FIELD OF THE INVENTION

The present invention relates to bacteria that have increased antibiotic resistance, particularly increased resistance to quinolone antibiotics. The invention also relates to a method of generating bacteria with increased antibiotic resistance, as well as methods of identifying a compound with antibiotic activity. The invention further relates to methods utilising such bacteria, including methods for the design, discovery or development of antibiotic compounds, including new antibiotic compounds.

BACKGROUND OF THE INVENTION

Antibiotics are naturally-derived or synthetic drugs that inhibit the proliferation of bacteria and so are used in medicine for the treatment of bacterial infections such as pneumonia, meningitis and tuberculosis. Multiple classes of antibiotics have been discovered over the past century. These classes are categorised based on the cellular mechanism-of-action and chemical structure. The quinolone class of antibiotics inhibits bacterial DNA synthesis by targeting the type II topoisomerase enzymes, DNA gyrase and topoisomerase IV. DNA gyrase is comprised of two protein subunits termed GyrA and GyrB. Topoisomerase IV is comprised of two subunits called ParC and ParE in the Gram-negative bacterium Escherichia coli, or GrIA and GrIB in the Gram-positive bacterium Staphylococcus aureus. Examples of quinolone antibiotics include nalidixic acid, ciprofloxacin, levofloxacin, moxifloxacin and delafloxacin.

Bacterial resistance to antibiotics is a significant and increasing public health concern. Bacteria may become resistant to antibiotics through one or more biological mechanisms. These mechanisms include: mutations in the target enzymes or proteins that alter the ability of the drug to interact with the target; the expression of enzymes or other factors that degrade the drug; or changes in the composition of the microbial cell envelope that limit permeability of the drug or enhance the rate of its efflux.

Given the increasing resistance to existing antibiotics there is a need to discover and develop new antibiotics to treat drug-resistant infections. This endeavour is aided by the availability of biological reagents that allow the researcher accurately to evaluate the potency, spectrum and mechanism-of-action of investigational and approved antibiotics. Such reagents include live whole strains of bacteria that demonstrate altered susceptibility properties to antibiotics. These strains may originate in the natural or clinical setting. Alternatively, they may be artificially prepared in the laboratory either by recombinant DNA technology or by exposing strains of bacteria to concentrations of inhibitory compounds in order to force the selection of resistant variants. One in vitro method of forcing resistant variants involves a process in which a wild-type bacterium is repeatedly cultured in the presence of a sub-inhibitory concentration of an antibiotic. This method is often referred to as serial passage.

Resistant bacterial strains may be characterised phenotypically and/or genetically. Genetic characterisation can include the nucleic acid sequencing of specific regions of the chromosome or whole-genome sequencing. Whole-genome sequencing is a powerful method for elucidating mutations within the DNA that might be associated with altered phenotypes because it allows for a precise understanding of the totality of mutations across the entire genome. Whole-genome sequencing of antibiotic-resistant variants has been described in diverse species of bacteria including S. aureus (Proc. Natl. Acad. Sci. USA 104, 9451-9456; J. Clin. Microbiol. 52, 1181-1191), Streptococcus pneumoniae (WO 2009/143614; Antimicrob. Agents Chemother. 57, 4911-4919), E. coli (J. Antimicrob. Chemother. 68, 2809-2819), Pseudomonas aeruginosa {Microb. Drug Res. 19, 428-436) and Mycobacterium tuberculosis (Science 307, 223-227).

There are reports in the prior art of the selection of resistant mutants by serial passage in the presence of quinolones. Eliopoulos et al (Antimicrob. Agents Chemother. 25, 331-335), Heiseg and Tschorny (Antimicrob. Agents Chemother. 38, 1284-1291) and Gilbert et al (Antimicrob. Agents Chemother. 45, 883-892) were able to select resistant derivatives of E. coli following repeat exposure to ciprofloxacin. Using DNA sequencing of gyrA, Heiseg and Tschorny identified one (Ser83Leu) or two (Ser83Leu and Asp87Gly) mutations within this gene in the resistant strains. Gilbert et al (Antimicrob. Agents Chemother. 45, 883-892) and Morrow et al (Antimicrob. Agents Chemother. 55, 5512-5521) selected resistant derivatives of S. aureus following repeat exposure to a variety of quinolones. Morrow et al characterised the gyrA, gyrB, parC, and parE genes of selected serial passage isolates by DNA sequence analysis. The observed mutations in these studies included GyrA Ser84Leu, GyrA Gly82Cys, ParC Ser80Phe and ParC Glu84Lys.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a bacterium that is resistant to quinolone antibiotics; that has altered susceptibility to at least one other class of antibiotic; and contains a combination of at least four mutations selected from the group consisting of: a mutation of GyrA; a mutation of OmpR; a mutation of the putative outer membrane protein YfeN; a mutation of ParC; a mutation of SoxR; a mutation of GrIA; a mutation of GrIB; an intergenic mutation upstream of NorA; a mutation of MgrA; a mutation of PBP2; a mutation of LyrA; a mutation of hypothetical probable lipoprotein (homologous to ORF SAOUHSC 01584 in S. aureus NCTC 8325); a mutation of hypothetical probable membrane protein (homologous to ORF SAOUHSC 00508 of S. aureus NCTC 8325); a mutation of MoaA; a mutation of NanM; a mutation of hypothetical glutathione ABC transporter ATP-binding protein (ORF DR76 1147 of E. coli ATCC 25922); a mutation of a hypothetical transcription regulator of the LysR-family (ORF DR76 208 of E. coli ATCC 25922); a mutation of hypothetical L-serine dehydratase (ORF DR76 3029 of E. coli ATCC 25922); a mutation of GntU; and a mutation of PstA.

In a second aspect the invention provides a bacterium comprising at least one mutation from the group consisting of:

a mutation of MoaA, such as a mutation causing a substitution at a position homologous to the valine residue at 124 of the gene product in E. coli; a mutation of NanM, such as a mutation causing a substitution at a position homologous to the serine residue at 99 of the gene product in E. coli; a mutation of hypothetical glutathione ABC transporter ATP-binding protein (ORF DR76 1147 of E. coli ATCC 25922), such as a mutation causing a substitution at a position homologous to the leucine residue at 355 of the gene product in E. coli; a mutation of hypothetical transcription regulator of the LysR-family (ORF DR76 208 of E. coli ATCC 25922), such as a mutation causing a truncation at a position homologous to the lysine residue at 230 of the gene product in E. coli; a mutation of hypothetical L-serine dehydratase (ORF DR76 3029 of E. coli ATCC 25922), such as a mutation causing a truncation at a position homologous to the leucine residue at 199 of the gene product in E. coli; and a mutation of GntU, such as a mutation causing a truncation at a position homologous to the leucine residue at 41 of the gene product in E. coli; and

at least one mutation from the list consisting of:

a mutation of GyrA, such as a mutation causing a substitution at a position homologous to the serine residue at 83 of the gene product in E. coli; a mutation of GyrA, such as a mutation causing a substitution at a position homologous to the aspartic acid residue at 87 of the gene product in E. coli; a mutation of OmpR, such as a mutation causing a substitution at a position homologous to the proline residue at 109 of the gene product in E. coli; a mutation of YfeN, such as a mutation causing a substitution at a position homologous to the methionine residue at 178 of the gene product in E. coli; a mutation of ParC, such as a mutation causing a substitution at a position homologous to the glutamic acid residue at 84 of the gene product in E. co r, and a mutation of SoxR, such as a mutation causing a deletion at a position homologous to the arginine residue at 127 of the gene product in E. coli.

Suitably a bacterium of the second aspect of the invention is an E. coli bacterium.

In a third aspect the invention provides a bacterium comprising a mutation of PstA, such as a mutation causing a frame shift at a position homologous to the glutamic acid residue at 90 of the gene product in S. aureus; and

at least one mutation from the group consisting of:

a mutation of GyrA, such as a mutation causing a substitution at a position homologous to the serine residue at 84 of the gene product in S. aureus; a mutation of GrIA, such as a mutation causing a substitution at a position homologous to the glutamic acid residue at 84 of the gene product in S. aureus; a mutation of GrIB, such as a mutation causing a substitution at a position homologous to the isoleucine residue at 473 of the gene product in S. aureus; an intergenic mutation upstream of NorA, such as a mutation at a position homologous to position 687333 of S. aureus NCTC 8325; a mutation of MgrA, such as a mutation causing a substitution at a position homologous to the proline residue at 71 of the gene product in S. aureus; a mutation of PBP2, such as a mutation causing a substitution at a position homologous to the alanine residue at 132 of the gene product in S. aureus; a mutation of LyrA, such as a mutation causing a substitution at a position homologous to the glycine residue at 8 of the gene product in S. aureus; a mutation of hypothetical probable lipoprotein (homologous to ORF SAOUHSC 01584 of S. aureus NCTC 8325), such as a mutation causing a substitution at a position homologous to the threonine residue at 38 of the gene product in S. aureus; and a mutation of hypothetical probable membrane protein (homologous to ORF SAOUHSC 00508 of S. aureus NCTC 8325), such as a mutation causing a truncation at a position homologous to the serine residue at 160 of the gene product in S. aureus.

Suitably a bacterium of the third aspect of the invention is a S. aureus bacterium.

In a fourth aspect the invention provides a method of producing a bacterium according to the first aspect of the invention, the method comprising modifying a bacterium such that at least four mutations selected from the group consisting of: a mutation of GyrA; a mutation of OmpR; a mutation of YfeN; a mutation of ParC; a mutation of SoxR; a mutation of GrIA; a mutation of GrIB; an intergenic mutation upstream of NorA; a mutation of MgrA; a mutation of PBP2; a mutation of LyrA; a mutation of hypothetical probable lipoprotein (homologous to ORF SAOUHSC 01584 of S. aureus NCTC 8325); a mutation of hypothetical probable membrane protein (homologous to ORF SAOUHSC 00508 of S. aureus NCTC 8325); a mutation of MoaA; a mutation of NanM; a mutation of hypothetical glutathione ABC transporter ATP-binding protein (ORF DR76 1147 of E. coli ATCC 25922); a mutation of hypothetical transcription regulator of the LysR-family (ORF DR76 208 of E. coli ATCC 25922); a mutation of hypothetical L-serine dehydratase (ORF DR76 3029 of E. coli ATCC 25922); a mutation of GntU; and a mutation of PstA;

are introduced into the genome of the bacterium.

In a fifth aspect the invention provides a method of producing a bacterium according to the second aspect of the invention, the method comprising modifying a bacterium such that at least one mutation selected from the group consisting of:

a mutation of MoaA, such as a mutation causing a substitution at a position homologous to the valine residue at 124 of the gene product in E. coli; a mutation of NanM, such as a mutation causing a substitution at a position homologous to the serine residue at 99 of the gene product in E. coli; a mutation of hypothetical glutathione ABC transporter ATP-binding protein (ORF DR76 1147 of E. coli ATCC 25922), such as a mutation causing a substitution at a position homologous to the leucine residue at 355 of the gene product in E. coli; a mutation of hypothetical transcription regulator of the LysR-family (ORF DR76 208 of E. coli ATCC 25922), such as a mutation causing a truncation at a position homologous to the lysine residue at 230 of the gene product in E. coli; a mutation of a hypothetical L-serine dehydratase (ORF DR76 3029 of E. coli ATCC 25922), such as a mutation causing a truncation at a position homologous to the leucine residue at 199 of the gene product in E. coli; and a mutation of GntU, such as a mutation causing a truncation at a position homologous to the leucine residue at 41 of the gene product in E. coli; and

at least one mutation from the list consisting of:

a mutation of GyrA, such as a mutation causing a substitution at a position homologous to the serine residue at 83 of the gene product in E. coli; a mutation of GyrA, such as a mutation causing a substitution at a position homologous to the aspartic acid residue at 87 of the gene product in E. coli; a mutation of OmpR, such as a mutation causing a substitution at a position homologous to the proline residue at 109 of the gene product in E. coli; a mutation of YfeN, such as a mutation causing a substitution at a position homologous to the methionine residue at 178 of the gene product in E. coli; a mutation of ParC, such as a mutation causing a substitution at a position homologous to the glutamic acid residue at 84 of the gene product in E. coli; and a mutation of SoxR, such as a mutation causing a deletion at a position homologous to the arginine residue at 127 of the gene product in E. coli; are introduced into the genome of the bacterium.

In a sixth aspect the invention provides a method of producing a bacterium according to the third aspect of the invention, the method comprising modifying a bacterium such that a mutation of PstA, such as a mutation causing a frame shift at a position homologous to the glutamic acid residue at 90 of the gene product in S. aureus; and

at least one mutation selected from the group consisting of: a mutation of GyrA, such as a mutation causing a substitution at a position homologous to the serine residue at 84 of the gene product in S. aureus; a mutation of GrIA, such as a mutation causing a substitution at a position homologous to the glutamic acid residue at 84 of the gene product in S. aureus; a mutation of GrIB, such as a mutation causing a substitution at a position homologous to the isoleucine residue at 473 of the gene product in S. aureus; an intergenic mutation upstream of NorA, such as a mutation at a position homologous to position 687333 of S. aureus NCTC 8325; a mutation of MgrA, such as a mutation causing a substitution at a position homologous to the proline residue at 71 of the gene product in S. aureus; a mutation of PBP2, such as a mutation causing a substitution at a position homologous to the alanine residue at 132 of the gene product in S. aureus; a mutation of LyrA, such as a mutation causing a substitution at a position homologous to the glycine residue at 8 of the gene product in S. aureus; a mutation of hypothetical probable lipoprotein (homologous to ORF SAOUHSC 01584 of S. aureus NCTC 8325), such as a mutation causing a substitution at a position homologous to the threonine residue at 38 of the gene product in S. aureus; and a mutation of hypothetical probable membrane protein (homologous to ORF SAOUHSC 00508 of S. aureus NCTC 8325), such as a mutation causing a truncation at a position homologous to the serine residue at 160 of the gene product in S. aureus;

are introduced into the genome of the bacterium.

Suitably the methods of the fourth, fifth, or sixth aspects of the invention may be effected by an essentially biological process, as discussed further elsewhere in the specification.

In a seventh aspect the invention provides the use of a bacterium of the invention in the design or development of antibiotic compounds. In an eighth aspect the invention provides the use of a bacterium of the invention in the design, discovery or development of novel antibiotic compounds. In a ninth aspect the invention provides a method of developing antibiotic compounds, the method comprising incubating one or more antibiotics in the presence of a bacterium of the invention. In a tenth aspect the invention provides a method of discovering and/or developing novel antibiotic compounds, the method comprising incubating one or more antibiotics in the presence of a bacterium of the invention. In an eleventh aspect the invention provides the use of a bacterium of the invention in a diagnostic and/or analytical method, assay or kit for the detection of antibiotic compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Minimum inhibitory concentration (MIC) of E. coli ATCC 25922 serially passaged in the presence of ciprofloxacin to yield exemplary bacterial strains of the invention ECCPX1-SP22 and ECCPX1-SP25 (products of the 22 ^nd and 25 ^th passages, respectively).

Figure 2. MIC of S. aureus ATCC 29213 serially passaged in the presence of ciprofloxacin to yield exemplary bacterial strains of the invention SACPX1-SP25 and SACPX1-SP28 (products of the 25 ^th and 28 ^th passages, respectively).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the identification that bacteria incorporating certain mutations exhibit an increase in antibiotic resistance. Bacteria incorporating these mutations represent reagents that are highly useful in the discovery and development of antibiotics.

Bacteria of the invention all exhibit increased resistance to quinolone antibiotics. To their surprise, the inventors have identified that a number of the mutations that characterise the bacteria of the invention fall outside of the quinolone-resistance determining region (QRDR), and so would not have been expected to arise in response to exposure to ciprofloxacin.

As described in more detail elsewhere within the present specification, many of the genes and gene products subject to mutation in the bacteria of the invention have no known link to mechanisms associated with antibiotic resistance. It is thus highly surprising that mutations such as the Val124lle mutation in the Molybdenum cofactor biosynthesis protein A are characteristic of bacteria exhibiting increased antibiotic resistance.

Some bacteria of the invention also exhibit increased resistance to antibiotics including β- lactams, aminoglycosides and polyketides.

The extent of the antibiotic resistance exhibited by the bacteria of the invention is very surprising. Indeed, the inventors have found that bacteria in accordance with the present invention are able to demonstrate resistance to quinolone antibiotics that is up to 2,048-fold increased as compared to the progenitor strains from which the bacteria of the invention are derived. This remarkable increase in resistance is highly surprising, in light of previous reports that have suggested that serial passage approaches to the generation of antibiotic- resistant bacteria have yielded approximately 32-fold increases in such resistance.

The markedly and unexpectedly improved antibiotic resistance demonstrated by the bacteria of the invention confers notable advantages in the discovery and development of new antibiotic compounds. The bacteria of the invention are of particular utility in the development of new antibiotics that are able to overcome the heightened antibiotic resistance exhibited by the bacteria disclosed herein.

A further surprising feature of certain of the bacteria of the invention is that, while they exhibit increased resistance to quinolone antibiotics, they also exhibit increased susceptibility to non-quinolone antibiotics. In particular, S. aureus bacteria of the invention may exhibit increased susceptibility to antibiotics including the β-lactams. That the same bacteria exhibit increased resistance in respect of quinolone antibiotics and increased susceptibility in respect of other antibiotics (for example the β-lactams) is unexpected, in that many mechanisms of antibiotic resistance, such as decreasing permeability to antibiotics or increasing efflux of antibiotics from bacteria, are common across multiple classes of antibiotics.

Furthermore, this unexpected profile of increased resistance to quinolone antibiotics and increased susceptibility to antibiotics such as the β-lactams confers notable practical advantages on the bacteria of the invention, and their use in the identification and development of antibiotic compounds. These advantages are described further elsewhere in the specification, and the advantages, along with the biological properties of the bacteria of the invention that give rise to them, are only made available by the present disclosure, and could not be predicted from the prior art.

A still further important property of bacteria of the invention is that they retain comparable growth characteristics to the progenitor strains from which they are derived. Thus, in the case of bacteria of the invention based upon progenitor strains that are Clinical Laboratory and Standards Institute (CLSI) reference type bacterial strains, such as the exemplary strains ECCPX1-SP22 and ECCPX1-SP25 (both derived from the CLSI reference E. coli strain ATCC 25922), or SACPX1-SP25 and SACPX1-SP28 (both derived from the CLSI S. aureus reference strain ATCC 29213), the bacteria of the invention retain similar characteristics in terms of growth rate, yield and morphology. This similarity to the parental strains makes the bacteria of the invention easy to propagate and manipulate in the laboratory, and also means that the bacteria of the invention are suitable for use in the same assays or models in which their progenitor strains may be employed. Accordingly, data generated using the bacteria of the invention can suitably be compared with data generated using the appropriate progenitor strain, which may be a CLSI reference strain.

DEFINITIONS

The following definitions may be helpful in understanding the disclosure of the present invention.

"Bacteria of the invention"

Except for where the context requires otherwise, for the purposes of the present disclosure, references to "bacteria of the invention" may be taken as encompassing any bacteria as defined herein, and particularly those bacteria in accordance with the first, second, or third aspects of the invention.

Bacteria of the invention may be characterised by whole-genome sequencing. Bacteria characterised in this manner offer benefits in contexts such as research uses in that their biological properties can often be predicted in light of the extensive literature regarding the impact of the bacterial genome, and mutations thereto, on antibiotic resistance.

As described further herein, bacteria of the invention may be produced by serial passage of non-modified bacteria in the presence of ciprofloxacin.

Bacteria of the invention may be based upon any non-modified bacteria. The bacteria of the invention are defined with respect to mutations at positions that correspond to amino acid residues in certain recited bacteria. It will be appreciated that the presence of mutations at corresponding positions in other bacterial species or strains will still provide bacteria of the invention even in strains other than those described or exemplified herein.

Bacteria of the invention may be based upon bacteria that are useful in research, and particularly in research into the identification and development of antibiotic compounds, such as those selected from the group consisting of: E. coli and S. aureus.

Bacteria of the invention may be Gram-negative bacteria, or they may be Gram-positive bacteria. Examples of Gram-negative bacteria of the invention include E. coli strains of the invention, such as the exemplary strains ECCPX1-SP22 and ECCPX1-SP25. Examples of Gram-positive bacteria of the invention include S. aureus strains of the invention, such as the exemplary strains SACPX1-SP25 and SACPX1-SP28. These exemplary strains of bacteria are discussed further below.

Gene and gene product mutations that may be present in bacteria of the invention

As set out above, the present invention is based upon the inventors' findings that bacteria incorporating certain mutations exhibit an increase in antibiotic resistance, as well as certain other beneficial phenotypes. The following provides examples of, and further information regarding, the genes, and gene products, mutations of which have been identified in embodiments of the bacteria of the invention.

"gyrA"

"gyrA" is the gene encoding the DNA gyrase A subunit. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to serine residue 83 of E. coli or serine residue 84 of S. aureus. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of serine 83, or a S. aureus bacterium of the invention may comprise a mutation within the gene product at position of serine 84. The mutation, whether replacing the specified serine residues or residues homologous thereto, may be a substitution with a leucine residue.

Additionally, or alternatively, a bacterium of the invention may comprise a genetic mutation within gyrA that causes a mutation within the gene product at a position homologous to aspartic acid residue 87 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of aspartic acid residue 87. The mutation, whether replacing the specific aspartic acid residue or a residue homologous thereto, may be a substitution with a glycine residue.

"ompR"

"ompR" is a gene encoding a transcriptional regulatory protein. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to proline residue 109 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of proline residue 109. The mutation, whether replacing the specific proline residue or a residue homologous thereto, may be a substitution with a glutamine residue. "YfeN"

Hypothetical outer membrane protein YfeN. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to methionine residue 178 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of methionine residue 178. The mutation, whether replacing the specific methionine residue or a residue homologous thereto, may be a substitution with an isoleucine residue.

Prior to the present disclosure, there has been no suggestion in the literature that this gene, or the protein that it encodes, plays any role in antibiotic resistance. Accordingly, it is highly surprising that this mutation has been found to be characteristic of bacteria of the invention, such as the exemplary E. coli strain ECCPX1-SP22.

"parC"

"parC" is the gene encoding the topoisomerase IV A subunit. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to glutamic acid residue 84 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of glutamic acid residue 84. The mutation, whether replacing the specific glutamic acid residue or a residue homologous thereto, may be a substitution with a lysine residue.

"soxR"

"soxR" is the gene encoding the redox-sensitive transcriptional regulator. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to arginine residue 127 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of arginine residue 127. The mutation, whether with respect to the specific arginine residue or a residue homologous thereto, may be a deletion of the residue in question.

"hypothetical glutathione ABC transporter ATP-binding protein"

The inventors have identified a mutation associated with a hypothetical glutathione ABC transporter ATP-binding protein (ORF DR76 1147 of E. coli AT C 25922) that is associated with the increased antibiotic resistance noted in bacteria of the invention. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to leucine residue 355 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of leucine residue 355. The mutation, whether replacing the specific leucine residue or a residue homologous thereto, may be a substitution with an isoleucine residue.

Prior to the present disclosure, there has been no suggestion in the literature that this gene, or the protein that it encodes, plays any role in antibiotic resistance. Accordingly, it is highly surprising that this mutation has been found to be characteristic of bacteria of the invention, such as the exemplary E. coli strains ECCPX1-SP22 and ECCPX1-SP25.

"Hypothetical L-serine dehydratase"

Hypothetical L-serine dehydratase. The inventors have identified a mutation associated with a hypothetical L-serine dehydratase (ORF DR76 3029 of E. coli ATCC 25922) that is associated with the increased antibiotic resistance noted in bacteria of the invention. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to leucine residue 199 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of leucine residue 199. The mutation, whether replacing the specific leucine residue or a residue homologous thereto, may be a truncation at this residue. In the case of the exemplary bacterial strains described below, the inventors believe the truncation to arise as a result of a genetic mutation causing a frame shift.

Prior to the present disclosure, there has been no suggestion in the literature that this gene, or the protein that it encodes, plays any role in antibiotic resistance. Accordingly, it is highly surprising that this mutation has been found to be characteristic of bacteria of the invention, such as the exemplary E. coli strains ECCPX1-SP22 and ECCPX1-SP25.

"Hypothetical transcriptional regulator, LysR-family HTH"

The inventors have identified a mutation associated with a hypothetical transcriptional regulator, LysR-family HTH (ORF DR76 208 of E. coli ATCC 25922) that is associated with the increased antibiotic resistance noted in bacteria of the invention. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to lysine residue 230 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of lysine residue 230. The mutation, whether replacing the specific lysine residue or a residue homologous thereto, may be a truncation at this residue. In the case of the exemplary bacterial strains described below, the inventors believe the truncation to arise as a result of a genetic mutation causing a frame shift. Prior to the present disclosure, there has been no suggestion in the literature that this gene, or the protein that it encodes, plays any role in antibiotic resistance. Accordingly, it is highly surprising that this mutation has been found to be characteristic of bacteria of the invention, such as the exemplary E. coli strains ECCPX1-SP22 and ECCPX1-SP25.

"moaA"

"moaA" is the gene encoding molybdenum cofactor biosynthesis protein A. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to valine residue 124 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of valine residue 124. The mutation, whether replacing the specific valine residue or a residue homologous thereto, may be a substitution with an isoleucine residue.

Prior to the present disclosure, there has been no suggestion in the literature that this gene, or the protein that it encodes, plays any role in antibiotic resistance. Accordingly, it is highly surprising that this mutation has been found to be characteristic of bacteria of the invention, such as the exemplary E. coli strains ECCPX1-SP22 and ECCPX1-SP25.

"nanM"

"nanM" is the gene encoding N-acetylneuraminate epimerase. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to serine residue 99 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of serine residue 99. The mutation, whether replacing the specific serine residue or a residue homologous thereto, may be a substitution with an arginine residue.

Prior to the present disclosure, there has been no suggestion in the literature that this gene, or the protein that it encodes, plays any role in antibiotic resistance. Accordingly, it is highly surprising that this mutation has been found to be characteristic of bacteria of the invention, such as the exemplary E. coli strains ECCPX1-SP22 and ECCPX1-SP25.

"gntU"

"gntU" is the gene encoding the gluconate transporter. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to leucine residue 41 of E. coli. In particular, an E. coli bacterium of the invention may comprise a mutation within the gene product at the position of leucine residue 41 may be a truncation at this residue. In the case of the exemplary bacterial strains described below, the inventors believe the truncation to arise as a result of a genetic mutation causing a frame shift.

Prior to the present disclosure, there has been no suggestion in the literature that this gene, or the protein that it encodes, plays any role in antibiotic resistance. Accordingly, it is highly surprising that this mutation has been found to be characteristic of bacteria of the invention, such as the exemplary E. coli strain ECCPX1-SP25.

"grIA"

"grIA" is the gene encoding the topoisomerase IV A subunit. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to glutamic acid residue 84 of S. aureus. In particular, a S. aureus bacterium of the invention may comprise a mutation within the gene product at the position of glutamic acid residue 84. The mutation, whether replacing the specific glutamic acid residue or a residue homologous thereto, may be a substitution with a glycine residue.

"grIB"

"grIB" is the gene encoding the topoisomerase IV B subunit. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to isoleucine residue 473 of S. aureus. In particular, a S. aureus bacterium of the invention may comprise a mutation within the gene product at the position of isoleucine residue 473. The mutation, whether replacing the specific isoleucine residue or a residue homologous thereto, may be a substitution with an asparagine residue.

"Intergenic mutation upstream of Nor A"

An "intergenic mutation upstream of NorA" for the purposes of the present invention should be taken as referring to a mutation within the promoter region of the multidrug resistance efflux pump NorA. NorA is the multidrug MFS protein. In particular, a bacterium of the invention may comprise an intergenic mutation at a position homologous to position 687333 of the S. aureus reference strain NCTC 8325.

"mgrA"

"mgrA" is the gene encoding the MarR-family transcriptional regulator. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to proline residue 71 of S. aureus. In particular, a S. aureus bacterium of the invention may comprise a mutation within the gene product at the position of proline residue 71. The mutation, whether replacing the specific proline residue or a residue homologous thereto, may be a substitution with a threonine residue.

"pbp2"

"pbp2" is the gene encoding the penicillin-binding protein 2. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to alanine residue 132 of S. aureus. In particular, a S. aureus bacterium of the invention may comprise a mutation within the gene product at the position of alanine residue 132. The mutation, whether replacing the specific alanine residue or a residue homologous thereto, may be a substitution with a threonine residue.

"lyrA"

"lyrA" is the gene encoding the lysostaphin resistance protein A. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to glycine residue 8 of S. aureus. In particular, a S. aureus bacterium of the invention may comprise a mutation within the gene product at the position of glycine residue 8. The mutation, whether replacing the specific glycine residue or a residue homologous thereto, may be a substitution with a valine residue.

"Hypothetical probable membrane protein"

The inventors have identified that mutation of a hypothetical probable membrane protein (homologous to ORF SAOUHSC 00508 of S. aureus NCTC 8325) is associated with the increased antibiotic resistance noted in bacteria of the invention. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to serine residue 160 of S. aureus. In particular, a S. aureus bacterium of the invention may comprise a mutation within the gene product at the position of serine residue 160. The mutation, whether replacing the specific serine residue or a residue homologous thereto, may be a truncation at this residue. In the case of the exemplary bacterial strains described below, the inventors believe the truncation to arise as a result of a genetic mutation causing a frame shift.

"Hypothetical probable lipoprotein"

The inventors have identified that mutation of a hypothetical probable lipoprotein (homologous to ORF SAOUHSC 01584 of S. aureus NCTC 8325) is associated with the increased antibiotic resistance noted in bacteria of the invention. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to threonine residue 38 of S. aureus. In particular, a S. aureus bacterium of the invention may comprise a mutation within the gene product at the position of threonine residue 38. The mutation, whether replacing the specific threonine residue or a residue homologous thereto, may be a substitution with an alanine residue.

"pstA"

"pstA" is the gene encoding the phosphate ABC transporter permease protein A. A bacterium of the invention may comprise a genetic mutation that causes a mutation within the gene product at a position homologous to glutamic acid residue 90 of S. aureus. In particular, a S. aureus bacterium of the invention may comprise a mutation within the gene product at the position of glutamic acid residue 90. The mutation, whether replacing the specific glutamic acid residue or a residue homologous thereto, may be a truncation at this residue. In the case of the exemplary bacterial strains described below, the inventors believe the truncation to arise as a result of a genetic mutation causing a frame shift.

Prior to the present disclosure, there has been no suggestion in the literature that this gene, or the protein that it encodes, plays any role in antibiotic resistance. Accordingly, it is highly surprising that this mutation has been found to be characteristic of bacteria of the invention, such as the exemplary S. aureus strains SACPX1-SP25 and SACPX1-SP28.

Bacteria in accordance with the first aspect of the invention comprise a combination of at least four mutations of the gene products discussed in the preceding paragraphs, and may comprise further mutations. For example, bacteria of the invention, whether in accordance with the first aspect or otherwise, may comprise at least five such mutations, at least six, such mutations, at least seven such mutations, or at least eight or more such mutations. Further guidance as to the number and nature of such mutations that may be found in bacteria of the invention is provided elsewhere in the specification.

Preferred groups of mutations:

Suitably a bacterium of the invention may comprise at least one mutation from among the group consisting of:

• A mutation of GyrA, such as a mutation causing a substitution at a position

homologous to the serine residue at 83 of the gene product in E. coli, such as a substitution with leucine; A mutation of GyrA, such as a mutation causing a substitution at a position homologous to the aspartic acid residue at 87 of the gene product in E. coli, such as a substitution with glycine;

A mutation of OmpR, such as a mutation causing a substitution at a position homologous to the proline residue at 109 of the gene product in E. coli, such as a substitution with glutamine;

A mutation of YfeN, such as a mutation causing a substitution at a position homologous to the methionine residue at 178 of the gene product in E. coli, such as a substitution with isoleucine;

A mutation of ParC, such as a mutation causing a substitution at a position homologous to the glutamic acid residue at 84 of the gene product in E. coli, such as a substitution with lysine;

A mutation of SoxR, such as a mutation causing a deletion at a position homologous to the arginine residue at 127 of the gene product in E. coli;

A mutation of GyrA, such as a mutation causing a substitution at a position homologous to the serine residue at 84 of the gene product in S. aureus, such as a substitution with leucine;

A mutation of GrIA, such as a mutation causing a substitution at a position homologous to the glutamic acid residue at 84 of the gene product in S. aureus, such as a substitution with glycine;

A mutation of GrIB, such as a mutation causing a substitution at a position homologous to the isoleucine residue at 473 of the gene product in S. aureus, such as a substitution with asparagine;

An intergenic mutation upstream of NorA, such as a mutation at a position homologous to position 687333 of S. aureus NCTC 8325;

A mutation of MgrA, such as a mutation causing a substitution at a position homologous to the proline residue at 71 of the gene product in S. aureus, such as a substitution with threonine;

A mutation of PBP2, such as a mutation causing a substitution at a position homologous to the alanine residue at 132 of the gene product in S. aureus, such as a substitution with threonine;

A mutation of LyrA, such as a mutation causing a substitution at a position homologous to the glycine residue at 8 of the gene product in S. aureus, such as a substitution with valine;

A mutation of hypothetical probable lipoprotein (homologous to ORF SAOUHSC 01584 of S. aureus NCTC 8325), such as a mutation causing a substitution at a position homologous to the threonine residue at 38 of the gene product in S. aureus such as a substitution with alanine; and

• A mutation of hypothetical probable membrane protein (homologous to ORF

SAOUHSC 00508 of S. aureus NCTC 8325), such as a mutation causing a truncation at a position homologous to the serine residue at 160 of the gene product in S.

aureus.

A bacterium in accordance with the invention may comprise a combination of two or more of the mutations referred to in the preceding paragraph, for example, a combination of three or more such mutations, four or more such mutations, five or more such mutations, six or more such mutations, seven or more such mutations, or eight or more such mutations. Such a bacterium may comprise a combination of nine or more of the mutations referred to in the preceding paragraph, for example, a combination of ten or more such mutations, eleven or more such mutations, twelve or more such mutations, thirteen or more such mutations, or fourteen or more such mutations. In a suitable embodiment a bacterium of the invention may comprise all fifteen of the mutations set out above.

Suitably a bacterium of the invention in accordance with the embodiment above may additionally comprise one or more mutations from among the group consisting of:

• A mutation of MoaA, such as a mutation causing a substitution at a position homologous to the valine residue at 124 of the gene product in E. coli, such as a substitution with isoleucine;

• A mutation of NanM, such as a mutation causing a substitution at a position homologous to the serine residue at 99 of the gene product in E. coli, such as a substitution with arginine;

• A mutation of hypothetical glutathione ABC transporter ATP-binding protein (ORF DR76 1 147 of E. coli ATCC 25922), such as a mutation causing a substitution at a position homologous to the leucine residue at 355 of the gene product in E. coli, such as a substitution with isoleucine;

• A mutation of hypothetical transcription regulator of the LysR-family (ORF DR76 208 of E. coli ATCC 25922), such as a mutation causing a truncation at a position homologous to the lysine residue at 230 of the gene product in E. coli;

• A mutation of hypothetical L-serine dehydratase (ORF DR76 3029 of E. coli ATCC 25922), such as a mutation causing a truncation at a position homologous to the leucine residue at 199 of the gene product in E. coli; • A mutation of GntU, such as a mutation causing a truncation at a position homologous to the leucine residue at 41 of the gene product in E. coli; and

• A mutation of PstA, such as a mutation causing a frame shift at a position homologous to the glutamic acid residue at 90 of the gene product in S. aureus.

None of the mutations described in the preceding paragraphs have previously been associated with an increase in antibiotic resistance. Suitably a bacterium in accordance with the invention may comprise a combination of two or more of the mutations referred to in the preceding paragraph, for example, a combination of three or more such mutations, four or more such mutations, five or more such mutations, six or more such mutations, or seven or more such mutations.

In a suitable embodiment, the bacterium of the invention is an E. coli bacterium, and comprises at least one mutation from among the group consisting of:

• A mutation of GyrA, such as a mutation causing a substitution at a position homologous to the serine residue at 83 of the gene product in E. coli, such as a substitution with leucine;

• A mutation of GyrA, such as a mutation causing a substitution at a position homologous to the aspartic acid residue at 87 of the gene product in E. coli, such as a substitution with glycine;

• A mutation of OmpR, such as a mutation causing a substitution at a position homologous to the proline residue at 109 of the gene product in E. coli, such as a substitution with glutamine;

• A mutation of YfeN, such as a mutation causing a substitution at a position homologous to the methionine residue at 178 of the gene product in E. coli, such as a substitution with isoleucine;

• A mutation of ParC, such as a mutation causing a substitution at a position homologous to the glutamic acid residue at 84 of the gene product in E. coli, such as a substitution with lysine; and

• A mutation of SoxR, such as a mutation causing a deletion at a position homologous to the arginine residue at 127 of the gene product in E. coli.

In a suitable embodiment such an E. coli bacterium of the invention may comprise at least two of the mutations set out above, optionally at least three of the recited mutations, suitably at least four of the mutations, or at least five of the mutations, or may even comprise all six of the mutations recited in the preceding paragraph.

Suitably an E. coli bacterium of the invention comprising one or more of the mutations set out above may further comprise at least one, at least two, at least three, at least four, at least five, or all six of the following mutations. These mutations have not previously been identified as having an impact upon antibiotic resistance.

• A mutation of MoaA, such as a mutation causing a substitution at a position homologous to the valine residue at 124 of the gene product in E. coli, such as a substitution with isoleucine;

• A mutation of NanM, such as a mutation causing a substitution at a position homologous to the serine residue at 99 of the gene product in E. coli, such as a substitution with arginine;

• A mutation of hypothetical glutathione ABC transporter ATP-binding protein (ORF DR76 1 147 of E. coli ATCC 25922), such as a mutation causing a substitution at a position homologous to the leucine residue at 355 of the gene product in E. coli, such as a substitution with isoleucine;

• A mutation of hypothetical transcription regulator of the LysR-family (ORF DR76 208 of E. coli ATCC 25922), such as a mutation causing a truncation at a position homologous to the lysine residue at 230 of the gene product in E. coli;

• A mutation of hypothetical L-serine dehydratase (ORF DR76 3029 of E. coli ATCC 25922), such as a mutation causing a truncation at a position homologous to the leucine residue at 199 of the gene product in E. coli; and

• A mutation of GntU, such as a mutation causing a truncation at a position homologous to the leucine residue at 41 of the gene product in E. coli.

In a suitable embodiment, the bacterium of the invention is a S. aureus bacterium, and comprises at least one mutation from among the group consisting of:

• A mutation of GyrA, such as a mutation causing a substitution at a position homologous to the serine residue at 84 of the gene product in S. aureus, such as a substitution with leucine;

• A mutation of GrIA, such as a mutation causing a substitution at a position homologous to the glutamic acid residue at 84 of the gene product in S. aureus, such as a substitution with glycine; • A mutation of GrIB, such as a mutation causing a substitution at a position homologous to the isoleucine residue at 473 of the gene product in S. aureus, such as a substitution with asparagine;

• An intergenic mutation upstream of NorA, such as a mutation at a position homologous to position 687333 of S. aureus NCTC 8325;

• A mutation of MgrA, such as a mutation causing a substitution at a position homologous to the proline residue at 71 of the gene product in S. aureus, such as a substitution with threonine;

• A mutation of PBP2, such as a mutation causing a substitution at a position homologous to the alanine residue at 132 of the gene product in S. aureus, such as a substitution with threonine;

• A mutation of LyrA, such as a mutation causing a substitution at a position homologous to the glycine residue at 8 of the gene product in S. aureus, such as a substitution with valine;

• A mutation of hypothetical probable lipoprotein (homologous to ORF SAOUHSC 01584 of S. aureus), such as a mutation causing a substitution at a position homologous to the threonine residue at 38 of the gene product in S. aureus such as a substitution with alanine; and

• A mutation of hypothetical probable membrane protein (homologous to ORF SAOUHSC 00508 of S. aureus, such as a mutation causing a truncation at a position homologous to the serine residue at 160 of the gene product in S. aureus.

In a suitable embodiment such a S. aureus bacterium of the invention may comprise at least three of the mutations set out above, at least four of the mutations, at least five of the mutations, or at least six of the recited mutations. A S. aureus bacterium of the invention may comprise at least seven of the mutations set out above, at least eight of these mutations, or even all nine of the recited mutations.

Suitably a S. aureus bacterium of the invention according to the preceding embodiment may additionally comprise a mutation of PstA, such as a mutation causing a frame shift at a position homologous to the glutamic acid residue at 90 of the gene product in S. aureus. As described elsewhere in the present specification, such mutations have not previously been associated with increased antibiotic resistance.

Exemplary strains of bacteria of the invention Particular exemplary strains of E. coli bacteria of the invention are herein referred to as ECCPX1-SP22 and ECCPX1-SP25, while exemplary strains of S. aureus bacteria of the invention are referred to as SACPX1-SP25, and SACPX1-SP28.

ECCPX1-SP22

For the purposes of the present disclosure, ECCPX1-SP22 is taken to be a strain of E. coli characterised by the following mutations as compared to the progenitor strain ATCC 25922:

GyrA Ser83Leu and Asp87Gly

MoaA Val124lle

NanM Ser99Arg

OmpR Pro109Gln

Hypothetical glutathione ABC transporter ATP-binding protein Leu355lle

Hypothetical outer membrane protein YfeN Met178lle

Hypothetical transcriptional regulator, LysR-family HTH frame-shift truncation at Lys230 Hypothetical L-serine dehydratase frame-shift truncation at Leu199

Whole genome sequencing indicates that these gene product mutations are the only differences between this bacterium of the invention and the progenitor strain.

ECCPX1-SP22 is an example of a bacterium in accordance with the first and second aspects of the invention. ECCPX1-SP22 may be produced by methods in accordance with the fourth and fifth aspects of the invention. ECCPX1-SP22 is suitable for use in the uses and methods of the seventh, eighth, ninth, tenth, or eleventh aspects of the invention.

ECCPX1-SP25

For the purposes of the present disclosure, ECCPX1-SP25 is taken to be a strain of E. coli characterised by the following mutations as compared to the progenitor strain ATCC 25922:

GyrA Ser83Leu and Asp87Gly

ParC Glu84Lys

MoaA Val124lle

NanM Ser99Arg

OmpR Pro109Gln

SoxR deletion of Arg127 GntU frame-shift truncation Leu41

Hypothetical glutathione ABC transporter ATP-binding protein Leu355lle

Hypothetical transcriptional regulator, LysR-family HTH frame-shift truncation at Lys230

Hypothetical L-serine dehydratase frame-shift truncation at Leu199

Whole genome sequencing indicates that these gene product mutations are the only differences between this bacterium of the invention and the progenitor strain.

ECCPX1-SP25 is an example of a bacterium in accordance with the first and second aspects of the invention. ECCPX1-SP25 may be produced by methods in accordance with the fourth and fifth aspects of the invention. ECCPX1-SP25 is suitable for use in the uses and methods of the seventh, eighth, ninth, tenth, or eleventh aspects of the invention.

SACPX1-SP25

For the purposes of the present disclosure, SACPX1 -SP25 is taken to be a strain of S. aureus characterised by the following mutations as compared to the progenitor strain ATCC 29213:

GyrA Ser84Leu

GrIA Glu84Gly

Intergenic (upstream of NorA, equivalent to position 687333 in S. aureus NCTC 8325)

MgrA Pro71Thr

PBP2 Ala132Thr

LyrA Gly8Val

PstA frame-shift at Glu90

Hypothetical probable membrane protein frame-shift truncation at Ser160

Whole genome sequencing indicates that these gene product mutations are the only differences between this bacterium of the invention and the progenitor strain.

SACPX1-SP25 is an example of a bacterium in accordance with the first and third aspects of the invention. SACPX1-SP25 may be produced by methods in accordance with the fourth and sixth aspects of the invention. SACPX1-SP25 is suitable for use in the uses and methods of the seventh, eighth, ninth, tenth, or eleventh aspects of the invention.

SACPX1-SP28 For the purposes of the present disclosure, SACPX1-SP28 is taken to be a strain of S. aureus characterised by the following mutations as compared to the progenitor strain ATCC 29213:

GyrA Ser84Leu

GrIA Glu84Gly

GrIB Me473Asn

Intergenic (upstream of NorA, equivalent to position 687333 in S. aureus NCTC 8325)

MgrA Pro71Thr

PBP2 Ala132Thr

PstA frame-shift at Glu90

Hypothetical probable lipoprotein Thr38Ala

Whole genome sequencing indicates that these gene product mutations are the only differences between this bacterium of the invention and the progenitor strain.

SACPX1-SP28 is an example of a bacterium in accordance with the first and third aspects of the invention. SACPX1-SP28 may be produced by methods in accordance with the fourth and sixth aspect of the invention. SACPX1-SP28 is suitable for use in the uses and methods of the seventh, eighth, ninth, tenth, or eleventh aspects of the invention.

Homologous genes and gene products

There may be natural variations in the primary nucleotide and amino acid sequences of the genes and gene products described in this invention in strains or species of bacteria related to those of the invention. As a consequence there may be differences in the numbering of specific bases or residues. Identical types of nucleotide or amino acid to those of the bacteria of the invention that align with them in related strains and species but are not necessarily located at the exact same position are considered to be homologous. References in this disclosure to homology, and to amino acid residues that are homologous to specific residues found in proteins of a bacterial species or strain, should be interpreted accordingly.

"Antibiotic resistance" Assessment of antibiotic resistance is important in confirming resistance of bacteria to an antibiotic of interest, and also in monitoring the activity and effectiveness of newly identified or developed antibiotics.

There are many measures by which antibiotic resistance of bacteria (such as bacteria of the invention, or comparator bacteria such as CLSI reference strains) may be assessed. These include measurement of the minimum inhibitory concentration (MIC) of a given antibiotic (or putative antibiotic) in respect of bacteria such as those of the invention. Antibiotic resistance, and particularly MIC, may be assessed by any suitable means known to those skilled in the art, including (but not limited to) those selected from the group consisting of: the broth dilution method; the agar dilution method; the disc diffusion method; the Etest ^® (J. Clin. Microbiol. 30, 2709-2713); and the M.I.C. Evaluator™ (J. Clin. Microbiol. 50, 1147-1 152).

The MIC is defined as the lowest concentration of an antibiotic that is able to inhibit visible growth of bacteria after incubation with the compound for an appropriate period of time. Such a period of time may, for example, be an overnight incubation, or equivalent.

Suitable methods by which the MIC may be determined in respect of bacteria of interest, such as bacteria of the invention or suitable comparator bacteria, are described in the Examples that follow later in the specification.

Except for where the context requires otherwise, antibiotic resistance may be demonstrated by an increase in any one of these assays as compared to relevant comparator bacteria. Suitably bacteria of the invention may demonstrate antibiotic resistance by virtue of increased values in at least one of the above assays.

Bacteria of the invention may exhibit at least a 4-fold, at least an 8-fold, or at least a 16-fold increase in antibiotic resistance, assessed by MIC, as compared to suitable comparator bacteria. Indeed, bacteria of the invention may exhibit at least a 32-fold or at least a 64-fold increase in antibiotic resistance, assessed by MIC, as compared to suitable comparator bacteria.

In a suitable embodiment, bacteria of the invention may exhibit at least a 128-fold or at least a 256-fold increase in antibiotic resistance, assessed by MIC, as compared to suitable comparator bacteria. Suitably, bacteria of the invention may exhibit at least a 512-fold increase in antibiotic resistance, assessed by MIC, as compared to suitable comparator bacteria. Bacteria of the invention may exhibit at least a 1 ,024-fold increase in antibiotic resistance, assessed by MIC, as compared to suitable comparator bacteria, or even an increase of 2,048-fold, or more.

Suitably comparator bacteria may include the progenitor bacteria from which bacteria of the invention are derived, including progenitor bacteria that are of CLSI reference strains.

As noted elsewhere in the specification, bacteria of the invention are resistant to quinolone antibiotics. Accordingly, suitable bacteria of the invention may demonstrate at least a 16-fold increase in MIC with respect to a quinolone antibiotic as compared to suitable comparator bacteria. In a suitable embodiment bacteria of the invention may demonstrate at least a 32- fold increase in MIC with respect to a quinolone antibiotic as compared to suitable comparator bacteria, or may demonstrate at least a 64-fold or at least a128-fold increase in MIC as compared to a comparator. Bacteria of the invention may exhibit at least a 256-fold or at least a 512-fold increase in resistance to a quinolone antibiotic, assessed by MIC, as compared to suitable comparator bacteria. Indeed, bacteria of the invention may exhibit at least a 1 ,024-fold or at least a 2,048-fold increase in resistance to a quinolone antibiotic, as assessed by MIC, when compared to suitable comparator bacteria.

Compared with some other reported quinolone resistant strains of E. coli and S. aureus, the bacteria of the invention demonstrate relatively higher-level resistance to quinolone antibiotics (see results set out in Tables 1 and 2 of the Examples). Merely by way of example, the MIC of ciprofloxacin against S. aureus SACPX1-SP28 is 64 μg/mL compared with 16 μg/mL versus S. aureus strain NRS1 (GyrA S84L and E409L, GrIA S80F). The MIC of delafloxacin against S. aureus SACPX1-SP28 is increased by a factor of 2,048-fold relative to the parental wild-type strain ATCC 29213, whereas S. aureus mutants generated by Remy et al (J. Antimicrob. Chemother. 67, 2814-2820) demonstrated a 32-fold increase in the MIC with delafloxacin relative to the parental wild-type strain. Also, Eliopoulos et al (Antimicrob. Agents Chemother. 25, 331-335) were only able to achieve resistance up to 8 μg/mL with ciprofloxacin in E. coli ATCC 25922 following serial passage. By comparison, E. coli ECCPX1-SP25 shows resistance up to 32 μg/mL with ciprofloxacin.

The bacteria of the invention demonstrate a number of surprising phenotypes that could not have been predicted a priori. There are interesting and unexpected differences in antibiotic susceptibilities between for example S. aureus SACPX1-SP25 and S. aureus SACPX1- SP28 with respect to ampicillin and penicillin. Accordingly, such strains may be useful in the discovery of new drugs of the penicillin class by providing a biological indicator of the potency of new penicillin analogues.

Compared with single type II topoisomerase mutants, the strains demonstrate cross- resistance with other antibiotic classes.

As will be expected, the antibiotic resistance of bacteria of the invention varies with reference to particular strains of the bacteria, and particular antibiotics of interest. For example, bacteria of the invention (such as the E. coli strain ECCPX1-SP22) may demonstrate at least a 32-fold increase in MIC to ciprofloxacin as compared to suitable comparators, while other bacteria of the invention (such as the S. aureus strain SACPX1- SP25) may demonstrate at least a 64-fold increase in MIC to ciprofloxacin. Still other bacteria of the invention (such as S. aureus strain SACPX1-SP28) may demonstrate at least a 128-fold increase in MIC to ciprofloxacin as compared to suitable comparators, while other bacteria of the invention (such as the E. coli strain ECCPX1-SP25) may demonstrate at least a 1 ,024-fold increase in MIC to ciprofloxacin.

Bacteria of the invention (such as the S. aureus strain SACPX1-SP25) may demonstrate at least a 16-fold increase in MIC to delafloxacin as compared to suitable comparators, while other bacteria of the invention (such as the E. coli strain ECCPX1-SP22) may demonstrate at least a 256-fold increase in MIC to delafloxacin. Still other bacteria of the invention (such as E. coli strain ECCPX1-SP25) may demonstrate at least a 512-fold increase in MIC to delafloxacin as compared to suitable comparators, while other bacteria of the invention (such as the S. aureus strain SACPX1-SP28) may demonstrate at least a 2,048-fold increase in MIC to delafloxacin.

Suitably bacteria of the invention (such as the S. aureus strain SACPX1-SP25) may demonstrate at least a 16-fold increase in MIC to levofloxacin as compared to suitable comparators, while other bacteria of the invention (such as the S. aureus strain SACPX1- SP28, or the E. coli strain ECCPX1-SP22) may demonstrate at least a 32-fold increase in MIC to levofloxacin. Still other bacteria of the invention (such as the E. coli strain ECCPX1- SP25) may demonstrate at least a 512-fold increase in MIC to levofloxacin as compared to suitable comparators.

Bacteria of the invention (such as the S. aureus strain SACPX1-SP28) may demonstrate at least a 16-fold increase in MIC to moxifloxacin as compared to suitable comparators, while other bacteria of the invention (such as the S. aureus strain SACPX1-SP25) may demonstrate at least a 32-fold increase in MIC to moxifloxacin. Still other bacteria of the invention (such as the E. coli strain ECCPX1-SP22) may demonstrate at least a 256-fold increase in MIC to moxifloxacin as compared to suitable comparators, while other bacteria of the invention (such as the E. coli strain ECCPX1-SP25) may demonstrate at least a 2,048- fold increase in MIC to moxifloxacin.

As described elsewhere in the present disclosure, certain bacteria of the invention have proven to have reduced resistance to β-lactam antibiotics, such as penicillin or ampicillin, as compared to parental bacterial strains from which they are derived. In a suitable embodiment, a bacterium of the invention may exhibit at least a 4-fold, at least an 8-fold, or at least a 16-fold decrease in resistance to β-lactam antibiotics, assessed by MIC, as compared to suitable comparator bacteria. Indeed, bacteria of the invention may exhibit at least a 32-fold decrease in antibiotic resistance, assessed by MIC, as compared to suitable comparator bacteria.

Bacteria of the invention (such as the exemplary S. aureus strain SACPX1-SP28) may demonstrate at least a 16-fold decrease in MIC to ampicillin as compared to suitable comparators.

Bacteria of the invention (such as the exemplary S. aureus strain SACPX1-SP28) may demonstrate at least a 32-fold decrease in MIC to penicillin as compared to suitable comparators.

Methods of producing bacteria of the invention

The fourth, fifth, and sixth aspects of the invention provide methods of producing a bacterium of the invention. In their broadest forms these aspects of the invention encompass any method by which the requisite mutations are introduced into a bacterium, thereby producing a bacterium of the invention (such as the exemplary E. coli strains ECCPX1-SP22 and ECCPX1-SP25, or the exemplary S. aureus strains SACPX1-SP25 and SACPX1-SP28).

In a suitable embodiment of the methods of the fourth, fifth, or sixth aspects of the invention the requisite mutations are introduced by means of targeted mutation of the bacterial genome. Suitably such a process may involve site-directed mutagenesis of the relevant DNA sequence followed by introduction of the mutated DNA directly or indirectly in to the host bacterium. Such mutated DNA may be introduced via methods including, but not limited to, transformation, transduction or conjugation. In an alternative embodiment of the methods of the fourth, fifth, or sixth aspects of the invention the requisite mutations are introduced by an essentially biological process. Suitably such an essentially biological process may involve culturing bacteria in the presence of a sub-inhibitory concentration of an antibiotic. Suitably bacteria of the invention may be generated by exposure of progenitor bacterial strains to quinolone antibiotics. In a suitable embodiment the quinolone antibiotic is ciprofloxacin.

Details of a biological process that may be used to produce bacteria of the invention are described in the Examples that follow later in this specification.

Uses of the bacteria of the invention, and methods using such bacteria

The bacteria of the invention, whether in accordance with the first, second, or third aspects 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.

As referred to above, the ninth aspect of the invention provides a method of developing antibiotic compounds, and the tenth aspect of the invention provides a method of discovering and/or developing novel antibiotic compounds. Both methods comprise incubating at least one antibiotic, or putative antibiotic, in the presence of a bacterium of the invention.

Suitably such a method further comprises 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, such methods may be useful in the discovery of novel antibiotic compounds. In the case of compounds that are based upon known antibiotics, the methods 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 a method of the ninth or tenth 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.

A method of the ninth or tenth aspect 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.

The methods of the invention may be used in 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.

In addition to the methods described above, the invention also provides the use of a bacterium of the invention in the design, discovery or development of antibiotic compounds such as novel antibiotic compounds.

In particular, since the bacteria of the invention exhibit increased resistance to quinolone antibiotics, they may be used in the discovery and development of new antibiotic compounds (such as topoisomerase inhibitors) that are effective against high-level resistance to quinolones. In such an embodiment, bacteria of the invention may be incubated with quinolone-based test compounds, and thus used for the discovery and development of new quinolone-based antibiotics (such as topoisomerase inhibitors). The ability of a test compound to inhibit growth of bacteria of the invention, and particularly to kill such bacteria, will indicate that it has antibiotic properties in respect of bacteria that possess high levels of resistance to quinolones.

Bacteria of the invention may also be used for the discovery and development of new non- quinolone-based topoisomerase inhibitors. For example, the bacteria of the invention may be used to demonstrate that novel antibiotic compounds do not suffer the same cross- resistance liabilities as do some bacterial type II topoisomerase inhibitors such as levofloxacin.

Bacteria of the invention may also be generally useful in the discovery and development of new antibiotics that are effective against β-lactam-, aminoglycoside- and polyketide-resistant phenotypes in addition to, or independent of, quinolone resistance phenotypes. Bacteria of the invention will be particularly useful in the discovery and development of new penicillin antibiotics. For example, strain SACPX1-SP28, because of its increased susceptibility to penicillin and ampicillin, may be used in the discovery of new penicillin and ampicillin analogues. In particular, strain SACPX1-SP28 could be useful in in vitro antibiotic susceptibility tests using any of the methods described above for the identification of analogues with modest antibiotic potency that may not be detected using wild-type reference strains with standard penicillin-susceptibility phenotypes.

Bacteria of the invention may, for example, be used in in vitro susceptibility testing of approved and/or investigational antibiotics. Suitably such a method may comprise the use of bacteria of the invention in the determination of the MIC with respect to such an approved and/or investigational 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 approved and/or investigational 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 will be particularly useful in such embodiments in antibiotic susceptibility testing with approved and/or investigational antibiotics selected from, or based upon, those of the group consisting of: the quinolones, β-lactams, aminoglycosides and polyketides. Bacteria of the invention are particularly useful in this method because they may be derived from the well-characterised CLSI reference strains E. coli ATCC 25922 and S. 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 approved and/or investigational 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 demonstrate particular advantages in identifying combinations of antibiotics that are independently or simultaneously effective against quinolone-, β-lactam-, aminoglycoside- and polyketide-resistance phenotypes. Bacteria of the invention may be used in methods to determine the bactericidal activity of approved and/or investigational 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 log ₁₀ decrease in CFU/mL following incubation of the agar plates for an appropriate duration in the relevant atmospheric conditions and temperature.

An alternative method in which bacteria of the invention may be used to determine bactericidal activity of approved and/or investigational antibiotics is 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 log ₁₀ decrease in the total number of CFU/mL indicates a bactericidal effect. Bacteria of the invention will be particularly useful in determining bactericidal activity antibiotic susceptibility testing with the quinolones, β- lactams, aminoglycosides and polyketides.

Bacteria of the invention may be used in the in vivo biological evaluation of approved and/or investigational 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 will be particularly useful in models where quinolone-, β-lactam-, aminoglycoside- or polyketide-resistance is relevant, for example in determining in vivo proof-of-concept of investigational antibiotics that have demonstrated in vitro potency against high-level quinolone resistance. Bacteria of the invention are particularly useful in this method because they may be derived from the well-characterised CLSI reference strains E. coli ATCC 25922 and S. 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 investigational 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 should be particularly advantageous in elucidating the mechanism-of-action of investigational antibiotics that target any of the gene products found to be mutated in strains ECCPX1-SP22, ECCPX1-SP25, SACPX1-SP25 and SACPX1-SP28.

Bacteria of the invention may be used for the in silico design of new antibiotics. The mutations identified in bacteria of the invention could be useful 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 are particularly useful in this instance because they simultaneously provide information on multiple resistance mechanisms that could be incorporated in to a multi-factorial model in order to facilitate the discovery of antibiotics effective against multi-drug resistance.

Bacteria of the invention may be used in the production of recombinant bacterial proteins. 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 vector for expression and purification of the corresponding gene products for use, for example in in vitro biochemical assays, crystallography studies, etc. Bacteria of the invention are expected to be particularly useful in providing proteins associated with multi-drug resistance and in particular proteins associated with high-level quinolone resistance.

Bacteria of the invention may be used in a diagnostic method. For example, bacteria of the invention could be used as a biosensor of antibiotic in environmental, clinical or other samples. Bacteria of the invention could be particularly useful in the detection of quinolone, β-lactam, aminoglycoside or polyketide antibiotics in samples. Bacteria of the invention could be particularly useful in the detection of high concentrations of quinolone antibiotics in samples. For example, a suitable diagnostic test may make use of a bacterial strain of the invention (such as E. coli strain ECCPX1-SP25) and a reference strain (such as the progenitor strain E. coli ATCC 25922), which are each independently cultured in samples. Growth of E. coli ECCPX1-SP25 but not E. coli ATCC 25922 in this case may indicate the presence of quinolone antibiotics in samples. Bacteria of the invention could be particularly useful in the detection of low concentrations of penicillin antibiotics in samples. For example, a suitable diagnostic test may comprise strain S. aureus SACPX1-SP28 and its reference progenitor strain, S. aureus ATCC 29213, which are each independently cultured in samples. Growth of S. aureus ATCC 29213 but not S. aureus SACPX1-SP28 in this case may indicate the presence of penicillin antibiotics in samples.

The invention also provides the use of a bacterium of the invention in a diagnostic and/or analytical method, assay or kit for the detection of antibiotic compounds. Such uses may be of particular benefit in the detection of quinolone, β-lactam, aminoglycoside or polyketide antibiotic compounds.

EXAMPLES

Example 1

Strain Isolation and Characterisation

Mutants were raised from Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 in the presence of a sub-inhibitory concentration of the quinolone antibiotic ciprofloxacin over multiple passages. 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 ciprofloxacin 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 ciprofloxacin. 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 E. coli strains ECCPX1-SP22 and ECCPX1-SP25 were isolated after 22 and 25 passages, respectively. The S. aureus strains SACPX1-SP25 and SACPX1-SP28 were isolated after 25 and 28 passages, respectively.

Genomic DNA (gDNA) was extracted from the resistant strains using the EdgeBio PurElute Bacterial Genomic Kit. The gDNA from the E. coli strains (ATCC 25922, ECCPX1-SP22 and ECCPX1-SP25) was purified exactly according to the manufacturer's instructions. S. aureus gDNA (ATCC 29213, SACPX1-SP25 and SACPX1-SP28) was purified using some modification of the manufacturer's methodology as follows: lysostaphin was incorporated into the spheroblast buffer (100 μg/mL) to ensure complete cell lysis; proteinase K was also included in the reaction when adding the extraction buffer (100 μg/mL), which was then incubated at 37°C for 15 minutes.

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.

Example 2

Antibiotic Susceptibility Testing

Minimum Inhibitory Concentrations (MICs) versus strains of Escherichia coli or 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 final concentration range of 0.001-64 μg/mL and a maximum final concentration of 1 % DMSO. Bacterial strains tested included E. coli ATCC 25922, E. coli ECCPX1-SP22, E. coli ECCPX1-SP25, S. aureus ATCC 29213, S. aureus SACPX1-SP25 and S. aureus SACPX1-SP28. Strains were grown in cation-adjusted Muller-Hinton broth or agar 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.

Results

Table 1. MICs of ciprofloxacin against strains of Escherichia coli and Staphylococcus aureus.

Table 2. MICs of selected quinolone antibiotics against strains of Escherichia coli and Staphylococcus aureus.

Table 3. MICs of selected non-quinolone antibiotics against strains of Escherichia coli and Staphylococcus aureus. Example 3

Further Antibiotic Susceptibility Testing

Use of exemplary strains of the bacteria of the invention in the discovery, design or development of new antibiotic compounds is described in the following example. Here, MICs versus planktonic bacteria are 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 dilution method involves a two-fold serial dilution of compounds in 96-well microtitre plates, giving a final concentration range of 0.25-128 μg/mL and a maximum final concentration of 1 % DMSO. The bacterial strains tested include the Gram-positive strains Staphylococcus aureus ATCC 29213, Staphylococcus aureus NRS1 , Staphylococcus aureus NRS74, Staphylococcus aureus NRS482, Staphylococcus epidermidis ATCC 12228, Streptococcus pneumoniae ATCC 49619 and the Gram-negative strains Escherichia coli ATCC 25922, E. coli MG1655 and the Gyrase A mutants E. coli MG1655 S83L and E. coli MG1655 D87G derived from the isogenic parent strain E. coli MG1655. Antibiotic resistance of these well-known and characterised bacteria is also compared to the exemplary bacterial strains of the invention SACPX1-SP25, SACPX1-SP28, ECCPX1-SP22, and ECCPX1- SP25.

Strains are grown in cation-adjusted Muller-Hinton broth (supplemented with 2% w/v NaCI in the case of methicillin-resistant S. aureus strains) or on Muller-Hinton agar at 37°C in an ambient atmosphere. The MIC is determined as the lowest concentration of compound that inhibits growth following a 16-20 h incubation period. The data reported correspond to the modes of three independent experiments.

In Tables 4 to 7 a MIC (in μg/mL) of less or equal to 1 is assigned the letter A; a MIC of from 1 to 10 is assigned the letter B; a MIC of from 10 to 100 is assigned the letter C; and a MIC of over 100 is assigned the letter D. Table 4. MIC values against wild-type strains.

n.d. = not determined

Thus, an experimental antibiotic compound A showed activity against both Gram-positive (S. aureus, S. epidermidis and S. pneumoniae) and Gram-negative (E. coli) bacteria of the prior art (including the progenitor strains of the exemplary bacteria of the invention). Two further experimental antibiotic compounds (B and C) also showed activity against both Gram- positive (S. aureus and S. pneumoniae) and Gram-negative (E. coli) bacteria.

Table 5. Potency of reference compounds and test compounds A, B and C against fluoroquinolone (FQ) susceptible and resistant Staphylococcus aureus strains including bacteria of the invention.

Vancomycin A B B A A A

Oxacillin A A D n.d. n.d. n.d.

A A A A A B A

B C B A A D C

C A A A A A A

MSSA = methicillin-susceptible S. aureus; MRSA = methicillin-resistant S. aureus; FQ = fluoroquinolone; AMG = aminoglycoside; TET = tetracycline ; n.d. = not determined

Thus, compounds A and C exhibit excellent activity against all strains of S. aureus tested, including those which are resistant to fluoroquinolone antibiotics and other antibiotics, such as the exemplary bacterial strains of the invention SACPX1-SP25 and SACPX1-SP28. Compound B also showed significant activity against all strains tested.

Table 6. Fold increase in MIC of compounds against fluoroquinolone-resistant single point mutation Escherichia coli strains (MG1655 S83L and MG1655 D87G) compared to the isogenic parent strain E. coli MG1655.

M 1 1

N 0.5 1

0 0.5 2

¹ Ser83Leu mutation on DNA gyrase subunit GyrA

2Asp87Gly mutation on DNA gyrase subunit GyrA

Compounds A-0 showed no significant loss of activity against the E. coli MG1655 mutant strains.

Table 7. Fold increase in MIC of compounds against fluoroquinolone-resistant multiple point mutation Escherichia coli strains of the invention (ECCPX1-SP22 and ECCPX1-SP25) compared to the isogenic parent strain E. coli ATCC 25922.

includes mutations as defined elsewhere in the specification, including DNA gyrase subunit GyrA: Ser83Leu and Asp87Gly

includes mutations as defined elsewhere in the specification, including DNA gyrase subunit GyrA: Ser83Leu and Asp87Gly, on DNA topoisomerase IV subunit ParC: E84K Thus, compounds B, C, D, E, F, J, and O showed an eight-fold increase in MIC against both mutant strains compared to the wild-type parent strain E. coli ATCC 25922 and were less susceptible to the gyrase Ser83Leu and Asp87Gly mutations than the fluoroquinolone antibiotics. Compound N showed a four-fold increase in MIC against both mutant strains compared to the wild-type parent strain E. coli ATCC 25922 and was less susceptible to the gyrase Ser83Leu and Asp87Gly mutations than the fluoroquinolone antibiotics and the other compounds tested.

Details of compounds A-0 described in these examples are found in the Applicant's copending International Patent Application PCT/GB2015/052303. The disclosure of this application, as it relates to the names, structures, manufacture and functions of compounds A-0 is incorporated by reference herein.