a , C(0)OR ^22a , C(0)NR ^22a R ^23a , C1-C3- alkylene-R ^24a and Ci-C ₃ -alkylene-R ^20a ; R ^20a is independently aryl, heteroaryl, 3-12-heterocycloalkyl, NR ^22a R ^23a , OR ^22a , C(0)OR ^22a , C(0)NR ^22a R ^23a ;

R ^{21 a} is independently selected from Ci-C4-alkyl, aryl, heteroaryl, 3-7-heterocycloalkyl, C3-C7- cycloalkyl, NR ^22a R ^23a , Ci-C ₃ -alkylene-R ^20a ;

R ^22a is independently selected from H, Ci-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, aryl, heteroaryl, 3-7-heterocycloalkyl, C3-C7-cycloalkyl; or R ^22a and R ^23a together with the nitrogen atom to which they are attached together form a 5-12-heterocycloalkyl group or heteroaromatic group;

R ^24a is independently selected from C(O)-Ci-C ₃ -alkylene-R ^20a and S(O) ₂ -Ci-C ₃ -alkylene-R ^20a ; y is independently at each occurrence an integer selected from 1 and 2;

where any of the alkyl, alkylene, alkenyl, alkynyl, haloalkyl, cycloalkyl, halocycloalkyl, heterocycloalkyl, aryl (e.g. phenyl) or heteroaryl groups mentioned above are optionally substituted, where chemically possible, by 1 to 5 substituents which are each independently at each occurrence selected from: oxo, =NR ^a1 , =NOR ^a1 , halo, nitro, cyano, NR ^a1 R ^a1 , NR ^a1 S(0) ₂ R ^a1 , NR ^a CONR ^a1 R ^a1 , NR ^a1 C0 ₂ R ^a1 , OR ^a1 ; SR ^a1 , S(0)R ^a1 , S(0) ₂ OR ^a1 , S(0) ₂ R ^a1 , S(0) ₂ NR ^a1 R ^a1 , C0 ₂ R ^a1 C(0)R ^a1 , CONR ^a1 R ^a1 , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, C1-C4- haloalkyl, CR ^a1 R ^a1 OR ^a1 , CR ^a1 R ^a1 NR ^a1 R ^a1 , CR ^a1 R ^a1 NR ^a1 C(0)R ^a1 and =CR ^b1 CR ^a1 R ^a1 NR ^a1 R ^a1 ; wherein R ^a1 is independently at each occurrence selected from: H and Ci-C4-alkyl; and R ^b1 is independently at each occurrence selected from: H, halogen, Ci-C4-alkyl and Ci-C4-haloalkyl; and wherein any alkylene group is optionally substituted with two substituents which together with the carbon atom or carbon atoms to which they are attached form a C3-C7-cycloalkyl ring or a 3-7-heterocycloalkyl ring; wherein any alkylene group is optionally substituted with a single Co-C3-alkylene-R ^c1 group, wherein R ^c1 is independently aryl, heteroaryl, 3-7-heterocycloalkyl, C ₃ -C ₇ -cycloalkyl, C(0)NR ^a1 R ^a1 and C(0)OR ^a1 .

The compounds set out above are examples of non-quinolone antibiotics. In particular, certain of the compounds (such as Example Compounds 1 , 2, and 5) are tricyclic non-quinolone antibiotics, and certain of the compounds (such as Example Compounds 3 and 4) are tetracyclic non-quinolone antibiotics. Accordingly, in a suitable embodiment, when the invention indicates the use of an antibiotic inhibitor of the type II topoisomerases, it may be desired to employ a non-quinolone antibiotic in a method or use of the invention. Suitably the non-quinolone antibiotic may be a tricyclic non-quinolone antibiotic. Suitably the non- quinolone antibiotic may be a tetracyclic non-quinolone antibiotic

The compounds set out in this section may also be characterised as non-dione antibiotics, or as non-coumarin antibiotics. Accordingly, in a suitable embodiment, when the invention indicates the use of an antibiotic inhibitor of the type II topoisomerases, it may be desired to employ a non-dione antibiotic as a therapeutic agent in a method or use of the invention. Alternatively, or additionally, a suitable embodiment may employ a non-coumarin antibiotic as a therapeutic agent in a method or use of the invention.

Antibiotics other than Example Compound 1 , Example Compound 2, Example Compound 3, Example Compound 4, Example Compound 5, Compound 6, Example Compound 7, Example Compound 8 or Example Compound 9 suitable for use in the methods or medical uses of the invention

The seventh, ninth, tenth, and twelfth aspects of the invention each refer to methods of uses of the invention in which the use of an antibiotic other than those selected from the group consisting of: Example Compound 1 ; Example Compound 2; Example Compound 3; Example Compound 4; Example Compound 5; Example Compound 6; Example Compound 7; Example Compound 8; and Example Compound 9 is indicated. The structures of these Example Compounds are set out in the Examples later in this specification.

Generally, the contexts in which it is desirable to use antibiotics other than these Example Compounds are those in which the presence of a gene encoding a GrIB protein comprising a mutation at the amino acid residue homologous to arginine 453 of the protein in Staphylococcus aureus has been identified in a bacterium (such as a bacterium associated with an infection of a subject).

The Example Compounds 1 to 9, are examples of a broader group of compounds disclosed in the applicant's co-pending International Patent Application number PCT/GB2015/051 107 (published as WO 2015/155549), PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7. In certain embodiments where the use of an antibiotic compound other than Example Compounds 1 to 9 is indicated, it may be further desirable to utilise an antibiotic compound other than those disclosed in the above-mentioned International Patent Application. Thus, in certain embodiments of the invention, it may be desired to use an antibiotic compound other than a compound of formula (I), or a pharmaceutically acceptable salt thereof: wherein X ¹ is independently selected from: N and CR ⁵ ;

X ² is independently selected from: N and CR ² ;

=A is independently selected from: =0, =S, =NR ⁶ and =NOR ⁶ ;

Y ¹ and Y ² are each independently selected from C and N; Z ¹ , Z ² and Z ³ are each independently selected from O, S, S(O), NR ¹¹ , CR ¹² and C=W; wherein W is selected from O, S or NR ⁶ ; with the proviso that if none of Z ¹ , Z ² and Z ³ is C=W, then the ring formed by Z ¹ , Z ² , Z ³ , Y ¹ and Y ² contains two endocyclic double bonds and, if one of Z ¹ , Z ² and Z ³ is C=W, then the ring formed by Z ¹ , Z ² , Z ³ , Y ¹ and Y ² contains a single endocyclic double bond; and with the further proviso that at least one of Z ¹ , Z ² , Z ³ , Y ¹ and Y ² is O, S, N or N R ¹¹ ;

R ¹ is independently selected from: H, F, NR ⁶ R ⁷ , NR ⁶ NR ⁶ R ⁷ and Ci-C ₄ -alkyl;

R ² is independently selected from: H and F;

R ³ is independently selected from: -(CR ⁸ R ⁸ ) _n -3-io heterocycloalkyl, -(CR ⁸ R ⁸ ) _n -aryl, -(CR ⁸ R ⁸ ) _n - heteroaryl, and -(CR ⁸ R ⁸ ) _n -C3-Cio cycloalkyl; wherein the aryl, heteroaryl, heterocycloalkyl or cycloalkyl group is optionally substituted with 1 , 2 or 3 R ¹⁵ groups; wherein R ¹⁵ is independently at each occurrence selected from: oxo, =NR ⁶ , =NOR ⁶ , 3-5-heterocycloalkyl, halo, nitro, cyano, NR ⁶ R ⁷ , NR ⁶ S(0) ₂ R ⁶ , NR ⁶ CONR ⁶ R ⁶ , NR ⁶ C0 ₂ R ⁶ , OR ⁶ ; SR ⁶ , SOR ⁶ , SO3R ⁶ , SO2R ⁶ , S0 ₂ NR ⁶ R ⁶ C0 ₂ R ⁶ C(0)R ⁶ , CONR ⁶ R ⁶ , C(0)NR ⁶ CR ⁶ R ⁶ C(0)OR ⁶ , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, Ci-C ₄ haloalkyl, CR ⁶ R ⁶ OR ⁶ , CR ⁶ R ⁶ NR ⁷ R ⁶ , and =CR ⁶ CR ⁶ R ⁶ NR ⁷ R ⁶ ;

R ⁴ is independently selected from: Ci-Cs alkyl, C ₂ -Cs alkenyl, C ₂ -Cs alkynyl, Ci-Cs haloalkyl, - (CR ⁸ R ⁸ ) _n -C ₃ -C ₆ cycloalkyl, -(CR ⁸ R ⁸ ) _n -C ₃ -C ₆ heterocycloalkyl, -(CR ⁸ R ⁸ ) _n -C ₃ -C ₆ halocycloalkyl, - (CR ⁸ R ⁸ ) _n -phenyl, and -(CR ⁸ R ⁸ ) _n - heteroaryl;

R ⁵ is independently selected from: H, O-d-Cs alkyl, halo, Ci-Cs alkyl, C ₂ -Cs alkenyl, C ₂ -Cs alkynyl, Ci-Cs haloalkyl, O-d-Cs haloalkyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C3-C6 halocycloalkyl; or R ⁴ and R ⁵ together form an alkylene or heteroalkylene chain of the form - (CR ⁸ R ⁸ )rW ¹ -(CR ⁸ R ⁸ )s-W ² -(CR ⁸ R ⁸ )t- and which is attached at its respective ends to the substitution point for R ⁴ and R ⁵ respectively; wherein W ¹ and W ² are each independently selected from: a bond, O, S and NR ⁹ ; wherein r, s, and t are each independently an integer selected from 0, 1 and 2 and wherein definitions of r, s, t, W ¹ and W ² are chosen such that the total length of the alkylene or heteroalkylene chain is 2, 3 or 4 atoms; R ⁶ , R ⁹ , R ¹⁰ and R ¹³ are independently at each occurrence selected from: H, C1-C4 alkyl, and C1-C4 haloalkyi;

R ⁷ and R ¹⁴ are each independently at each occurrence selected from: H, C1-C4 alkyl, C1-C4 haloalkyi, S(0) ₂ -Ci-C ₄ alkyl, C(0)-Ci-C ₄ alkyl, C(0)-0-Ci-C ₄ alkyl and CH ₂ -phenyl;

R ⁸ is independently at each occurrence selected from: H, Me, CF3 and F;

where the nitrogen to which R ¹¹ is attached has a formal double bond to one of its neighbouring atoms in the heteroaromatic ring, R ^{1 1} is absent; or, where the nitrogen to which R ^{1 1} is attached is attached via formal single bonds to both of its neighbouring atoms in the heteroaromatic ring, R ^{1 1} is independently selected from: H , C1-C4 alkyl, and C1-C4 haloalkyi;

R ¹² may be independently at each occurrence selected from: H, halo, nitro, cyano, NR ¹³ R ¹⁴ ,

NR ¹³ S(0) ₂ R ¹³ , NR ¹³ CONR ¹³ R ¹³ , NR ¹³ C0 ₂ R ¹³ , OR ¹³ ; SR ¹³ , SOR ¹³ , SO3R ¹³ , S0 ₂ R ¹³ ,

S0 ₂ NR ¹³ R ¹³ C0 ₂ R ¹³ C(0)R ¹³ , CONR ¹³ R ¹³ , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, C1-C4 haloalkyi, CR ¹³ R ¹³ OR ¹³ , CR ¹³ R ¹³ OC(0)R ¹³ ; and CR ¹³ R ¹³ NR ¹³ R ¹⁴ ;and

n is an integer independently selected at each occurrence from 0, 1 , 2 and 3; and

wherein each of the aforementioned aryl, heteroaryl, C3-C10 heterocycloalkyi or C3-C10 cycloalkyi groups is monocyclic or bicyclic; and

where the groups R ¹ , R ⁴ , R ⁶ , R ⁷ , R ⁹ , R ¹⁰ and R ¹⁵ groups is an alkyl, alkenyl, alkynyl, haloalkyi, cycloalkyi, halocycloalkyi, heterocycloalkyi, aryl (e.g. phenyl) or heteroaryl groups , that group is optionally substituted, where chemically possible, by 1 to 5 substituents which are each independently at each occurrence selected from: oxo, =NR ^a , =NOR ^a , halo, nitro, cyano, NR ^a R ^a , NR ^a S(0) ₂ R ^a , NR ^a CONR ^a R ^a , NR ^a C0 ₂ R ^a , OR ^a ; SR ^a , S(0)R ^a , S(0) ₂ OR ^a , S(0) ₂ R ^a , S(0) ₂ NR ^a R ^a , C0 ₂ R ^a C(0)R ^a , CONR ^a R ^a , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, C1-C4 haloalkyi, CR ^b R ^b OR ^a , CR ^b R ^b NR ^a R ^a , and =CR ^b CR ^b R ^b NR ^a R ^a ; wherein R ^a is independently at each occurrence selected from: H, C1-C4 alkyl and C1-C4 haloalkyi; and R ^b is independently at each occurrence selected from: H, halogen, C1-C4 alkyl and C1-C4 haloalkyi.

Suitably both Y ¹ and Y ² are C and Z ¹ , Z ² and Z ³ are selected from CR ¹² , O, S and N; wherein a single one of Z ¹ , Z ² and Z ³ is N and that N must form part of a C=N endocyclic double bond; and wherein a single one of Z ¹ , Z ² and Z ³ is CR ¹² .

In a suitable embodiment the compound of formula (I) is a compound of formula (VII): (VII), wherein Z ¹ is selected from O and S.

For example, in a suitable embodiment of such a compound, wherein Z ¹ may be O.

In a suitable embodiment of such a compound R ¹² is independently at each occurrence selected from: H. Ci-C ₄ -alkyl, CR ¹³ R ¹³ OR ¹³ , CR ¹³ R ¹³ OC(0)R ¹³ and CR ¹³ R ¹³ NR ¹³ R ¹⁴ .

Merely by way of example, in any such compound A may be O.

By the same token, in any such compound R ¹ may be H.

Alternately or additionally, in any such compound X ² may be CR ² .

Merely by way of example, in any such compound X ¹ may be CR ⁵ .

In a suitable example of such a compound R ⁵ is independently selected from: CI, 0-Ci-C ₄ alkyl, and Ci-C ₄ alkyl.

In an alternative example of such a compound R ⁴ and R ⁵ may together form an alkylene or heteroalkylene chain of the form -0-(CR ⁸ R ⁸ )2- and which is attached at its respective ends to the substitution point for R ⁴ and R ⁵ respectively.

Suitably R ⁴ may be independently selected from Ci-C ₄ alkyl, Ci-C ₄ haloalkyi, cyclopropyl and halocyclopropyl.

Alternatively, R ³ may be selected from phenyl and 6- or 9-membered heteroaryl comprising at least one nitrogen. In a suitable embodiment, of such a compound R ³ i is ; wherein R ¹⁶ is R or wherein two R ¹⁶ groups together with the carbon or carbons to which they are attached form a 3-6 membered cycloalkyi, a 3-6 membered heterocycloalkyi ring or a 6-membered aryl or heteroaryl ring; wherein where two R ¹⁶ groups form a heterocycloalkyi ring, that ring will comprise 1 or 2 heteroatoms selected from N, O and S in the ring system; wherein where two R ¹⁶ groups form a cycloalkyi or heterocycloalkyi ring, that ring is optionally substituted with one or two R ¹⁵ groups; wherein R ¹⁵ is independently selected from oxo, =NOR ⁶ , N R ⁶ R ⁷ , OR ⁶ , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, CR ⁶ R ⁶ N R ⁶ R ⁷ and =CR ⁶ CR ⁶ R ⁶ N R ⁶ R ⁷ ; and m is an integer independently selected from 0, 1 , 2, 3 and 4.

In certain embodiments of the invention, it may be desired to use an antibiotic compound other than a compound of formula (II), or a pharmaceutically acceptable salt thereof:

(ii);

wherein X ^{1 a} is independently selected from: N and CR ^5a ;

X ^2a is independently selected from: N and CR ^6a ;

=A ^a is independently selected from: =0, =S, (-F) ₂ , =N R ^7a and =NOR ^7a ;

Y ^{1 a} and Y ^2a are each independently selected from C and N ; Z ^{1 a} , Z ^2a and Z ^3a are each independently selected from O, S, S(0) ₂ , S(O), N R ^8a , CR ^9a and C=W ^a ; wherein W ^a is selected from O, S or N R ^7a ; with the proviso that if none of Z ^{1 a} , Z ^2a and Z ^3a is C=W ^a , then the ring formed by Z ^{1 a} , Z ^2a , Z ^3a , Y ^{1 a} and Y ^2a contains two endocyclic double bonds and, if one of Z ^{1 a} , Z ^2a and

Z ^3a is C=W ^a , then the ring formed by Z ^{1 a} , Z ^2a , Z ^3a , Y ^{1 a} and Y ^2a contains a single endocyclic double bond; with the further provisos that at least one of Z ^{1 a} , Z ^2a , Z ^3a , Y ^{1 a} and Y ^2a is O, S, N or N R ^8a and that no more than one of Z ^{1 a} , Z ^2a and Z ^3a is C=W; and with the yet further proviso that at least one of Z ^{1 a} , Z ^2a and Z ^3a is N R ^8a or CR ^9a ;

R ^{1 a} is independently selected from: H, F, N R ^7a R ^10a , N R ^7a N R ^7a R ^10a and Ci-C ₄ -alkyl;

R ^2a is Co-C ₃ -alkylene-R ^{1 1 a} ;

R ^3a is -W ^{1 a} -Co-C3-alkylene-R ^12a ; wherein W ^{1 a} is selected from a bond, Ci-C3-alkylene, acetylene, -0-, -S(0)y- (wherein y is an integer selected from 0, 1 and 2), -N R ^7a -, -N R ^7a S(0) ₂ - , -S(0) ₂ N R ^7a -, -C(0)N R ^7a , -N R ^7a C(0)-, -OC(O)-, -C(0)0-, -OC(0)N R ^7a -, -N R ^7a C(0)0 and - NR ^7a C(0)NR ^7a - ; and wherein R ^12a is independently selected from phenyl, monocyclic heteroaryl, monocyclic 3-10 heterocycloalkyl, monocyclic C3-Cio-cycloalkyl and a bicyclic group comprising two fused rings each independently selected from phenyl, heteroaryl, 3-7- heterocycloalkyl and C3-C7-cycloalkyl; wherein R ^12a is optionally substituted with 1 , 2 or 3 R ^13a groups; wherein R ^13a is independently at each occurrence selected from: oxo, =NR ^7a , =NOR ^7a , 3-5-heterocycloalkyl, halo, nitro, cyano, NR ^7a R ^10a , NR ^7a S(0) ₂ R ^7a , NR ^7a CONR ^7a R ^7a , NR ^7a C0 ₂ R ^7a , OR ^7a , SR ^7a , SOR ^7a , S0 ₃ R ^7a , S0 ₂ R ^7a , S0 ₂ NR ^7a R ^7a , C0 ₂ R ^7a , C(0)R ^7a , CONR ^7a R ^7a , C(0)NR ^7a CR ^7a R ^7a C(0)OR ^7a , CrC ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, Ci-C ₄ -haloalkyl, Ci-C ₄ - alkylene-OR ^7a , Ci-C ₄ -alkylene-NR ^7a R ^10a , and =CR ^7b CR ^7a R ^7a NR ^7a R ^10a ;

R ^4a is independently selected from: d-Cs-alkyl, C ₂ -Cs-alkenyl, C ₂ -Cs-alkynyl, d-Cs-haloalkyl and Co-C3-alkylene-R ^14a ; wherein R ^14a is selected from C3-C6-cycloalkyl, 3-6-heterocycloalkyl, C3-C6-halocycloalkyl, -phenyl and -heteroaryl;

R ^5a is independently selected from: H, O-d-Cs-alkyl, halo, Ci-Cs-alkyl, C ₂ -Cs-alkenyl, C ₂ -Cs- alkynyl, d-Cs-haloalkyl, O-d-Cs-haloalkyl, C3-C6-cycloalkyl, 3-6-heterocycloalkyl, C3-C6- halocycloalkyl; or R ^4a and R ^5a together form an alkylene or heteroalkylene chain of the form - (CR ^7a R ^7a ) _ra -W ^2a -(CR ^7a R ^7a )sa-W ^3a -(CR ^7a R ^7a )ta- and which is attached at its respective ends to the substitution point for R ^4a and R ^5a respectively; wherein W ²³ and W ³³ are each independently selected from: a bond, O, S and NR ^15a ; wherein ra, sa, and ta are each independently an integer selected from 0, 1 and 2 and wherein definitions of r, s, t, W ²³ and W ³³ are chosen such that the total length of the alkylene or heteroalkylene chain is 2, 3 or 4 atoms;

or R ^3a and R ^5a , together with the carbons to which they are attached, form a 5-7-heterocycloalkyl ring which is optionally substituted with a single R ^11a group and/or from 1 to 5 R ^13a groups; R ^6a is independently selected from: H , Ci-C ₄ -alkyl and halo;

or R ^3a and R ^6a , together with the carbons to which they are attached, form a 5-7heterocycloalkyl ring which is optionally substituted with a single R ^11a group and/or from 1 to 5 R ^13a groups; R ^7a , R ^15a , R ^16a and R ^23a are independently at each occurrence selected from: H and Ci-C ₄ - alkyl;

R ^7b is independently selected from: H, halogen and Ci-C ₄ -alkyl;

where the nitrogen to which R ^8a is attached has a formal double bond to one of its neighbouring atoms in the ring comprising Z ^{1 a} and Z ^2a , R ^8a is absent; or, where the nitrogen to which R ^8a is attached is attached via formal single bonds to both of its neighbouring atoms in the ring comprising Z ^{1 a} and Z ^2a , R ^8a is independently selected from: R ^2a , H, Ci-C ₄ -alkyl, and Ci-C ₄ - haloalkyl;

R ^9a is independently at each occurrence selected from: R ^2a , H , halo, nitro, cyano, Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl and Ci-C ₄ -haloalkyl; providing that Z ^1a , Z ^2a , Z ^3a , Y ^1a , Y ^2a , R ^8a and R ^9a are selected such that the compound comprises either no R ^2a group or a single R ^2a group;

R ^10a is independently at each occurrence selected from: H, Ci-C4-alkyl, Ci-C4-haloalkyl, S(0)2- Ci-C ₄ -alkyl, C(0)-Ci-C ₄ -alkyl, C(0)-0-Ci-C ₄ -alkyl and CH ₂ -phenyl;

R ^{1 1 a} is independently selected from: aryl, C3-C7-cycloalkyl, heteroaryl, 3-12-heterocycloalkyl, CN, NR ^16a R ^17a , OR ^18a , SR ^18a , C(0)R ^19a , S(0) _y R ^19a (wherein y is 1 or 2) and S(0)(NR ^7a )R ^19a ; R ^17a is independently selected from: H, Ci-C ₄ -alkyl, aryl, heteroaryl, 3-7-heterocycloalkyl, C3- C ₇ -cycloalkyl, C(0)R ^19a , C(S)R ^19a , C(NR ^6a )R ^19a ; S(0) ₂ R ^19a ; Ci-C ₃ -alkylene-R ^20a ; or R ^16a and R ^17a , togetherwith the nitrogen to which they are attached togetherform a 5-12-heterocycloalkyl group or a heteroaromatic group;

R ^18a is independently selected from Ci-C ₄ -alkyl, C2-C ₄ -alkenyl, C2-C ₄ -alkynyl, aryl, heteroaryl, 3-7-heterocycloalkyl, C ₃ -C ₇ -cycloalkyl, C(0)R ^{21 a} and Ci-C ₄ -alkylene-R ^20a ;

R ^19a is independently selected from Ci-C ₄ -alkyl, C2-C ₄ -alkenyl, C2-C ₄ -alkynyl, heteroaryl, 3-12- heterocycloalkyl, C ₃ -C ₇ -cycloalkyl, aryl, NR ^22a R ^23a , OR ^22a , C(0)OR ^22a , C(0)NR ^22a R ^23a , C1-C3- alkylene-R ^24a and Ci-C ₃ -alkylene-R ^20a ;

R ^20a is independently aryl, heteroaryl, 3-12-heterocycloalkyl, NR ^22a R ^23a , OR ^22a , C(0)OR ^22a , C(0)NR ^22a R ^23a ;

R ^{21 a} is independently selected from Ci-C ₄ -alkyl, aryl, heteroaryl, 3-7-heterocycloalkyl, C3-C7- cycloalkyl, NR ^22a R ^23a , Ci-C ₃ -alkylene-R ^20a ;

R ^22a is independently selected from H, Ci-C ₄ -alkyl, C2-C ₄ -alkenyl, C2-C ₄ -alkynyl, aryl, heteroaryl, 3-7-heterocycloalkyl, C3-C7-cycloalkyl; or R ^22a and R ^23a together with the nitrogen atom to which they are attached together form a 5-12-heterocycloalkyl group or heteroaromatic group;

R ^24a is independently selected from C(O)-Ci-C ₃ -alkylene-R ^20a and S(O) ₂ -Ci-C ₃ -alkylene-R ^20a ; y is independently at each occurrence an integer selected from 1 and 2;

where any of the alkyl, alkylene, alkenyl, alkynyl, haloalkyl, cycloalkyl, halocycloalkyl, heterocycloalkyl, aryl (e.g. phenyl) or heteroaryl groups mentioned above are optionally substituted, where chemically possible, by 1 to 5 substituents which are each independently at each occurrence selected from: oxo, =NR ^a1 , =NOR ^a1 , halo, nitro, cyano, NR ^a1 R ^a1 , NR ^a1 S(0) ₂ R ^a1 , NR ^a CONR ^a1 R ^a1 , NR ^a1 C0 ₂ R ^a1 , OR ^a1 ; SR ^a1 , S(0)R ^a1 , S(0) ₂ OR ^a1 , S(0) ₂ R ^a1 , S(0) ₂ NR ^a1 R ^a1 , C0 ₂ R ^a1 C(0)R ^a1 , CONR ^a1 R ^a1 , Ci-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ -alkynyl, Ci-C ₄ - haloalkyl, CR ^a1 R ^a1 OR ^a1 , CR ^a1 R ^a1 NR ^a1 R ^a1 , CR ^a1 R ^a1 NR ^a1 C(0)R ^a1 and =CR ^b1 CR ^a1 R ^a1 NR ^a1 R ^a1 ; wherein R ^a1 is independently at each occurrence selected from: H and Ci-C ₄ -alkyl; and R ^b1 is independently at each occurrence selected from: H, halogen, Ci-C ₄ -alkyl and Ci-C ₄ -haloalkyl; and wherein any alkylene group is optionally substituted with two substituents which together with the carbon atom or carbon atoms to which they are attached form a C3-C7-cycloalkyl ring or a 3-7-heterocycloalkyl ring; wherein any alkylene group is optionally substituted with a single Co-C3-alkylene-R ^c1 group, wherein R ^c1 is independently aryl, heteroaryl, 3-7-heterocycloalkyl, Cs-Cy-cycloalkyl, C(0)NR ^a1 R ^a1 and C(0)OR ^a1 .

Each of these examples referred to above (the Example Compounds 1 to 9, and the compounds disclosed in WO 2015/155549, PCT/GB2016/052899, PCT/GB2016/052898, PCT/GB2016/052896 and GB1615829.7) are examples of inhibitors of type II topoisomerases. In certain embodiments where the use of an antibiotic compound other than Example Compounds 1 to 9 is indicated, it may be desirable to utilise an alternative antibiotic compound other than an inhibitor of type II topoisomerases.

The compounds set out above are examples of non-quinolone antibiotics. Certain of the compounds (such as Example Compounds 1 , 2, and 5) are tricyclic non-quinolone antibiotics. Other compounds (such as Example Compounds 3 and 4) are tetracyclic non-quinolone antibiotics. Accordingly, in a suitable embodiment where the use of an antibiotic compound other than Example Compounds 1 to 9 is indicated, the relevant method or use of the invention may employ an antibiotic other than a non-quinolone antibiotic. In particular, such embodiments of the invention may employ an antibiotic other than a tricyclic non-quinolone antibiotic, or an antibiotic other than a tetracyclic non-quinolone antibiotic.

The compounds set out in the preceding paragraphs of this section may also be characterised as non-dione antibiotics, or as non-coumarin antibiotics. Accordingly, a suitable embodiment of a method or use of the invention, where the use of an antibiotic compound other than Example Compounds 1 to 9 is indicated, may employ an antibiotic other than a non-dione antibiotic as a therapeutic agent. Alternatively, or additionally, a suitable embodiment of a method or use of the invention, where the use of an antibiotic compound other than Example Compounds 1 to 9 is indicated, may employ an antibiotic other than a non-coumarin antibiotic as a therapeutic agent.

The invention will now be further described in the following Examples. EXAMPLES

Example 1

Table 1. Identities of the Example Compounds used in this study.

Example 2

Mutant Isolation by Serial Passage

Mutants were raised from Staphylococcus aureus ATCC 29213 over multiple passages in the presence of a sub-inhibitory concentration of Example Compound 1. Specifically, cation- adjusted Muller-Hinton broth (CA-MHB) was inoculated with the bacterial strains and allowed to grow for 18 hours at 37°C. Following incubation, a doubling-dilution series of the compound was prepared in CA-MHB (concentration range 0.06 to 8 μg/mL). Broths were inoculated using the previously grown cultures at a ratio of 1 : 100 and incubated for another 18 hours at 37°C. After incubation, the lowest concentration that inhibited visible growth (the minimum inhibitory concentration, MIC) was recorded and the culture equivalent to one-quarter of the MIC was used to inoculate a further set of broths containing the compound. The concentration range was increased with each passage to span the MIC from the previous passage. This process was repeated until the desired level of resistance had been achieved. The Staphylococcus aureus strain SA4139-SP40 was isolated after 40 passages.

Example 3

Spontaneous Mutant Isolation

The isolation of spontaneously-arising resistant derivatives of Staphylococcus aureus ATCC 29213 to Compound 3 was achieved as follows. Staphylococcus aureus ATCC 29213 was cultured using CA-MHB and Muller Hinton agar (MHA). Bacterial cultures containing ~10 ⁹ CFU/mL were plated onto agar containing 4 ^χ or 8 ^χ MIC of the compound to allow enumeration of resistant mutants. Cultures were also diluted in phosphate-buffered saline (PBS) and dilutions containing ~10 ² CFU/mL were plated on to compound-free agar to allow enumeration of total viable cells. Plates were incubated at 37 °C for 48 h for Staphylococcus aureus. Representative clones, including SA6485-A2, were selected for genotypic and phenotypic characterisation.

Example 4

Antibiotic Susceptibility Testing

Minimum Inhibitory Concentration (MIC) of antibiotics versus strains of Staphylococcus aureus were determined by the broth microdilution procedure according to the guidelines of the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Ninth Edition. CLSI document M07-A9, 2012). The broth microdilution method involves a two-fold serial dilution of compounds in 96-well microtitre plates, giving a typical final concentration range of 0.001-64 μg/mL and a maximum final concentration of 1 % DMSO. Bacterial strains tested included Staphylococcus aureus ATCC 29213, Staphylococcus aureus SA4139-SP40 and Staphylococcus aureus SA6845-A2. Strains were grown in CA-MHB or MHA at 37°C in an ambient atmosphere. The MIC is determined as the lowest concentration of compound that inhibits visible growth following a 16-20 hour incubation period. A difference in MIC equivalent to one doubling dilution in either direction is conventionally considered to be within the standard variability of this method.

Table 2. MICs of antibiotic compounds against strains of Staphylococcus aureus.

Trimethoprim 4 2 4

Vancomycin 0.5 1 1

Example Compound 1 0.03 0.25 0.12

Example Compound 2 0.5 4 4

Example Compound 3 0.5 4 4

Example Compound 4 0.5 2 2

Example Compound 5 0.06 1 0.25

Example Compound 6 0.12 0.5 0.5

Example Compound 7 0.25 4 1

Example Compound 8 2 16 16

Example Compound 9 0.25 2 1

The susceptibility of the Staphylococcus aureus strains SA4139-SP40 and SA6485-A2 to ampicillin, ciprofloxacin, delafloxacin, erythromycin, gentamicin, levofloxacin, linezolid, novobiocin, penicillin G, rifampicin, tetracycline, trimethoprim and vancomycin was at least as much as for the wild-type strain, Staphylococcus aureus ATCC 29213. In contrast, the Staphylococcus aureus strains SA4139-SP40 and SA6485-A2 had reduced susceptibility (greater than or equal to a four-fold increase in the MIC) to the Example Compounds 1-9. This is consistent with a selective and specific marker for this class of novel small-molecule inhibitor of the bacterial type II topoisomerases.

Example 5

DNA sequencing

Genomic DNA (gDNA) was extracted from the bacterial strains using the EdgeBio PurElute Bacterial Genomic Kit (Gaithersburg, MD, USA). Staphylococcus aureus gDNA (ATCC 29213, SA4139-SP40 and SA6485-A2) was purified using a modified protocol of the manufacturer's methodology as follows: lysostaphin (Sigma-Aldrich, Dorset, UK) was incorporated into the spheroblast buffer (100 μg/mL) to ensure complete cell lysis; proteinase K (Sigma-Aldrich, Dorset, UK) was also included in the reaction when adding the extraction buffer (100 μg/mL), which was then incubated at 37°C for 15 minutes.

The topoisomerase genes gyrA, gyrB, grIA and grIB, were amplified from Staphylococcus aureus ATCC 29213, SA4139-SP40 and SA6485-A2 gDNA by polymerase chain reaction (PCR). The sequences of the oligonucleotide primers used for PCR and for DNA sequence determination are shown in Table 3. Phusion high-fidelity polymerase (New England Biolabs, Ipswich, MA, USA) was used to amplify target genes using the reaction conditions and cycling parameters shown in Tables 3 and 4.

Amplicons were purified using the QIAquick PCR purification kit (QIAGEN, Manchester, UK) according to the manufacturer's instructions and were visualised by agarose gel electrophoresis. Specifically, molten agarose (1 % agarose [w/v] in tris-acetate-EDTA [TAE] buffer) was combined with SYBRsafe DNA gel stain (Invitrogen, Paisley, UK) and allowed to set. PCR amplicons (5 μΙ_) and DNA markers (Hyperladder I [Bioline, London, UK]) were loaded onto the gel which was then run at 90 V for 40 min. DNA bands were visualised using a UVP Chemidoc-it 2 system (Upland, CA, USA). DNA quantity was determined by assessing the absorbance at 260 nm; a value of 1 represented a DNA concentration of 50 μg/mL. DNA purity was also evaluated by deducing the ratio of the absorbance values at 260:280 nm and 260:230 nm. Ratios of around 1.8 indicated a sample free of contaminating protein and other organic compounds.

Topoisomerase genes DNA sequence determination was elucidated by Sanger sequencing at Beckman Coulter Genomics Inc (Takeley, UK). Sequences were aligned and analysed using DNASTAR SeqMan Pro software (Version 12.2.0).

Table 3. Oligonucleotide primers used in the PCR amplification and DNA sequencing of gyrA, gyrB, grIA and grIB of Staphylococcus aureus mutant strains.

AAGCGGTTATGAGTGCCACA 66 °C gyrA_Rev

SEQ ID NO:6

CGCAGCAGCAATGCGTTATAC

gyrA_Seq1_Fwd

SEQ ID NO:7

ATTGCAGAGCTCGTTCGTGA

gyrA_Seq2_Fwd

SEQ ID NO:8

GCTCAAGCTATTTTAGACATGCGT

gyrA_Seq3_Fwd

SEQ ID N0:9

TGCAACTAAACGTGGTGTCG

gyrA_Seq4_Fwd

SEQ ID NO:10

TCTCAAAATGGTCGTGCAGC

gyrA_Seq5_Fwd

SEQ ID N0:11

CGATTCAGCATAAAGTACAAACATTTGTC 62 °C gyrB_Fwd

SEQ ID N0:12

GCACGAGCAACGATAACAC 62 °C gyrB_Rev

SEQ ID N0:13

CTGGTGGTTTACATGGTGTTGG

gyrB_Seq1_Fwd

SEQ ID N0:14

AGGTGGTACGCATGAAGACG

gyrB_Seq2_Fwd

SEQ ID N0:15

TTGGTACAGGAATCGGTGGC

gyrB_Seq3_Fwd

SEQ ID N0:16

AAATGATCAATTTGATGAGGAGGAA 66 °C grlA_Fwd

SEQ ID N0:17

TTATCATTTAATTTCGTGATTGCATATAG 66 °C grlA_Rev

SEQ ID N0:18

CTGCAGGTTACGCGACAGAT

grlA_Seq1_Fwd

SEQ ID N0:19

ATCACCAAATTGAGGTTGTTGC

grlA_Seq2_Fwd

SEQ ID NO:20

AGCTTTAATGCTAGCGGTGT

grlA_Seq3_Fwd

SEQ ID N0:21

TGACCAATAACATCACCGACTGT

grlA_Seq4_Rev

SEQ ID NO:22

GCATTTTACGCTGATTTATATAAGAATAACTATTG 55 °C grlB_Fwd

SEQ ID NO:23 GACTGTTTTCGCACTTTTA 55 °C grlB_Rev

SEQ ID NO:24

GTGGTTCGCCATCTTCTGGT

grlB_Seq1_Fwd

SEQ ID NO:25

ACAGCTGTTGTGTCTGTTCGT

grlB_Seq2_Fwd

SEQ ID NO:26

TGGTGCGCATATTCAAGTGC

grlB_Seq3_Fwd

SEQ ID NO:27

Table 4. Composition of the reaction mixtures used in the PCR amplification of Staphylococcus aureus gyrA, gyrB, grIA and grIB.

Table 5. Reaction conditions used for PCR amplification of Staphylococcus aureus gyrA, gyrB, grIA and grIB.

Example 6 Sequences of Staphylococcus aureus topoisomerase genes and gene products

Single nucleotide polymorphisms (SNPs) identified by the DNA sequencing of the gyrA, gyrB, grIA and grIB genes of Staphylococcus aureus strains SA4139-SP40 and SA6485-A2 are summarised in Table 6.

Table 6. Mutations identified in the DNA sequences of the topoisomerase genes of Staphylococcus aureus strains relative to Staphylococcus aureus strain ATCC 29213 (nominally designated as wild-type).

Each strain contained the same single SNP in grIB, namely an adenine to guanine substitution at nucleotide number 1358. The full nucleotide sequence of the grIB coding sequence from Staphylococcus aureus strains SA4139-SP40 and SA6485-A2 is shown in SEQ ID NO: 1.

Amino acid substitutions in the topoisomerase gene products resulting from the mutations identified by the DNA sequencing of the gyrA, gyrB, grIA and grIB genes of Staphylococcus aureus strains SA4139-SP40 and SA6485-A2 are summarised in Table 7.

Table 7. Mutations identified in the amino acid sequences of the topoisomerase gene products of Staphylococcus aureus strains relative to Staphylococcus aureus strain ATCC 29213 (nominally designated as wild-type).

Both strains contained the same single amino acid substitution, namely an arginine to histidine substitution at residue number 453 of the GrIB gene product. The full primary amino acid sequence of the GrIB gene product from Staphylococcus aureus strains SA4139-SP40 and SA6485-A2 is shown in SEQ ID NO: 2.

Example 7

Whole genome Sequencing of Staphylococcus aureus strains

Genomic DNA sequence determination was performed at the Next Generation Sequencing facility in the Leeds Institute of Molecular Medicine at the University of Leeds. Purified genomic DNA was used to create whole genome libraries using NEBNext Ultra kit and 150 bp paired end read sequence data were produced using an lllumina MiSeq. Read data were stored as FASTQ files and then adaptor sequences were removed using cutadapt software (Version 1.8). Data for the wild-type strains were used to construct reference genome sequences using the CLCBio genome assembler (Version 8.0.1). Sequence data for each sample, including the progenitor strains, were aligned to the relevant genome using BWA (Version 0.7.12); aligned data were sorted using Samtools6 (Version 1.2). Variants were identified using VarScan (Version 2.3.7) using the appropriate assembled genome as the reference sequence. The resulting data provided a read depth of >100 across the genome. Single nucleotide polymorphisms (SNPs), insertions and deletions were identified that were prevalent in≥95% of the reads compared with the progenitor strains.

The SNPs identified in both mutant strains of Staphylococcus aureus include the mutation in grIB, namely an adenine to guanine substitution at nucleotide number 1358. No further SNPs were identified in strain SA6485-A2. However three further SNPs were identified in strain SA4139-SP40. The gene products containing these mutations are listed below in table 8. None of the further mutations identified in strain SA4139-SP40 are related to the GrIB topoisomerase proteins as used in the present invention and are not believed to influence the resistance of bacteria to inhibitors of type II topoisomerases. However, the further mutations may be used to better characterise the SA4139-SP40 strain.

Table 8. Mutations identified in the gene products of the genome sequences of the Staphylococcus aureus strains relative to Staphylococcus aureus strain ATCC 29213 (nominally designated as wild-type). Gene Product Sequence

organic

autolysin/mann hydroperoxide

NagD-like osyl- resistance

Strain phosphatase/H glycoprotein transcriptional

GrIB

AD family endo-beta-N- regulator/MarR

hydrolase acetylglucosa family

minidase transcriptional

regulator

Pro185Ser Ala79Val Ser70Leu Arg453His

SA4139-SP40

Wild-type Wild-type W\ Id-type Arg453His

SA6485-A2

SEQ ID NO: 1

AT GAATAAACAAAATAAT TAT T CAGAT GAT T CAATACAGGT T T TAGAGGGGT TAGAAGCAGT T C G T AAAAGAC CTGGTATGTATATTG GAT C AAC T GAT AAAC G G G GAT T AC AT CAT C TAG TAT AT GAAAT T G T C GAT AAC TCCGTCGAT GAAG T AT T GAAT G G T T AC G G T AAC GAAAT AGAT G T A AC AAT TAAT AAAGAT G G TAG T AT T T C T AT AGAAGAT AAT G GAC GTGGTATGC C AAC AG G T AT AC AT AAAT C AG G T AAAC C GAC AG T C GAAG TTATCTTTACTGTTT T AC AT G C AG GAG G TAAAT TTGGACAAGGCGGCTATAAAACTTCAGGTGGTCTTCACGGTGTTGGTGCTTCAGTTGTAA AT G C AT T GAG T GAAT G G C T T GAAG T T GAAAT C C AT C GAGAT GGTAGTATATATCAT CAAAG T T T TAAAAACGGTGGTTCGCCATCTTCTGGTTTAGTGAAAAAAGGTAAAACTAAGAAAACAGG TA C CAAAG T AAC AT T T AAAC C T GAT GAC AC AAT T T T TAAAG C AT C T AC AT CATTTAATTTTGAT G T T T T AAG C GAAC GAC T AC AAGAG T C T G C G T T C T T AT T GAAAAAT T T AAAAAT AAC G C T T AA T GAT T T AC G C AG T G G T AAAGAG C G T C AAGAG CAT T AC CAT TAT GAAGAAG GAAT C AAAGAG T TTGTTAGTTATGTCAATGAAGGAAAAGAAGTTTTGCATGACGTGGCTACATTTTCAGGTG AA G C AAAT G G T AT AGAG G TAGAC GTAGCTTTC C AAT AT AAT GAT C AAT AT T CAGAAAG T AT T T T AAG T T T T G TAAAT AAT G T AC G T AC T AAAGAT G G T G G T AC AC AT GAAG T T G G T T T T AAAAC AG C AAT GAC AC G T G T AT T TAAT GAT T AT G C AC GTCGTATTAAT GAAC T T AAAAC AAAAGAT AAA AACTTAGATGGTAATGATATTCGTGAAGGTTTAACAGCTGTTGTGTCTGTTCGTATTCCA GA AGAAT TAT TACAAT T T GAAG GAC AAAC GAAAT C TAAAT TGGGTAC T T C T GAAG C T AGAAG T G C T G T T GAT T C AG T T G T T G C AGAC AAAT T G C C AT T C T AT T T AGAAGAAAAAG GAC AAT T G T C T AAAT C AC T T G T GAAAAAAG C GAT TAAAG C AC AAC AAG C AAG G GAAG C T G C AC G TAAAG C T C G T GAAGAT G C T C G T T C AG G T AAGAAAAAC AAG C G T AAAGAC AC TTTGCTATCTGG T AAAT T AA C AC C T G C AC AAAG T AAAAAC AC T GAAAAAAAT GAAT TGTATTTAGTC GAAG GTGATTCTGCG G GAG G T T C AG C AAAAC T T G GAC GAGAC C G C AAAT T C C AAG C GAT AT T AC CAT T AC AT G G T AA G G T AAT T AAT AC AGAGAAAG C AC G T C T AGAAGAT AT T T T TAAAAAT GAAGAAAT T AAT AC AA T T AT C C AC AC AAT C G G G G C AG G C G T T G G T AC T GAC T T T AAAAT T GAAGAT AG T AAT T AT AAT CGTGTAATTATTATGACTGATGCTGATACTGATGGTGCGCATATTCAAGTGCTATTGTTA AC ATTCTTCTTCAAATATATGAAACCGCTTGTTCAAGCAGGTCGTGTATTTATTGCTTTACC TC CAC T T TAT AAAT T G GAAAAAG GT AAAG GC AAAAC AAAG C GAG T T GAAT ACGC T T GGACAGAC GAAGAG C T T AAT AAAT T G C AAAAAGAAC T T G G T AAAG G C T T C AC G T T AC AAC G T T AC AAAG G T T T G G G T GAGAT GAAC C C T GAAC AAT T AT G G GAAAC GAC GAT GAAC C C AGAAAC AC GAAC T T TAATTCGTG T AC AAG T T GAAGAT GAAG TGCGTTCATC TAAAC G T G T AAC AAC AT T AAT G G G T GAC AAAG T AC AAC C T AGAC G T GAAT G GAT T GAAAAG CAT G T T GAG TTTGGTATG C AAGAG GA C C AAAG T AT T T T AGAT AAT T C T GAAG T AC AAG T G C T T G AAAAT GAT C AAT T T GAT GAG GAG G AAAT C TAG

SEQ ID NO: 2

MNKQNNYS DDS I QVLEGLEAVRKRPGMY I GS TDKRGLHHLVYE I VDNS VDEVLNGYGNE I DV TINKDGS IS IEDNGRGMPTGIHKSGKPTVEVI FTVLHAGGKFGQGGYKTSGGLHGVGASWN ALSEWLEVEIHRDGS I YHQSFKNGGSPSSGLVKKGKTKKTGTKVTFKPDDTI FKASTSFNFD VLSERLQESAFLLKNLKITLNDLRSGKERQEHYHYEEGIKEFVSYVNEGKEVLHDVATFS GE ANGIEVDVAFQYNDQYSES ILSFVNNVRTKDGGTHEVGFKTAMTRVFNDYARRINELKTKDK NLDGNDIREGLTAWSVRIPEELLQFEGQTKSKLGTSEARSAVDSWADKLPFYLEEKGQLS KSLVKKAIKAQQAREAARKAREDARSGKKNKRKDTLLSGKLTPAQSKNTEKNELYLVEGD SA GGSAKLGRDRKFQAILPLHGKVINTEKARLEDI FKNEEINTI IHTIGAGVGTDFKIEDSNYN RVI IMTDADTDGAHIQVLLLTFFFKYMKPLVQAGRVFIALPPLYKLEKGKGKTKRVEYAWTD EELNKLQKELGKGFTLQRYKGLGEMNPEQLWETTMNPETRTLIRVQVEDEVRSSKRVTTL MG DKVQPRREWIEKHVEFGMQEDQS ILDNSEVQVLENDQFDEEEI

SEQ ID NO:3

AT GAAT AAACAAAAT AAT TAT T CAGAT GAT T C AAT AC AG G T T T TAGAGGGGT TAGAAGCAGT T C G T AAAAGAC CTGGTATGTATATTG GAT C AAC T GAT AAAC G G G GAT T AC AT CAT C TAG TAT AT GAAAT T G T C GAT AAC TCCGTCGAT GAAG T AT T GAAT G G T T AC G G T AAC GAAAT AGAT G T A AC AAT T AAT AAAGAT G G TAG T AT T T C TAT AGAAGAT AAT G GAC GTGGTATGC C AAC AG G T AT AC AT AAAT C AG G TAAAC C GAC AG T C GAAG TTATCTTTACTGTTT T AC AT G C AG GAG G T AAAT TTGGACAAGGCGGCTATAAAACTTCAGGTGGTCTTCACGGTGTTGGTGCTTCAGTTGTAA AT G C AT T GAG T GAAT G G C T T GAAG T T GAAAT C C AT C GAGAT GGTAGTATATATCAT CAAAG T T T TAAAAACGGTGGTTCGCCATCTTCTGGTTTAGTGAAAAAAGGTAAAACTAAGAAAACAGG TA C CAAAG T AAC AT T TAAAC C T GAT GAC AC AAT T T T TAAAG C AT C T AC AT CATTTAATTTTGAT G T T T T AAG C GAAC GAC T AC AAGAG T C T G C G T T C T T AT T GAAAAAT T T AAAAAT AAC G C T T AA T GAT T T AC G C AG T G G T AAAGAG C G T C AAGAG CAT T AC CAT TAT GAAGAAG GAAT C AAAGAG T TTGTTAGTTATGTCAATGAAGGAAAAGAAGTTTTGCATGACGTGGCTACATTTTCAGGTG AA G C AAAT G G T AT AGAG G TAGAC GTAGCTTTC C AAT AT AAT GAT C AAT AT T CAGAAAG T AT T T T AAG T T T T G T AAAT AAT G T AC G T AC T AAAGAT G G T G G T AC AC AT GAAG T T G G T T T T AAAAC AG C AAT GAC AC G T G T AT T T AAT GAT T AT G C AC GTCGTATTAAT GAAC T T AAAAC AAAAGAT AAA AACTTAGATGGTAATGATATTCGTGAAGGTTTAACAGCTGTTGTGTCTGTTCGTATTCCA GA AGAAT TAT TACAAT T T GAAG GAC AAAC GAAAT C TAAAT TGGGTAC T T C T GAAG C T AGAAG T G C T G T T GAT T C AG T T G T T G C AGAC AAAT T G C C AT T C T AT T T AGAAGAAAAAG GAC AAT T G T C T AAAT C AC T T G T GAAAAAAG C GAT TAAAG C AC AAC AAG C AAG G GAAG C T G C AC G TAAAG C T C G T GAAGAT G C T C G T T C AG G T AAGAAAAAC AAG C G T AAAGAC AC TTTGCTATCTGG TAAAT T AA C AC C T G C AC AAAG T AAAAAC AC T GAAAAAAAT GAAT TGTATTTAGTC GAAG GTGATTCTGCG G GAG G T T C AG C AAAAC T T G GAC GAGAC C G C AAAT T C C AAG C GAT AT T AC CAT T AC G T G G T AA G G T AAT T AAT AC AGAGAAAG C AC G T C T AGAAGAT AT T T T T AAAAAT GAAGAAAT T AAT AC AA T T AT C C AC AC AAT C G G G G C AG G C G T T G G T AC T GAC T T T AAAAT T GAAGAT AG T AAT TAT AAT CGTGTAATTATTATGACTGATGCTGATACTGATGGTGCGCATATTCAAGTGCTATTGTTA AC ATTCTTCTTCAAATATATGAAACCGCTTGTTCAAGCAGGTCGTGTATTTATTGCTTTACC TC CAC T T TAT AAAT T G GAAAAAG GT AAAG GC AAAAC AAAG C GAG T T GAAT ACGC T T GGACAGAC GAAGAG C T T AAT AAAT T G C AAAAAGAAC T T G G TAAAG G C T T C AC G T T AC AAC G T T AC AAAG G T T T G G G T GAGAT GAAC C C T GAAC AAT T AT G G GAAAC GAC GAT GAAC C C AGAAAC AC GAAC T T TAATTCGTG T AC AAG T T GAAGAT GAAG TGCGTTCATC TAAAC G T G T AAC AAC AT T AAT G G G T GAC AAAG T AC AAC C TAGAC G T GAAT G GAT T GAAAAG CAT G T T GAG TTTGGTATG C AAGAG GA C CAAAG T AT T T T AGAT AAT T C T GAAG T AC AAG T G C T T G AAAAT GAT C AAT T T GAT GAG GAG G AAAT C TAG

SEQ ID NO:4

MNKQNNYS DDS I QVLEGLEAVRKRPGMY I GS TDKRGLHHLVYE I VDNS VDEVLNGYGNE I DV TINKDGS IS IEDNGRGMPTGIHKSGKPTVEVI FTVLHAGGKFGQGGYKTSGGLHGVGASWN ALSEWLEVEIHRDGS I YHQSFKNGGSPSSGLVKKGKTKKTGTKVTFKPDDTI FKASTSFNFD VLSERLQESAFLLKNLKITLNDLRSGKERQEHYHYEEGIKEFVSYVNEGKEVLHDVATFS GE ANGIEVDVAFQYNDQYSESILSFVNNVRTKDGGTHEVGFKTAMTRVFNDYARRINELKTK DK NLDGNDIREGLTAWSVRIPEELLQFEGQTKSKLGTSEARSAVDSWADKLPFYLEEKGQLS KSLVKKAIKAQQAREAARKAREDARSGKKNKRKDTLLSGKLTPAQSKNTEKNELYLVEGD SA GGSAKLGRDRKFQAILPLRGKVINTEKARLEDIFKNEEINTI IHTIGAGVGTDFKIEDSNYN RVIIMTDADTDGAHIQVLLLTFFFKYMKPLVQAGRVFIALPPLYKLEKGKGKTKRVEYAW TD EELNKLQKELGKGFTLQRYKGLGEMNPEQLWETTMNPETRTLIRVQVEDEVRSSKRVTTL MG DKVQPRREWIEKHVEFGMQEDQSILDNSEVQVLENDQFDEEEI