The present invention relates to a method of determining an infection of a patient with Proteus species potentially re ^¬ sistant to antimicrobial drug treatment, a method of select ^¬ ing a treatment of a patient suffering from an infection with a potentially resistant Proteus strain, and a method of de ^¬ termining an antimicrobial drug, e.g. antibiotic, resistance profile for bacterial microorganisms of Proteus species, as well as computer program products used in these methods.

Antibiotic resistance is a form of drug resistance whereby a sub-population of a microorganism, e.g. a strain of a bacterial species, can survive and multiply despite exposure to an antibiotic drug. It is a serious and health concern for the individual patient as well as a major public health issue. Timely treatment of a bacterial infection requires the analy- sis of clinical isolates obtained from patients with regard to antibiotic resistance, in order to select an efficacious therapy. Generally, for this purpose an association of the identified resistance with a certain microorganism (i.e. ID) is necessary.

Antibacterial drug resistance (ADR) represents a major health burden. According to the World Health Organization's antimicrobial resistance global report on surveillance, ADR leads to 25,000 deaths per year in Europe and 23,000 deaths per year in the US. In Europe, 2.5 million extra hospital days lead to societal cost of 1.5 billion euro. In the US, the di ^¬ rect cost of 2 million illnesses leads to 20 billion dollar direct cost. The overall cost is estimated to be substantial- ly higher, reducing the gross domestic product (GDP) by up to 1.6% .

Proteus is a genus of Gram-negative Proteobacteria . Proteus bacilli are widely distributed in nature as saprophytes, be ^¬ ing found in decomposing animal matter, in sewage, in manure soil, and in human and animal feces. They are opportunistic pathogens, commonly responsible for urinary and septic infec ^¬ tions .

In general the mechanisms for resistance of bacteria against antimicrobial treatments rely to a very substantial part on the organism's genetics. The respective genes or molecular mechanisms are either encoded in the genome of the bacteria or on plasmids that can be interchanged between different bacteria. The most common resistance mechanisms include:

1) Efflux pumps are high-affinity reverse transport systems located in the membrane that transports the antibiotic out of the cell, e.g. resistance to tetracycline.

2) Specific enzymes modify the antibiotic in a way that it loses its activity. In the case of streptomycin, the an ^¬ tibiotic is chemically modified so that it will no long ^¬ er bind to the ribosome to block protein synthesis.

3) An enzyme is produced that degrades the antibiotic,

thereby inactivating it. For example, the penicillinases are a group of beta-lactamase enzymes that cleave the beta lactam ring of the penicillin molecule.

In addition, some pathogens show natural resistance against drugs. For example, an organism can lack a transport system for an antibiotic or the target of the antibiotic molecule is not present in the organism. In the case of Gram-negative bacteria, the cell wall is covered with an outer membrane that may establish a permeability barrier against the antibi ^¬ otic.

Pathogens that are in principle susceptible to drugs can be ^¬ come resistant by modification of existing genetic material (e.g. spontaneous mutations for antibiotic resistance, hap ^¬ pening in a frequency of one in about 100 mio bacteria in an infection) or the acquisition of new genetic material from another source. One example is horizontal gene transfer, a process where genetic material contained in small packets of DNA can be transferred between individual bacteria of the same species or even between different species. Horizontal gene transfer may happen by transduction, transformation or conj ugation .

Generally, testing for susceptibility/resistance to antimi ^¬ crobial agents is performed by culturing organisms in differ ^¬ ent concentration of these agents. In brief, agar plates are inoculated with patient sample

(e.g. urine, sputum, blood, stool) overnight. On the next day individual colonies are used for identification of organisms, either by culturing or using mass spectroscopy. Based on the identity of organisms new plates containing increasing con- centration of drugs used for the treatment of these organisms are inoculated and grown for additional 12 - 24 hours. The lowest drug concentration which inhibits growth (minimal inhibitory concentration - MIC) is used to determine suscepti ^¬ bility/resistance for tested drugs. The process takes at least 2 to 3 working days during which the patient is treated empirically. A significant reduction of time-to-result is needed especially in patients with life-threatening disease and to overcome the widespread misuse of antibiotics. Recent developments include PCR based test kits for fast bac ^¬ terial identification (e.g. Biomerieux Biofire Tests, Curetis Unyvero Tests) . With these test the detection of selected re ^¬ sistance loci is possible for a very limited number of drugs, but no correlation to culture based AST is given. Mass spec ^¬ troscopy is increasingly used for identification of pathogens in clinical samples (e.g. Bruker Biotyper) , and research is ongoing to establish methods for the detection of susceptibility/resistance against antibiotics.

For some drugs such it is known that at least two targets are addressed, e.g. in case of Ciprofloxacin (drug bank ID 00537; http://www.drugbank.ca/drugs/DB00537) targets include DNA Topoisomerase IV, DNA Topoisomerase II and DNA Gyrase. It can be expected that this is also the case for other drugs alt ^¬ hough the respective secondary targets have not been identi ^¬ fied yet. In case of a common regulation, both relevant ge ^¬ netic sites would naturally show a co-correlation or redundancy .

It is known that drug resistance can be associated with ge ^¬ netic polymorphisms. This holds for viruses, where resistance testing is established clinical practice (e.g. HIV genotyp- ing) . More recently, it has been shown that resistance has also genetic causes in bacteria and even higher organisms, such as humans where tumors resistance against certain cyto ^¬ static agents can be linked to genomic mutations.

Wozniak et al . (BMC Genomics 2012, 13 (Suppl 7):S23) disclose genetic determinants of drug resistance in Staphylococcus aureus based on genotype and phenotype data. Stoesser et al . disclose prediction of antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data (J Antimicrob Chemother 2013; 68: 2234-2244) .

Chewapreecha et al (Chewapreecha et al (2014) Comprehensive Identification of single nucleotid polymorphisms associated with beta-lactam resistance within pneumococcal mosaic genes. PLoS Genet 10(8) : el004547) used a comparable approach to identify mutations in gram-positive Streptococcus Pneumonia. The fast and accurate detection of infections with Proteus species and the prediction of response to anti-microbial therapy represent a high unmet clinical need.

This need is addressed by the present invention.

Summary of the Invention

The present inventors addressed this need by carrying out whole genome sequencing of a large cohort of Proteus clinical isolates and comparing the genetic mutation profile to clas ^¬ sical culture based antimicrobial susceptibility testing with the goal to develop a test which can be used to detect bacte ^¬ rial susceptibility/resistance against antimicrobial drugs using molecular testing.

The inventors performed extensive studies on the genome of bacteria of Proteus species either susceptible or resistant to antimicrobial, e.g. antibiotic, drugs. Based on this in ^¬ formation, it is now possible to provide a detailed analysis on the resistance pattern of Proteus strains based on indi ^¬ vidual genes or mutations on a nucleotide level. This analy ^¬ sis involves the identification of a resistance against indi ^¬ vidual antimicrobial, e.g. antibiotic, drugs as well as clus ^¬ ters of them. This allows not only for the determination of a resistance to a single antimicrobial, e.g. antibiotic, drug, but also to groups of antimicrobial drugs, e.g. antibiotics such as lactam or quinolone antibiotics, or even to all rele ^¬ vant antibiotic drugs. Therefore, the present invention will considerably facilitate the selection of an appropriate antimicrobial, e.g. antibi ^¬ otic, drug for the treatment of a Proteus infection in a pa ^¬ tient and thus will largely improve the quality of diagnosis and treatment.

According to a first aspect, the present invention discloses a diagnostic method of determining an infection of a patient with Proteus species potentially resistant to antimicrobial drug treatment, which can be also described as a method of determining an antimicrobial drug, e.g. antibiotic, resistant Proteus infection of a patient, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 1 or

Table 2 below, wherein the presence of said at least two mu ^¬ tations is indicative of an infection with an antimicrobial drug resistant, e.g. antibiotic resistant, Proteus strain in said patient.

An infection of a patient with Proteus species potentially resistant to antimicrobial drug treatment herein means an in ^¬ fection of a patient with Proteus species wherein it is un ^¬ clear if the Proteus species is susceptible to treatment with a specific antimicrobial drug or if it is resistant to the antimicrobial drug.

In step b) above, as well as corresponding steps, at least one mutation in at least two genes is determined, so that in total at least two mutations are determined, wherein the two mutations are in different genes.

Table 1: List of genes

According to a second aspect, the present invention relates to a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Proteus stain, e.g. from an antimicrobial drug, e.g. antibiotic, resistant Proteus infection, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 1 or Table 2 above, wherein the presence of said at least two mu ^¬ tations is indicative of a resistance to one or more antimi ^¬ crobial, e.g. antibiotic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection.

A third aspect of the present invention relates to a method of determining an antimicrobial drug, e.g. antibiotic, re- sistance profile for bacterial microorganisms of Proteus spe ^¬ cies, comprising:

obtaining or providing a first data set of gene sequences of a plurality of clinical isolates of Proteus species;

providing a second data set of antimicrobial drug, e.g. anti ^¬ biotic, resistance of the plurality of clinical isolates of Proteus species;

aligning the gene sequences of the first data set to at least one, preferably one, reference genome of Proteus, and/or as- sembling the gene sequence of the first data set, at least in part ;

analyzing the gene sequences of the first data set for genet ^¬ ic variants to obtain a third data set of genetic variants; correlating the third data set with the second data set and statistically analyzing the correlation; and

determining the genetic sites in the genome of Proteus asso ^¬ ciated with antimicrobial drug, e.g. antibiotic, resistance.

In addition, the present invention relates in a fourth aspect to a method of determining an antimicrobial drug, e.g. anti ^¬ biotic, resistance profile for a bacterial microorganism be ^¬ longing to the species Proteus comprising the steps of a) obtaining or providing a sample containing or suspected of containing the bacterial microorganism;

b) determining the presence of a mutation in at least one gene of the bacterial microorganism as determined by the method according to the third aspect of the present inven ^¬ tion;

wherein the presence of a mutation is indicative of a re- sistance to an antimicrobial, e.g. antibiotic, drug.

Furthermore, the present invention discloses in a fifth as ^¬ pect a diagnostic method of determining an infection of a pa- tient with Proteus species potentially resistant to antimi ^¬ crobial drug treatment, which can, like in the first aspect, also be described as method of determining an antimicrobial drug, e.g. antibiotic, resistant Proteus infection of a pa- tient, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the species Proteus from the patient;

b) determining the presence of at least one mutation in at least one gene of the bacterial microorganism belonging to the species Proteus as determined by the method according to the third aspect of the present invention, wherein the pres ^¬ ence of said at least one mutation is indicative of an anti ^¬ microbial drug, e.g. antibiotic, resistant Proteus infection in said patient.

Also disclosed is in a sixth aspect a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Proteus strain, e.g. from an antimicrobi- al drug, e.g. antibiotic, resistant Proteus infection, com ^¬ prising the steps of:

a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the species Proteus from the patient;

b) determining the presence of at least one mutation in at least one gene of the bacterial microorganism belonging to the species Proteus as determined by the method according to the third aspect of the present invention, wherein the pres ^¬ ence of said at least one mutation is indicative of a re- sistance to one or more antimicrobial, e.g. antibiotic, drugs ;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection. A seventh aspect of the present invention relates to a method of acquiring, respectively determining, an antimicrobial drug, e.g. antibiotic, resistance profile for a bacterial mi ^¬ croorganisms of Proteus species, comprising:

obtaining or providing a first data set of gene sequences of a clinical isolate of Proteus species;

providing a second data set of antimicrobial drug, e.g. anti ^¬ biotic, resistance of a plurality of clinical isolates of Proteus species;

aligning the gene sequences of the first data set to at least one, preferably one, reference genome of Proteus, and/or as ^¬ sembling the gene sequence of the first data set, at least in part ;

analyzing the gene sequences of the first data set for genet ^¬ ic variants to obtain a third data set of genetic variants of the first data set;

correlating the third data set with the second data set and statistically analyzing the correlation; and

determining the genetic sites in the genome of Proteus of the first data set associated with antimicrobial drug, e.g. anti- biotic, resistance.

According to an eighth aspect, the present invention disclos ^¬ es a computer program product comprising executable instruc ^¬ tions which, when executed, perform a method according to the third, fourth, fifth, sixth or seventh aspect of the present invention . Further aspects and embodiments of the invention are dis ^¬ closed in the dependent claims and can be taken from the fol ^¬ lowing description, figures and examples, without being limited thereto.

Figures

The enclosed drawings should illustrate embodiments of the present invention and convey a further understanding thereof. In connection with the description they serve as explanation of concepts and principles of the invention. Other embodi ^¬ ments and many of the stated advantages can be derived in re ^¬ lation to the drawings. The elements of the drawings are not necessarily to scale towards each other. Identical, function- ally equivalent and acting equal features and components are denoted in the figures of the drawings with the same refer ^¬ ence numbers, unless noted otherwise.

Fig. 1 shows schematically a read-out concept for a diagnos- tic test according to a method of the present invention.

Detailed description of the present invention

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. An "antimicrobial drug" in the present invention refers to a group of drugs that includes antibiotics, antifungals, antiprotozoals, and antivirals. According to certain embodi ^¬ ments, the antimicrobial drug is an antibiotic. The term "nucleic acid molecule" refers to a polynucleotide molecule having a defined sequence. It comprises DNA mole ^¬ cules, RNA molecules, nucleotide analog molecules and combi ^¬ nations and derivatives thereof, such as DNA molecules or RNA molecules with incorporated nucleotide analogs or cDNA.

The term "nucleic acid sequence information" relates to an information which can be derived from the sequence of a nu ^¬ cleic acid molecule, such as the sequence itself or a varia- tion in the sequence as compared to a reference sequence.

The term "mutation" relates to a variation in the sequence as compared to a reference sequence. Such a reference sequence can be a sequence determined in a predominant wild type or- ganism or a reference organism, e.g. a defined and known bac ^¬ terial strain or substrain. A mutation is for example a deletion of one or multiple nucleotides, an insertion of one or multiple nucleotides, or substitution of one or multiple nu ^¬ cleotides, duplication of one or a sequence of multiple nu- cleotides, translocation of one or a sequence of multiple nu ^¬ cleotides, and, in particular, a single nucleotide polymor ^¬ phism (SNP) .

In the context of the present invention a "sample" is a sam- pie which comprises at least one nucleic acid molecule from a bacterial microorganism. Examples for samples are: cells, tissue, body fluids, biopsy specimens, blood, urine, saliva, sputum, plasma, serum, cell culture supernatant, swab sample and others. According to certain embodiments, the sample is a patient sample (clinical isolate) .

New and highly efficient methods of sequencing nucleic acids referred to as next generation sequencing have opened the possibility of large scale genomic analysis. The term "next generation sequencing" or "high throughput sequencing" refers to high-throughput sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at once. Examples include Massively Parallel Signa ^¬ ture Sequencing (MPSS) , Polony sequencing, 454

pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ion semiconductor sequencing, DNA nanoball sequencing, Helioscope (TM) single molecule sequencing, Single Molecule SMRT(TM) sequencing, Single Molecule real time (RNAP) se ^¬ quencing, Nanopore DNA sequencing, Sequencing By Hybridization, Amplicon Sequencing, GnuBio.

Within the present description the term "microorganism" com- prises the term microbe. The type of microorganism is not particularly restricted, unless noted otherwise or obvious, and, for example, comprises bacteria, viruses, fungi, micro ^¬ scopic algae und protozoa, as well as combinations thereof. According to certain aspects, it refers to one or more Pro- teus species, particularly Proteus mirabilis, Proteus penneri and/or Proteus vulgaris.

A reference to a microorganism or microorganisms in the pre ^¬ sent description comprises a reference to one microorganism as well a plurality of microorganisms, e.g. two, three, four, five, six or more microorganisms.

A vertebrate within the present invention refers to animals having a vertebrae, which includes mammals - including hu- mans, birds, reptiles, amphibians and fishes. The present in ^¬ vention thus is not only suitable for human medicine, but al ^¬ so for veterinary medicine. According to certain embodiments, the patient in the present methods is a vertebrate, more preferably a mammal and most preferred a human patient. Before the invention is described in exemplary detail, it is to be understood that this invention is not limited to the particular component parts of the process steps of the meth ^¬ ods described herein as such methods may vary. It is also to be understood that the terminology used herein is for purpos- es of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include singular and/or plural referents unless the context clearly dictates otherwise. For example, the term "a" as used herein can be understood as one single entity or in the meaning of "one or more" entities. It is al ^¬ so to be understood that plural forms include singular and/or plural referents unless the context clearly dictates other ^¬ wise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

Regarding the dosage of the antimicrobial, e.g. antibiotic, drugs, it is referred to the established principles of phar- macology in human and veterinary medicine. For example, Forth, Henschler, Rummel "Allgemeine und spezielle

Pharmakologie und Toxikologie" , 9th edition, 2005 might be used as a guideline. Regarding the formulation of a ready-to- use medicament, reference is made to "Remington, The Science and Practice of Pharmacy", 22 ^nd edition, 2013.

Assembling of a gene sequence can be carried out by any known method and is not particularly limited. According to certain embodiments, mutations that were found using alignments can also be compared or matched with align ^¬ ment-free methods, e.g. for detecting single base exchanges, for example based on contigs that were found by assemblies. For example, reads obtained from sequencing can be assembled to contigs and the contigs can be compared to each other.

According to a first aspect, the present invention relates to a diagnostic method of determining an infection of a patient with Proteus species potentially resistant to antimicrobial drug treatment, which can also be described as method of de ^¬ termining an antimicrobial drug, e.g. antibiotic, resistant Proteus infection of a patient, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes consisting of parC, secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, and gpmB, wherein the presence of said at least two mutations is indicative of an infection with an an ^¬ timicrobial, e.g. antibiotic, resistant Proteus strain in said patient.

In this method, as well as the other methods of the inven- tion, the sample can be provided or obtained in any way, preferably non-invasive, and can be e.g. provided as an in vitro sample or prepared as in vitro sample.

According to certain aspects, mutations in at least two, three, four, five, six, seven, eight, nine or ten genes are determined in any of the methods of the present invention, e.g. in at least two genes or in at least three genes. In ^¬ stead of testing only single genes or mutants, a combination of several variant positions can improve the prediction accu ^¬ racy and further reduce false positive findings that are in ^¬ fluenced by other factors. Therefore, it is in particular preferred to determine the presence of a mutation in 2, 3, 4, 5, 6, 7, 8 or 9 (or more) genes selected from Table 1 or 2.

For the above genes, i.e. the genes also denoted in Tables 1 and 2, the highest probability of a resistance to at least one antimicrobial drug, e.g. antibiotic, could be observed, with p-values smaller than 10 , particularly smaller than

10 ^~40 , further particularly smaller than 10 ^~60 , indicating the high significance of the values (n= 583; a = 0.05) . Details regarding Tables 1 and 2 can be taken from Tables 3 and 4 (4a, 4b, 4c) disclosed in the Examples. Having at least two genes with mutations determined, a high probability of an an ^¬ timicrobial drug, e.g. antibiotic, resistance could be deter ^¬ mined. The genes in Table 1 thereby represent the best genes for which a mutation was observed in the genomes of Proteus species, whereas the genes in Table 2 represent the best genes for which a cross-correlation could be observed for the antimicrobial drug, e.g. antibiotic, susceptibility testing for Proteus species as described below.

According to certain embodiments, the obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient in this method - as well as the other methods of the invention - can comprise the follow ^¬ ing :

A sample of a vertebrate, e.g. a human, e.g. is provided or obtained and nucleic acid sequences, e.g. DNA or RNA sequenc ^¬ es, are recorded by a known method for recording nucleic ac ^¬ id, which is not particularly limited. For example, nucleic acid can be recorded by a sequencing method, wherein any se- quencing method is appropriate, particularly sequencing methods wherein a multitude of sample components, as e.g. in a blood sample, can be analyzed for nucleic acids and/or nucle ^¬ ic acid fragments and/or parts thereof contained therein in a short period of time, including the nucleic acids and/or nu ^¬ cleic acid fragments and/or parts thereof of at least one mi ^¬ croorganism of interest, particularly of at least one Proteus species. For example, sequencing can be carried out using polymerase chain reaction (PCR) , particularly multiplex PCR, or high throughput sequencing or next generation sequencing, preferably using high-throughput sequencing. For sequencing, preferably an in vitro sample is used.

The data obtained by the sequencing can be in any format, and can then be used to identify the nucleic acids, and thus genes, of the microorganism, e.g. of Proteus species, to be identified, by known methods, e.g. fingerprinting methods, comparing genomes and/or aligning to at least one, or more, genomes of one or more species of the microorganism of inter- est, i.e. a reference genome, etc., forming a third data set of aligned genes for a Proteus species - discarding addition ^¬ al data from other sources, e.g. the vertebrate. Reference genomes are not particularly limited and can be taken from several databases. Depending on the microorganism, different reference genomes or more than one reference genomes can be used for aligning. Using the reference genome - as well as also the data from the genomes of the other species, e.g. Proteus species - mutations in the genes for each species and for the whole multitude of samples of different species, e.g. Proteus species, can be obtained.

For example, it is useful in genome-wide association studies to reference the points of interest, e.g. mutations, to one constant reference for enhanced standardization. In case of the human with a high consistency of the genome and 99% iden ^¬ tical sequences among individuals this is easy and represents the standard, as corresponding reference genomes are availa- ble in databases. In case of organisms that trigger infec ^¬ tious diseases (e.g. bacteria and viruses) this is much more difficult, though. One possibility is to fall back on a vir ^¬ tual pan genome which contains all sequences of a certain ge ^¬ nus. A further possibility is the analysis of all available references, which is much more complex. Therein all n refer ^¬ ences from a database (e.g. RefSeq) are extracted and com ^¬ pared with the newly sequenced bacterial genomes k. After this, matrices (% of mapped reads, % of covered genome) are applied to estimate which reference is best suited to all new bacteria. However, n x k complete alignments are carried out. Having a big number of references, though, stable results can be obtained, as is the case for Proteus.

According to certain embodiments, the genomes of Proteus spe- cies are referenced to one reference genome. However, it is not excluded that for other microorganisms more than one ref ^¬ erence genome is used. In the present methods, the reference genome of Proteus is NC_010554 as annotated at the NCBI ac ^¬ cording to certain embodiments. The reference genome is at- tached to this application as sequence listing.

The reference sequence was obtained from Proteus strain

NC_010554 (http : //www . genome . jp/dbget- bin/www_bget?refseq+NC_010554)

LOCUS NC_010554 4063606 bp DNA circular CON 07-FEB-2015 DEFINITION Proteus mirabilis strain HI4320, complete genome. ACCESSION NC_010554

VERSION NC 010554.1 GI:197283915 DBLINK BioProject: PRJNA224116

Assembly: GCF_000069965.1

KEYWORDS RefSeq; complete genome.

SOURCE Proteus mirabilis HI4320

ORGANISM Proteus mirabilis HI4320

Bacteria; Proteobacteria; Gammaproteobacteria;

Enterobacteriales ; Enterobacteriaceae ; Proteus.

REFERENCE 1

AUTHORS Pearson, M.M. , Sebaihia,M., Churcher,C,

Quail, M. A., Seshasayee, A. S . , Luscombe, N . M. , Abdellah,Z., Arrosmith, C . , Atkin,B., Chillingworth, . , Hauser,H., Ja- gels,K., Moule,S., Mungall,K., Norbertczak, H . , Rabbino- witsch,E., Walker, D., Whithead,S., Thomson, N . R . , Rather, P.N. , Parkhill,J. and Mobley,H.L.

TITLE Complete genome sequence of uropathogenic Proteus mirabilis, a master of both adherence and motility

JOURNAL J. Bacteriol. 190 (11), 4027-4037 (2008)

PUBMED 18375554

REFERENCE 2 (bases 1 to 4063606)

AUTHORS Sebaihia,M.

TITLE Direct Submission

JOURNAL Submitted (18-FEB-2008) Sebaihia M., Sulston La ^¬ boratories, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 ISA, UNITED KINGDOM

Alternatively or in addition, the gene sequence of the first data set can be assembled, at least in part, with known meth ods, e.g. by de-novo assembly or mapping assembly. The se ^¬ quence assembly is not particularly limited, and any known genome assembler can be used, e.g. based on Sanger, 454, Solexa, Illumina, SOLid technologies, etc., as well as hy ^¬ brids/mixtures thereof. According to certain embodiments, the data of nucleic acids of different origin than the microorganism of interest, e.g. Proteus species, can be removed after the nucleic acids of interest are identified, e.g. by filtering the data out. Such data can e.g. include nucleic acids of the patient, e.g. the vertebrate, e.g. human, and/or other microorganisms, etc. This can be done by e.g. computational subtraction, as devel ^¬ oped by Meyerson et al . 2002. For this, also aligning to the genome of the vertebrate, etc., is possible. For aligning, several alignment-tools are available. This way the original data amount from the sample can be drastically reduced.

Also after such removal of "excess" data, fingerprinting and/or aligning, and/or assembly, etc. can be carried out, as described above, forming a third data set of aligned or as ^¬ sembled genes for a Proteus species.

Using these techniques, genes with mutations of the microor ^¬ ganism of interest, e.g. Proteus species, can be obtained for various species.

When testing these same species for antimicrobial drug, e.g. antibiotic, susceptibility of a number of antimicrobial drugs, e.g. antibiotics, e.g. using standard culturing meth- ods on dishes with antimicrobial drug, e.g. antibiotic, in ^¬ take, as e.g. described below, the results of these antimi ^¬ crobial drug, e.g. antibiotic, susceptibility tests can then be cross-referenced/correlated with the mutations in the ge ^¬ nome of the respective microorganism, e.g. Proteus. Using several, e.g. 50 or more than 50, 100 or more than 100, 200 or more than 200, 300 or more than 300, 400 or more than 400, or 450 or more than 450 different species of a microorganism, e.g. different Proteus species, statistical analysis can be carried out on the obtained cross-referenced data between mu- tations and antimicrobial drug, e.g. antibiotic, susceptibil ^¬ ity for these number of species, using known methods.

Regarding culturing methods, samples can be e.g. cultured overnight. On the next day individual colonies can be used for identification of organisms, either by culturing or using mass spectroscopy. Based on the identity of organisms new plates containing increasing concentration of antibiotics used for the treatment of these organisms are inoculated and grown for additional 12 - 24 hours. The lowest drug concen ^¬ tration which inhibits growth (minimal inhibitory concentration - MIC) can be used to determine susceptibil ^¬ ity/resistance for tested antibiotics. Correlation of the nucleic acid / gene mutations with antimi ^¬ crobial drug, e.g. antibiotic, resistance can be carried out in a usual way and is not particularly limited. For example, resistances can be correlated to certain genes or certain mu ^¬ tations, e.g. SNPs, in genes. After correlation, statistical analysis can be carried out.

In addition, statistical analysis of the correlation of the gene mutations with antimicrobial drug, e.g. antibiotic, re ^¬ sistance is not particularly limited and can be carried out, depending on e.g. the amount of data, in different ways, for example using analysis of variance (ANOVA) or Student's t- test, for example with a sample size n of 50, 100, 200, 300, 400 or 450, and a level of significance ( -error-level ) of e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller. A statistical value can be obtained for each gene and/or each position in the genome as well as for all antibiotics tested, a group of antibiotics or a single antibiotic. The obtained p-values can also be adapted for statistical errors, if need ^¬ ed . For statistically sound results a multitude of individuals should be sampled, with n = 50, 100, 200, 300, 400, 500 or 550, and a level of significance ( -error-level ) of e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller. According to certain embodiments, particularly significant results can be obtained for n = 200, 200, 400, 500 or 450.

According to certain embodiments, a multitude of individuals can be sampled, with n = 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more or 550 or more, and a level of significance (a-error-level) of e.g. 0.05 or smaller, e.g. 0.05, preferably 0.01 or smaller. According to certain embodiments, particularly significant results can be obtained for n = 200 or more, 300 or more, 400 or more, 500 or more or 550 or more.

After the above procedure has been carried out for more than 550, e.g. 583, individual species of Proteus, the data dis ^¬ closed in Tables 1 and 2 were obtained for the statistically best correlations between gene mutations and antimicrobial drug, e.g. antibiotic, resistances. Thus, mutations in these genes were proven as valid markers for antimicrobial drug, e.g. antibiotic, resistance. According to a further aspect, the present invention relates in a second aspect to a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Proteus stain, e.g. from an antimicrobial drug, e.g. antibiotic, resistant Proteus infection, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes consisting of parC, secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, and gpmB, wherein the presence of said at least two mutations is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection.

In this method, the steps a) of obtaining or providing a sample and b) of determining the presence of at least one muta- tion are as in the method of the first aspect.

The identification of the at least one or more antimicrobial, e.g. antibiotic, drug in step c) is then based on the results obtained in step b) and corresponds to the antimicrobial, e.g. antibiotic, drug(s) that correlate (s) with the muta ^¬ tions. Once these antimicrobial drugs, e.g. antibiotics, are ruled out, the remaining antimicrobial drugs, e.g. antibiotic drugs/antibiotics, can be selected in step d) as being suita ^¬ ble for treatment.

In the description, references to the first and second aspect also apply to the 14 ^th , 15 ^th , 16 ^th and 17 ^th aspect, referring to the same genes, unless clear from the context that they don't apply.

According to certain embodiments in the method of the first or second aspect, at least a mutation in parC, particularly in position 2562578 with regard to reference genome NC_010554 as annotated at the NCBI, is determined. For such mutation, a particularly relevant correlation with antimicrobial drug, e.g. antibiotic, resistance could be determined. In particu ^¬ lar, the mutation in position 2562578 with regard to refer- ence genome NC_010554 as annotated at the NCBI is a non- synonymous coding, particularly a codon change aGc/aTc.

According to certain embodiments, the antimicrobial drug, e.g. antibiotic, in the method of the first or second aspect, as well as in the other methods of the invention, is at least one selected from the group of β-lactams, β-lactam inhibi ^¬ tors, quinolines and derivatives thereof, aminoglycosides, polyketides, respectively tetracyclines, and folate synthesis inhibitors .

In the methods of the invention the resistance of Proteus to one or more antimicrobial, e.g. antibiotic, drugs can be de ^¬ termined according to certain embodiments. According to certain embodiments of the first and/or second aspect of the invention the antimicrobial, e.g. antibiotic, drug is selected from lactam antibiotics and the presence of a mutation in the following genes is determined: parC, secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, and/or gpmB .

According to certain embodiments of the first and/or second aspect of the invention the antimicrobial, e.g. antibiotic, drug is selected from quinolone antibiotics, preferably fluoroquinolone antibiotics, and the presence of a mutation in the following genes is determined: parC, secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, and/or gpmB. According to certain embodiments of the first and/or second aspect of the invention the antimicrobial, e.g. antibiotic, drug is selected from aminoglycoside antibiotics, and the presence of a mutation in the following genes is determined: parC.

According to certain embodiments of the first and/or second aspect of the invention the antimicrobial, e.g. antibiotic, drug is selected from polyketide antibiotics, preferably tet- racycline antibiotics, and the presence of a mutation in the following genes is determined: secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, and/or gpmB .

According to certain embodiments of the first and/or second aspect of the invention the antimicrobial, e.g. antibiotic, drug is selected from benzene derived/sulfonamide antibiot ^¬ ics, and the presence of a mutation in the following genes is determined: parC and/or fdoG. According to certain embodiments, the antimicrobial drug is an antibiotic/antibiotic drug.

According to certain embodiments of the first and/or second aspect of the invention, determining the nucleic acid se- quence information or the presence of a mutation comprises determining the presence of a single nucleotide at a single position in a gene. Thus the invention comprises methods wherein the presence of a single nucleotide polymorphism or mutation at a single nucleotide position is detected.

According to certain embodiments, the antibiotic drug in the methods of the present invention is selected from the group consisting of Amoxicillin/K Clavulanate (AUG) , Ampicillin (AM), Aztreonam (AZT) , Cefazolin (CFZ) , Cefepime (CPE),

Cefotaxime (CFT) , Ceftazidime (CAZ) , Ceftriaxone (CAX) , Ce- furoxime (CRM) , Cephalotin (CF) , Ciprofloxacin (CP) ,

Ertapenem (ETP) , Gentamicin (GM) , Imipenem (IMP), Levofloxa- cin (LVX) , Meropenem (MER) , Piperacillin/Tazobactam (P/T) , Ampicillin/Sulbactam (A/S), Tetracycline (TE) , Tobramycin (TO), and Trimethoprim/Sulfamethoxazole (T/S).

The inventors have surprisingly found that mutations in cer- tain genes are indicative not only for a resistance to one single antimicrobial, e.g. antibiotic, drug, but to groups containing several drugs .

According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from lactam antibiotics and a mutation in at least one of the following genes is detected with regard to reference genome NC_010554: parC, secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, and/or gpmB .

According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from quinolone antibiotics, preferably fluoroquinolone antibiotics, and a mutation in at least one of the following genes is detected with regard to reference genome NC_010554: parC, secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, and/or gpmB .

According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from aminoglycoside antibiot- ics and a mutation in at least one of the following genes is detected with regard to reference genome NC_010554: parC.

According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from polyketide antibiotics, preferably tetracycline antibiotics, and a mutation in at least one of the following genes is detected with regard to reference genome NC_010554: secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, and/or gpmB .

According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from benzene de- rived/sulfonamide antibiotics and a mutation in at least one of the following genes is detected with regard to reference genome NC_010554: parC and/or fdoG.

For specific antimicrobial drugs, e.g. antibiotics, specific positions in the above genes can be determined where a high statistical significance is observed. The inventors found that, apart from the above genes indicative of a resistance against antibiotics, also single nucleotide polymorphisms (= SNP's) may have a high significance for the presence of a re- sistance against defined antibiotic drugs. The analysis of these polymorphisms on a nucleotide level may further improve and accelerate the determination of a drug resistance to an ^¬ timicrobial drugs, e.g. antibiotics, in Proteus. According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from lactam antibiotics and a mutation in at least one of the following nucleotide posi- tions is detected with regard to reference genome NC_010554: 2562578, 3741905, 131826, 1482764, 1771087, 1771119, 1918241, 1968294, 2238063, 2238072, 2238088, 2238090, 2454709, 3039125, 3221491, 3221494, 3422635, 4059624, 4059634, 4060202, 131835.

According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from quinolone antibiotics, preferably fluoroquinolone antibiotics, and a mutation in at least one of the following nucleotide positions is detected with regard to reference genome NC_010554: 2562578, 3741905, 131826, 1482764, 1771087, 1771119, 1918241, 1968294, 2238063, 2238072, 2238088, 2238090, 2454709, 3039125, 3221491,

3221494, 3422635, 4059624, 4059634, 4060202, 131835.

According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from aminoglycoside antibiot- ics and a mutation in at least one of the following nucleo ^¬ tide positions is detected with regard to reference genome NC_010554: 2562578.

According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from polyketide antibiotics, preferably tetracycline antibiotics, and a mutation in at least one of the following nucleotide positions is detected with regard to reference genome NC_010554: 3741905, 131826, 1482764, 1771087, 1771119, 1918241, 1968294, 2238063,

2238072, 2238088, 2238090, 2454709, 3039125, 3221491,

3221494, 3422635, 4059624, 4059634, 4060202, 131835. According to certain embodiments of the first and/or second aspect of the invention, the gene is from Table 1 or Table 2, the antibiotic drug is selected from benzene de- rived/sulfonamide antibiotics and a mutation in at least one of the following nucleotide positions is detected with regard to reference genome NC_010554: 2562578, 3422635.

According to certain embodiments of the first and/or second aspect of the invention, the antibiotic drug is at least one of CF, CFZ, CRM, CP, CAX, AM, A/S, LVX and AUG, and a muta ^¬ tion in at least one of the following nucleotide positions is detected with regard to reference genome NC_010554: 2562578, 3741905, 131826, 1482764, 1771087, 1771119, 1918241, 1968294, 2238063, 2238072, 2238088, 2238090, 2454709, 3039125,

3221491, 3221494, 3422635, 4059624, 4059634, 4060202, 131835.

According to certain embodiments of the first and/or second aspect of the invention, the antibiotic drug is TE and a mu ^¬ tation in at least one of the following nucleotide positions is detected with regard to reference genome NC_010554:

3741905, 131826, 1482764, 1771087, 1771119, 1918241, 1968294, 2238063, 2238072, 2238088, 2238090, 2454709, 3039125,

3221491, 3221494, 3422635, 4059624, 4059634, 4060202, 131835. According to certain embodiments of the first and/or second aspect of the invention, the antibiotic drug is CFT and a mu ^¬ tation in at least one of the following nucleotide positions is detected with regard to reference genome NC_010554:

2562578, 3741905, 131826, 1482764, 1771087, 1771119, 1918241, 1968294, 2238063, 2238072, 2238088, 2238090, 3221491,

3221494, 4059624, 4059634, 4060202, 131835. According to certain embodiments of the first and/or second aspect of the invention, the antibiotic drug is T/S and a mu ^¬ tation in at least one of the following nucleotide positions is detected with regard to reference genome NC_010554:

2562578, 3422635.

According to certain embodiments of the first and/or second aspect of the invention, the antibiotic drug is at least one of GM and CPE and a mutation in at least one of the following nucleotide positions is detected with regard to reference ge ^¬ nome NC_010554: 2562578.

According to certain embodiments of the first and/or second aspect of the invention, the resistance of a bacterial micro- organism belonging to the species Proteus against 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, 17, 18, 19, 20 or 21 antibiotic drugs is determined.

According to certain embodiments of the first and/or second aspect of the invention, a detected mutation is a mutation leading to an altered amino acid sequence in a polypeptide derived from a respective gene in which the detected mutation is located. According to this aspect, the detected mutation thus leads to a truncated version of the polypeptide (wherein a new stop codon is created by the mutation) or a mutated version of the polypeptide having an amino acid exchange at the respective position.

According to certain embodiments of the first and/or second aspect of the invention, determining the nucleic acid se ^¬ quence information or the presence of a mutation comprises determining a partial sequence or an entire sequence of the at least two genes. According to certain embodiments of the first and/or second aspect of the invention, determining the nucleic acid se ^¬ quence information or the presence of a mutation comprises determining a partial or entire sequence of the genome of the Proteus species, wherein said partial or entire sequence of the genome comprises at least a partial sequence of said at least two genes. According to certain embodiments of the first and/or second aspect of the invention, determining the nucleic acid se ^¬ quence information or the presence of a mutation comprises using a next generation sequencing or high throughput sequencing method. According to preferred embodiments of the first and/or second aspect of the invention, a partial or en ^¬ tire genome sequence of the bacterial organism of Proteus species is determined by using a next generation sequencing or high throughput sequencing method. In a further, third aspect, the present invention relates to a method of determining an antimicrobial drug, e.g. antibi ^¬ otic, resistance profile for bacterial microorganisms of Pro ^¬ teus species, comprising:

obtaining or providing a first data set of gene sequences of a plurality of clinical isolates of Proteus species;

providing a second data set of antimicrobial drug, e.g. anti ^¬ biotic, resistance of the plurality of clinical isolates of Proteus species;

aligning the gene sequences of the first data set to at least one, preferably one, reference genome of Proteus, and/or as ^¬ sembling the gene sequence of the first data set, at least in part ; analyzing the gene sequences of the first data set for genet ^¬ ic variants to obtain a third data set of genetic variants; correlating the third data set with the second data set and statistically analyzing the correlation; and

determining the genetic sites in the genome of Proteus asso ^¬ ciated with antimicrobial drug, e.g. antibiotic, resistance.

The different steps can be carried out as described with re ^¬ gard to the method of the first aspect of the present inven- tion.

When referring to the second data set, wherein the second da ^¬ ta set e.g. comprises, respectively is, a set of antimicrobi ^¬ al drug, e.g. antibiotic, resistances of a plurality of clin- ical isolates, this can, within the scope of the invention, also refer to a self-learning data base that, whenever a new sample is analyzed, can take this sample into the second data set and thus expand its data base. The second data set thus does not have to be static and can be expanded, either by ex- ternal input or by incorporating new data due to self- learning. This is, however, not restricted to the third as ^¬ pect of the invention, but applies to other aspects of the invention that refer to a second data set, which does not necessarily have to refer to antimicrobial drug resistance. The same applies, where applicable, to the first data set, e.g. in the third aspect.

According to certain embodiments, statistical analysis in the present methods is carried out using Fisher' s test with p < 10 ^~6 , preferably p < 10 ^~9 , particularly p < 10 ^~10 .

The method of the third aspect of the present invention, as well as related methods, e.g. according to the 7 ^th and 10 ^th aspect, can, according to certain embodiments, comprise cor ^¬ relating different genetic sites to each other. This way even higher statistical significance can be achieved. According to certain embodiments of the method of the third aspect and related methods - as above, the second data set is provided by culturing the clinical isolates of Proteus spe ^¬ cies on agar plates provided with antimicrobial drugs, e.g. antibiotics, at different concentrations and the second data is obtained by taking the minimal concentration of the plates that inhibits growth of the respective Proteus species.

According to certain embodiments of the method of the third aspect and related methods, the antibiotic is at least one selected from the group of β-lactams, β-lactam inhibitors, quinolines and derivatives thereof, aminoglycosides,

tetracyclines, and folate synthesis inhibitors, preferably Amoxicillin/K Clavulanate, Ampicillin, Aztreonam, Cefazolin, Cefepime, Cefotaxime, Ceftazidime, Ceftriaxone, Cefuroxime, Cephalothin, Ciprofloxacin, Ertapenem, Gentamicin, Imipenem, Levofloxacin, Meropenem, Piperacillin/Tazobactam, Ampicil- lin/Sulbactam, Tetracycline, Tobramycin, and Trimethoprim/Sulfamethoxazole . According to certain embodiments of the method of the third aspect and related methods, the gene sequences in the third data set are comprised in at least one gene from the group of genes consisting of parC, secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, gpmB, or from the genes listed in Table 5.

According to certain embodiments of the method of the third aspect and related methods, the genetic variant has a point mutation, an insertion and or deletion of up to four bases, and/or a frameshift mutation, particularly a non-synonimous coding in YP_002152062.1. A fourth aspect of the present invention relates to a method of determining an antimicrobial drug, e.g. antibiotic, re ^¬ sistance profile for a bacterial microorganism belonging to the species Proteus comprising the steps of

a) obtaining or providing a sample containing or suspected of containing the bacterial microorganism;

b) determining the presence of a mutation in at least one gene of the bacterial microorganism as determined by the method of the third aspect of the invention;

wherein the presence of a mutation is indicative of a re- sistance to an antimicrobial drug, e.g. antibiotic, drug.

Steps a) and b) can herein be carried out as described with regard to the first aspect, as well as for the following as ^¬ pects of the invention.

With this method, any mutations in the genome of Proteus spe ^¬ cies correlated with antimicrobial drug, e.g. antibiotic, re ^¬ sistance can be determined and a thorough antimicrobial drug, e.g. antibiotic, resistance profile can be established.

A simple read out concept for a diagnostic test as described in this aspect is shown schematically in Fig. 1.

According to Fig. 1, a sample 1, e.g. blood from a patient, is used for molecular testing 2, e.g. using next generation sequencing (NGS) , and then a molecular fingerprint 3 is taken, e.g. in case of NGS a sequence of selected ge- nomic/plasmid regions or the whole genome is assembled. This is then compared to a reference library 4, i.e. selected se ^¬ quences or the whole sequence are/is compared to one or more reference sequences, and mutations (SNPs, sequence- gene ad ^¬ ditions/deletions, etc.) are correlated with susceptibility/ reference profile of reference strains in the reference li ^¬ brary. The reference library 4 herein contains many genomes and is different from a reference genome. Then the result 5 is reported comprising ID (pathogen identification), i.e. a list of all (pathogenic) species identified in the sample, and AST (antimicrobial susceptibility testing), i.e. a list including a susceptibility /resistance profile for all spe ^¬ cies listed

A fifth aspect of the present invention relates to a diagnos- tic method of determining an infection of a patient with Pro ^¬ teus species potentially resistant to antimicrobial drug treatment, which also can be described as method of determin ^¬ ing an antimicrobial drug, e.g. antibiotic, resistant Proteus infection in a patient, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the species Proteus from the patient;

b) determining the presence of at least one mutation in at least one gene of the bacterial microorganism belonging to the species Proteus as determined by the method of the third aspect of the present invention, wherein the presence of said at least one mutation is indicative of an antimicrobial drug, e.g. antibiotic, resistant Proteus infection in said patient.

Again, steps a) and b) can herein be carried out as described with regard to the first aspect of the present invention. According to this aspect, a Proteus infection in a patient can be determined using sequencing methods as well as a re ^¬ sistance to antimicrobial drugs, e.g. antibiotics, of the Proteus species be determined in a short amount of time com- pared to the conventional methods.

In a sixth aspect the present invention relates to a method of selecting a treatment of a patient suffering from an infection with a potentially resistant Proteus strain, e.g. an antimicrobial drug, e.g. antibiotic, resistant Proteus infec ^¬ tion, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing a bacterial microorganism belonging to the species Proteus from the patient;

b) determining the presence of at least one mutation in at least one gene of the bacterial microorganism belonging to the species Proteus as determined by the method of the third aspect of the invention, wherein the presence of said at least one mutation is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection.

This method can be carried out similarly to the second aspect of the invention and enables a fast was to select a suitable treatment with antibiotics for any infection with an unknown Proteus species.

A seventh aspect of the present invention relates to a method of acquiring, respectively determining, an antimicrobial drug, e.g. antibiotic, resistance profile for a bacterial mi ^¬ croorganisms of Proteus species, comprising:

obtaining or providing a first data set of gene sequences of a clinical isolate of Proteus species;

providing a second data set of antimicrobial drug, e.g. anti ^¬ biotic, resistance of a plurality of clinical isolates of Proteus species;

aligning the gene sequences of the first data set to at least one, preferably one, reference genome of Proteus, and/or as- sembling the gene sequence of the first data set, at least in part ;

analyzing the gene sequences of the first data set for genet ^¬ ic variants to obtain a third data set of genetic variants of the first data set;

correlating the third data set with the second data set and statistically analyzing the correlation; and

determining the genetic sites in the genome of Proteus of the first data set associated with antimicrobial drug, e.g. anti ^¬ biotic, resistance.

With this method, antimicrobial drug, e.g. antibiotic, re ^¬ sistances in an unknown isolate of Proteus can be determined.

According to certain embodiments, the reference genome of Proteus is NC_010554 as annotated at the NCBI . According to certain embodiments, statistical analysis in the present methods is carried out using Fisher's test with p < 10 ^~6 , preferably p < 10 ^~9 , particularly p < 10 ^~10 . Also, according to certain embodiments, the method further comprises corre- lating different genetic sites to each other.

An eighth aspect of the present invention relates to a com ^¬ puter program product comprising computer executable instruc- tions which, when executed, perform a method according to the third, fourth, fifth, sixth or seventh aspect of the present invention . In certain embodiments the computer program product is one on which program commands or program codes of a computer program for executing said method are stored. According to certain embodiments the computer program product is a storage medium. The same applies to the computer program products of the as- pects mentioned afterwards, i.e. the eleventh aspect of the present invention. As noted above, the computer program prod ^¬ ucts of the present invention can be self-learning, e.g. with respect to the first and second data sets. In order to obtain the best possible information from the highly complex genetic data and develop an optimum model for diagnostic and therapeutical uses as well as the methods of the present invention - which can be applied stably in clinical routine - a thorough in silico analysis can be necessary. The proposed principle is based on a combination of different approaches, e.g. alignment with at least one, preferably more reference genomes and/or assembly of the genome and correla ^¬ tion of mutations found in every sample, e.g. from each pa ^¬ tient, with all references and drugs, e.g. antibiotics, and search for mutations which occur in several drug and several strains .

Using the above steps a list of mutations as well of genes is generated. These can be stored in databases and statistical models can be derived from the databases. The statistical models can be based on at least one or more mutations at least one or more genes. Statistical models that can be trained can be combined from mutations and genes. Examples of algorithms that can produce such models are association

Rules, Support Vector Machines, Decision Trees, Decision For ^¬ ests, Discriminant-Analysis, Cluster-Methods, and many more. The goal of the training is to allow a reproducible, stand ^¬ ardized application during routine procedures.

For this, for example, a genome or parts of the genome of a microorganism can be sequenced from a patient to be diag- nosed. Afterwards, core characteristics can be derived from the sequence data which can be used to predict resistance. These are the points in the database used for the final mod ^¬ el, i.e. at least one mutation or at least one gene, but also combinations of mutations, etc.

The corresponding characteristics can be used as input for the statistical model and thus enable a prognosis for new pa ^¬ tients. Not only the information regarding all resistances of all microorganisms, e.g. of Proteus species, against all drugs, e.g. antibiotics, can be integrated in a computer de ^¬ cision support tool, but also corresponding directives (e.g. EUCAST) so that only treatment proposals are made that are in line with the directives. A ninth aspect of the present invention relates to the use of the computer program product according to the eighth aspect for acquiring an antimicrobial drug, e.g. antibiotic, re ^¬ sistance profile for bacterial microorganisms of Proteus spe ^¬ cies or in a method of the third aspect of the invention.

In a tenth aspect a method of selecting a treatment of a pa ^¬ tient having an infection with a bacterial microorganism of Proteus species, comprising: obtaining or providing a first data set comprising a gene sequence of at least one clinical isolate of the microorganism from the patient;

providing a second data set of antimicrobial drug, e.g. anti- biotic, resistance of a plurality of clinical isolates of the microorganism;

aligning the gene sequences of the first data set to at least one, preferably one, reference genome of the microorganism, and/or assembling the gene sequence of the first data set, at least in part;

analyzing the gene sequences of the first data set for genet ^¬ ic variants to obtain a third data set of genetic variants of the first data set;

correlating the third data set with the second data set of antimicrobial drug, e.g. antibiotic, resistance of a plurali ^¬ ty of clinical isolates of the microorganism and statistical ^¬ ly analyzing the correlation;

determining the genetic sites in the genome of the clinical isolate of the microorganism of the first data set associated with antimicrobial drug, e.g. antibiotic, resistance; and selecting a treatment of the patient with one or more antimi ^¬ crobial, e.g. antibiotic, drugs different from the ones iden ^¬ tified in the determination of the genetic sites associated with antimicrobial drug, e.g. antibiotic, resistance is dis- closed.

Again, the steps can be carried out as similar steps before. In this method, as well as similar ones, no aligning is nec ^¬ essary, as the unknown sample can be directly correlated, af- ter the genome or genome sequences are produced, with the se ^¬ cond data set and thus mutations and antimicrobial drug, e.g. antibiotic, resistances can be determined. The first data set can be assembled, for example, using known techniques. According to certain embodiments, statistical analysis in the present method is carried out using Fisher' s test with p < 10 ^~6 , preferably p < 10 ^~9 , particularly p < 10 ^~10 . Also, ac ^¬ cording to certain embodiments, the method further comprises correlating different genetic sites to each other.

An eleventh aspect of the present invention is directed to a computer program product comprising computer executable instructions which, when executed, perform a method according to the tenth aspect.

According to a twelfth aspect of the present invention, a diagnostic method of determining an infection of a patient with Proteus species potentially resistant to antimicrobial drug treatment, which can also be described as a method of deter ^¬ mining an antimicrobial drug, e.g. antibiotic, resistant Pro ^¬ teus infection of a patient is disclosed, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 5, wherein the presence of said at least two mutations is indic ^¬ ative of an antimicrobial drug, e.g. antibiotic, resistant Proteus infection in said patient.

A thirteenth aspect of the invention discloses a method of selecting a treatment of a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Proteus infection, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 5, wherein the presence of said at least two mutations is indic ^¬ ative of a resistance to one or more antimicrobial, e.g. an- tibiotic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection.

Again, the steps can be carried out as in similar methods be ^¬ fore, e.g. as in the first and second aspect of the inven ^¬ tion. In the twelfth and thirteenth aspect of the invention, all classes of antibiotics considered in the present method are covered.

Herein, the genes in Table 5 are the following:

parC, secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, gpmB, dnaK, nhaA, ribF, ileS, carA, hybO, hybA, hybB, hybD, cpdB, yajC, secD, secF, dxs, cyoE, cyoD, cyoB, tig, acrA, priC, dnaX, PMI0140, recR, dksA, pyrG, eno, epd, fbaA, PMI0341, nqrC, rimM, trmD, rplS, PMI0392, lipA, lipB, PMI3693, ompF, PMI3449, msbB, nagC, gyrB,

PMI2908, rpoC, PMI2124, PMI0936, mgtE, PMI1294, dmsA, gabD, PMI1896, PMI2380, hpmA, cscA, PMI2922, PMI1221, PMI0910, sucC, caiA, PMI3369, hemA, holC, gppA, PMI2178, gpsA, argl, PMI2961, PMI2783, kdsC, dacA, galK, emrB, fabF, pheT, cheB, nuoL, nuoJ, fixC, PMI2772, kefB, pstS, frdB, rpoN, tatA, yfbB, PMI2201, PMI0191, pre, fliK, nuoG, nuoC, atpA, and ilvB .

According to certain embodiments, mutations in at least two, three, four, five, six, seven, eight, nine or ten genes are determined in any of the methods of the present invention, e.g. in at least two genes or in at least three genes. In ^¬ stead of testing only single genes or mutants, a combination of several variant positions can improve the prediction accu ^¬ racy and further reduce false positive findings that are in- fluenced by other factors. Therefore, it is in particular preferred to determine the presence of a mutation in 2, 3, 4, 5, 6, 7, 8 or 9 (or more) genes selected from Table 5.

Table 5: List of genes

Further, according to certain embodiments, the reference ge ^¬ nome of Proteus is again NC_010554 as annotated at the NCBI . According to certain embodiments, statistical analysis in the present methods is carried out using Fisher' s test with p < 10 ^~6 , preferably p < 10 ^~9 , particularly p < 10 ^~10 . Also, ac ^¬ cording to certain embodiments, the method further comprises correlating different genetic sites to each other. Also the other aspects of the embodiments of the first and second as ^¬ pect of the invention apply.

Table 6: List for lactam antibiotics

FDR: determined according to FDR (Benjamini Hochberg) method (Benjamini Hochberg, 199

According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antimicrobial drug is an antibiotic. According to certain em- bodiments, the antibiotic is a lactam antibiotic and a muta ^¬ tion in at least one of the genes listed in Table 6 is de ^¬ tected, or a mutation in at least one of the positions (de ^¬ noted POS in the tables) listed in Table 6. According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is at least one of CF, CFT, CFZ, CRM, CAX, AM, A/S and AUG and a mutation in at least one of the genes of parC, PMI3693, ompF, PMI3449, msbB, nagC, gyrB, secG, cyoC, pykF, flhB, dedA, err, murF, PMI2939, PMI3715, gpmB is detected, or a mutation in at least one of the positions of 2562578, 4032998, 849533, 3777669, 1214898, 521806, 3450194, 3741905, 131826, 1482764, 1771087, 1771119, 1918241, 1968294, 2238063, 2238072, 2238088, 2238090, 3221491, 3221494, 4059624, 4059634, 4060202, 131835.

According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is CPE and a mutation in at least one of the genes of parC, PMI3449, msbB, nagC is detected, or a mutation in at least one of the positions of 2562578, 3777669, 1214898, 521806.

According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is P/T and a mutation in at least one of the genes of PMI3693, ompF, gyrB is detected, or a mutation in at least one of the positions of 4032998, 849533, 3450194. According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is a quinolone antibiotic and a mutation in at least one of the genes listed in Table 7 is detected, or a mutation in at least one of the positions (denoted POS in the tables) listed in Table 7.

According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is at least one of CP and LVX and a mutation in at least one of the genes of parC, secG, cyoC, pykF, flhB, dedA, err, murF, PMI2939, PMI3715, gpmB, gmhB, purH, fdoG is de ^¬ tected, or a mutation in at least one of the positions of 2562578, 3741905, 131826, 1482764, 1771087, 1771119, 1918241, 1968294, 2238063, 2238072, 2238088, 2238090, 3221491, 3221494, 4059624, 4059634, 4060202, 2454709, 3039125, 3422635, 131835. According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is an aminoglycoside antibiotic and a mutation in at least one of the genes listed in Table 8 is detected, or a mutation in at least one of the positions (denoted POS in the tables) listed in Table 8. Table 7: List for quinolone antibiotics

gene POS antibiotic p-value genbank protein name (FDR) accession number

parC 2562578 CF; /S ; CP; CF ; GM; CFZ ; CRM; CAX; CPE ; AM; A/S ; LVX; AUG 4, 65979E-71 YP_002152062.1 secG 3741905 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 5, 11728E-63 YP_002153099.1 cyoC 131826 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002149890.1 pykF 1482764 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002151136.1 flhB 1771087 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002151391.1 flhB 1771119 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002151391.1 dedA 1918241 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002151518.1 err 1968294 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002151557.1 murF 2238063 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002151793.1 murF 2238072 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002151793.1 murF 2238088 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002151793.1 murF 2238090 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002151793.1

PMI2939 3221491 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002152640.1

PMI2939 3221494 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002152640.1

PMI3715 4059624 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002153390.1

PMI3715 4059634 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002153390.1 gpmB 4060202 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002153391.1 gmhB 2454709 CF; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002151976.1 purH 3039125 CF; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002152469.1 fdoG 3422635 CF; T/S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP_002152801.1 cyoC 131835 CF; TE ; CFT ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 8, 38542E-63 YP_002149890.1

Table 8: List of aminoglycoside antibiotics

According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is at least one of GM and TO and a mutation in at least one of the genes of PMI2908, rpoC, PMI2124, PMI0936, mgtE, PMI1294, dmsA, gabD, PMI1896, PMI2380, hpmA, cscA, PMI2922, PMI1221, PMI0910 is detected, or a mutation in at least one of the positions of 3189475, 3053893, 2299533, 1013893, 2281052, 1367519, 1823348, 3708304, 2041811, 2603984, 2218536, 2376673, 3206198, 1290778, 994331.

According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is an polyketide antibiotic and a mutation in at least one of the genes listed in Table 9 is detected, or a mutation in at least one of the positions (denoted POS in the tables) listed in Table 9. According to certain embodiments of the method of the twelfth and/or thirteenth aspect of the present invention, as well as also of the eighteenth aspect of the present invention, the antibiotic is TE and a mutation in at least one of the genes of secG, cyoC, pykF, flhB, dedA, err, murF, PMI2939, PMI3715, gpmB, gmhB, purH, fdoG is detected, or a mutation in at least one of the positions of 3741905, 131826, 1482764, 1771087, 1771119, 1918241, 1968294, 2238063, 2238072, 2238088, 2238090, 3221491, 3221494, 4059624, 4059634, 4060202, 2454709, 3039125, 3422635, 131835.

According to certain embodiments of the method of the seven ^¬ teenth and/or eighteenth aspect of the present invention, the antibiotic is T/S and a mutation in at least one of the genes listed in Table 10 is detected, or a mutation in at least one of the positions (denoted POS in the tables) listed in Table 10.

Table 9: List of polyketides, preferably tetracycline

gene name POS antibiotic p-value (FDR) genbank protein accession number secG 3741905 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 5, 11728E-63 YP 002153099.1 cyoC 131826 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002149890.1 pykF 1482764 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002151136.1 flhB 1771087 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002151391.1 flhB 1771119 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002151391.1 dedA 1918241 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002151518.1 err 1968294 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002151557.1 murF 2238063 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002151793.1 murF 2238072 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002151793.1 murF 2238088 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002151793.1 murF 2238090 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002151793.1

PMI2939 3221491 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002152640.1

PMI2939 3221494 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002152640.1

PMI3715 4059624 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002153390.1

PMI3715 4059634 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002153390.1 gpmB 4060202 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002153391.1 gmhB 2454709 CF; TE ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002151976.1 purH 3039125 CF; TE ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002152469.1 fdoG 3422635 CF; T/S ; TE ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 7, 38724E-63 YP 002152801.1 cyoC 131835 CF; TE ; CFT ; CFZ ; CRM; CP ; CAX; LVX; AM; A/ S ; AUG 8, 38542E-63 YP 002149890.1

Table 10: List of others antibiotics ((benzene derived) /sulfonamide)

gene name POS antibiotic p-value genbank protein

(FDR) accession number parC 2562578 C ; T/ S ; CP ; CFT ; GM; CFZ ; CRM; CAX; CPE ; AM; A/S ; LVX; AUG 4, 65979E-71 YP 002152062.1 fdoG 3422635 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 7, 38724E-63 YP 002152801.1 dnaK 19958 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002149796.1 nhaA 21872 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002149798.1 fabF 952747 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002150620.1 pheT 1104454 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002150789.1 cheB 1773746 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002151394.1 nuoL 1876979 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002151482.1 nuoJ 1879024 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002151484.1 fixC 2898978 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002152352.1

PMI2772 3042468 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002152473.1 kefB 3076139 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002152506.1 pstS 3174532 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002152594.1 frdB 3918248 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 04565E-62 YP 002153262.1 fixC 2898937 CF; T/ S ; TE ; CFZ ; CRM; CP; CAX; LVX; AM; A/S ; AUG 1, 73077E-62 YP 002152352.1

A fourteenth aspect of the present invention is directed to a diagnostic method of determining an infection of a patient with Proteus species potentially resistant to antimicrobial drug treatment, which can also be described as method of de- termining an antimicrobial drug, e.g. antibiotic, resistant Proteus infection of a patient, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least one gene from the group of genes consisting of secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, gpmB, particularly secG, cyoC, pykF, flhB, dedA, err, purH, PMI2939, fdoG, PMI3715, gpmB, wherein the presence of said at least one mutation is indicative of an antimicro- bial drug, e.g. antibiotic, resistant Proteus infection in said patient.

A fifteenth aspect of the present invention is directed to a method of selecting a treatment of a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Proteus in ^¬ fection, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least one gene from the group of genes consisting of secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, gpmB, particularly secG, cyoC, pykF, flhB, dedA, err, purH, PMI2939, fdoG, PMI3715, gpmB, wherein the presence of said at least one mutation is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection. Again, in the fourteenth and the fifteenth aspect the steps correspond to those in the first or second aspect, although only a mutation in at least one gene is determined.

A sixteenth aspect of the present invention is directed to a method of treating a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Proteus infection, compris ^¬ ing the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least one gene from the group of genes consisting of secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, gpmB, particularly secG, cyoC, pykF, flhB, dedA, err, purH, PMI2939, fdoG, PMI3715, gpmB, wherein the presence of said at least one mutation is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs;

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection; and e) treating the patient with said one or more antimicrobi ^¬ al, e.g. antibiotic, drugs. A seventeenth aspect of the present invention is directed to method of treating a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Proteus infection, compris ^¬ ing the steps of: a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes consisting of parC, secG, cyoC, pykF, flhB, dedA, err, murF, gmhB, purH, PMI2939, fdoG, PMI3715, gpmB, wherein the presence of said at least two mutations is indicative of a resistance to one or more antimicrobial, e.g. antibiotic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs;

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection; and e) treating the patient with said one or more antimicrobi- al, e.g. antibiotic, drugs.

An eighteenth aspect of the present invention is directed to method of treating a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Proteus infection, compris- ing the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 5, wherein the presence of said at least two mutations is indic ^¬ ative of a resistance to one or more antimicrobial, e.g. an ^¬ tibiotic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs;

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection; and e) treating the patient with said one or more antimicrobi ^¬ al, e.g. antibiotic, drugs.

A nineteenth aspect of the present invention is directed to method of treating a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Proteus infection, compris ^¬ ing the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least one gene from the group of genes listed in Table 11, preferably from the group of genes listed in Table 12, where ^¬ in the presence of said at least one mutation is indicative of a resistance to one or more antimicrobial, e.g. antibi- otic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs;

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection; and e) treating the patient with said one or more antimicrobi ^¬ al, e.g. antibiotic, drugs.

Also in the sixteenth to nineteenth aspect of the invention, steps a) to d) are analogous to the steps in the method of the second aspect of the present invention. Step e) can be sufficiently carried out without being restricted and can be done e.g. non-invasively . A twentieth aspect of the present invention is directed to a diagnostic method of determining an infection of a patient with Proteus species potentially resistant to antimicrobial drug treatment, which can also be described as method of de- termining an antimicrobial drug, e.g. antibiotic, resistant Proteus infection of a patient, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least one gene from the group of genes listed in Table 11, preferably from the group of genes listed in Table 12, where ^¬ in the presence of said at least one mutation is indicative of an antimicrobial drug, e.g. antibiotic, resistant Proteus infection in said patient.

A twenty-first aspect of the present invention is directed to a method of selecting a treatment of a patient suffering from an antimicrobial drug, e.g. antibiotic, resistant Proteus in- fection, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least one gene from the group of genes listed in Table 11, preferably from the group of genes listed in Table 12, where ^¬ in the presence of said at least one mutation is indicative of a resistance to one or more antimicrobial, e.g. antibi ^¬ otic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection. Again, in the twentieth and the twenty-first aspect the steps correspond to those in the first or second aspect, although only a mutation in at least one gene is determined. Table 11: List of genes

Table 12: List of genes

According to a twenty-second aspect of the present invention, a diagnostic method of determining an infection of a patient with Proteus species potentially resistant to antimicrobial drug treatment, which can also be described as a method of determining an antimicrobial drug, e.g. antibiotic, resistant Proteus infection of a patient is disclosed, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 13, wherein the presence of said at least two mutations is indic- ative of an antimicrobial drug, e.g. antibiotic, resistant Proteus infection in said patient.

A twenty-third aspect of the invention discloses a method of selecting a treatment of a patient suffering from an antimi- crobial drug, e.g. antibiotic, resistant Proteus infection, comprising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 13, wherein the presence of said at least two mutations is indic ^¬ ative of a resistance to one or more antimicrobial, e.g. an ^¬ tibiotic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs; and

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection. Again, the steps can be carried out as in similar methods be ^¬ fore, e.g. as in the first and second aspect of the inven ^¬ tion. In the twenty-second and twenty-third aspect of the in ^¬ vention, as well as the twenty-fourth aspect, all classes of antibiotics considered in the present method are covered, the reference genome is again NC_010554 as annotated at the NCBI, and the statistical analysis is carried out using Fisher' s test with p < 10 ^~6 , preferably p < 10 ^~9 , particularly p < 10 ^{~ 10} .

A twenty-fourth aspect of the present invention is directed to method of treating a patient suffering from an antimicro ^¬ bial drug, e.g. antibiotic, resistant Proteus infection, com- prising the steps of:

a) obtaining or providing a sample containing or suspected of containing at least one Proteus species from the patient; b) determining the presence of at least one mutation in at least two genes from the group of genes listed in Table 13, wherein the presence of said at least two mutations is indic ^¬ ative of a resistance to one or more antimicrobial, e.g. an ^¬ tibiotic, drugs;

c) identifying said at least one or more antimicrobial, e.g. antibiotic, drugs;

d) selecting one or more antimicrobial, e.g. antibiotic, drugs different from the ones identified in step c) and being suitable for the treatment of a Proteus infection; and e) treating the patient with said one or more antimicrobi ^¬ al, e.g. antibiotic, drugs.

Also in the twenty-fourth aspect of the invention, steps a) to d) are analogous to the steps in the method of the second aspect of the present invention. Step e) can be sufficiently carried out without being restricted and can be done e.g. non-invasively . The genes in Table 13 thereby cover still p-values with very high probability, with the last gene in Table 13 still having a p-value of 1, 06789 E ^~62 , with the same n and a as before. Table 13: List of genes

parC secG cyoC pykF flhB dedA err murF gmhB purH PMI2939 fdoG PMI3715 gpmB dnaK nhaA ribF ileS carA hybO hybA hybB hybD cpdB yajC secD secF dxs cyoE cyoD cyoB tig acrA priC dnaX PMI0140 recR dksA dksA pyrG eno epd fbaA PMI0341 nqrC rimM trmD rplS

PMI0392 lipA lipB corC miaB ubiF gltA sdhC sdhA sdhB sucA sucB sucC PMI0580 tolQ tolB pal PMI0586 gpmA PMI0648 clpA serS pflA pflB rpsA aspC ompF asnC pncB rlmL ompA PMI0855

PMI0856 rpmF plsX fabH fabG fabF lolC PMI1014 proQ thrS rpml rplT pheS pheT prsA ipk prfA hemK znuA pykA fumC nth rnb tyrS gapA pgsA uvrC guaB xseA dapA upp purM

PMI1580 fliZ fliA fliG fliK fliL fliN flgB flhA cheY cheB cheR PMI1665 PMI1666 motB gyrA nrdB yfbB nuoM nuoL nuoK nuoJ nuol nuoH nuoG nuoF nuoE nuoC nuoA PMI1763 PMI1767 ackA purF cvpA fabB ptsl PMI1846 iscS iscR acnB lpdA aceF ace lpxC ftsZ ftsA ftsQ PMI2068 murC mraZ fold ppiB PMI2252 fabZ lpxD yaeT ecfE uppS pyrH tsf rpsB PMI2361 dnaG rpoD deoC PMI2417 hyfD hyfC hyfB hyfA PMI2531 groL groS fixC caiT PMI2717 PMI2719 PMI2720 PMI2721 PMI2722 potA PMI2745 uvrA ssb lexA dgkA plsB PMI2770

PMI2772 rpoC rpoB rplL rplJ rplA rplK nusG secE fusA rpsL PMI2796 slyD PMI2804 kefB kefG gmk spoT envZ pstS glpG glpD PMI2937 PMI2938

PMI2940 PMI2941 prlC damX gidA atpl atpB atpF atpH atpA atpG atpD atpC glmU fdhD trmE oxaA rnpA dnaA recF gyrB rpmB rpmG rimO

PMI3182 secB hslV ftsN rpmE argC murl coaA bfd bfr rplC rplD rplV rplP rpsQ rplX rpsN rplF rplR rpsE rpmD secY rpmj rpsM rpoA hdfR PMI3296 ilvL ilvG trxA rffT rffM hemX cyaY PMI3335 miaA hflX hflK PMI3369 purA rpsF priB rpll argR PMI3402 ispB rplU rpmA obgE PMI3410 rrmj ftsH glmM PMI3416 nusA infB pnp nlpl deaD PMI3465 ivbL ilvB nark frdC frdB frdA poxA ftsY dusB accB aroQ PMI3637 tldD PMI3641 tldE ptsN rplM diaA PMI3691 PMI3692

PMI3693 PMI3694 cyoB nuoM zipA dnaG hyfF murA

According to certain embodiments of the twenty-second, twenty- third, and/or twenty-fourth aspect, at least one of the following gene positions, preferably at least two, three, four, five, six, seven, eight, nine or more gene positions, is/are determined:

2562578, 3741905, 131826, 1482764, 1771087, 1771119, 1918241, 1968294, 2238063, 2238072, 2238088, 2238090, 2454709, 3039125, 3221491, 3221494, 3422635, 4059624, 4059634, 4060202, 131835, 19958, 21872, 25572, 25764, 25783, 26206, 26284, 32335, 32353, 53405, 53487, 53505, 53661, 53871, 54003, 54004, 54213, 54276, 54303, 54468, 54605, 55215, 55677, 55735, 56361, 56639, 58576, 58578, 68312, 105758, 106230, 106253, 107791, 122332, 131111, 131404, 131446, 132270, 132397, 141518, 141519, 142045, 142682, 165937, 166053, 166099, 166157, 166159, 166167, 166180, 171360, 171546, 171658, 171662, 174515, 174869, 175036, 236145, 236466, 262760, 263053, 289363, 291016, 291155, 291404, 390045, 390048, 404430, 438564, 438569, 438583, 438625, 438977, 439953, 447813, 488253, 488635, 489064, 512855, 515772, 516200, 609454, 609606, 610448, 610581, 610582, 610593, 610594, 612627, 612638, 612639, 612694, 612843, 613447, 613821, 613863, 613937, 613949, 614135, 614251, 616159, 616177, 616207, 616215, 616221, 616298, 616300, 616305, 616353, 616661, 616780, 616841, 616890, 616939, 617048, 618013, 618168, 628162, 628863, 631669, 631775, 632026, 632027, 632122, 632126, 632133, 632140, 632142, 632197, 632399, 632442, 632560, 632720, 637570, 704521, 749216, 764862, 764899, 764902, 764903, 764904, 764954, 769675, 771332, 771477, 771623, 771729, 771731, 784768, 785214, 785336, 848303, 849825, 851231, 852902, 860929, 873905, 947084, 947158, 947163, 947167, 947173, 947182, 947210, 947236, 947252, 947254, 947296, 947564, 947656, 947738, 947807, 947816, 947821, 947926, 947927, 947928, 948092, 948099, 948576, 948742, 949052, 949151, 949185, 949189, 949198, 949339, 949415, 949416, 949441, 949454, 949458, 949467, 949474, 949544, 949559, 949663, 951242, 951244, 951248, 951254, 951360, 951854, 951902, 952495, 952747, 952828, 952836, 968923, 1077386, 1077419, 1077468,

1077618, 1077724, 1078112, 1100144, 1100989, 1101393, 1101438, 1101919,

1101980, 1101994, 1104454, 1104820, 1104839, 1104869, 1105039, 1105097,

1105100, 1146632, 1146937, 1147549, 1147625, 1147626, 1147771, 1150615,

1150731, 1152456, 1212590, 1212620, 1212736, 1212743, 1212788, 1217061,

1371149, 1385767, 1385770, 1385787, 1386122, 1386278, 1464863, 1464864,

1464866, 1464868, 1465085, 1482775, 1596063, 1596215, 1596441, 1614151,

1614206, 1614352, 1614378, 1614858, 1615063, 1615065, 1615072, 1640778,

1640933, 1640990, 1641451, 1662486, 1673731, 1673848, 1674401, 1680613,

1731860, 1732817, 1732858, 1745743, 1748563, 1749691, 1749694, 1749702,

1749817, 1749878, 1750174, 1750252, 1750255, 1750318, 1750335, 1750351,

1750378, 1750387, 1750467, 1750477, 1750489, 1750499, 1750500, 1750515,

1750516, 1751645, 1764702, 1770887, 1771129, 1771251, 1771265, 1771266,

1773358, 1773359, 1773362, 1773396, 1773580, 1773746, 1775351, 1777128,

1777130, 1777396, 1777408, 1777703, 1782242, 1853651, 1853822, 1857896,

1866823, 1875173, 1875189, 1875301, 1876427, 1876529, 1876536, 1876822,

1876979, 1876980, 1876981, 1876996, 1877004, 1877005, 1878333, 1878585,

1878600, 1878732, 1878856, 1879024, 1879091, 1879137, 1879282, 1879349,

1879919, 1880000, 1880555, 1880557, 1881003, 1881096, 1881108, 1881129,

1881144, 1881154, 1881155, 1881168, 1881171, 1883439, 1883753, 1883897,

1883933, 1883937, 1884932, 1885231, 1885531, 1885549, 1885601, 1887186,

1887195, 1887200, 1887371, 1888236, 1888332, 1888582, 1888605, 1888971,

1894922, 1897741, 1914231, 1914232, 1914319, 1914381, 1914385, 1914632,

1925721, 1967765, 1967784, 1983874, 2000892, 2002419, 2183724, 2199454,

2200254, 2200493, 2200898, 2201059, 2201120, 2201122, 2201708, 2202174,

2202175, 2202457, 2202981, 2203212, 2203250, 2203904, 2203927, 2203955,

2205132, 2205184, 2227683, 2228236, 2229346, 2229439, 2229621, 2229622,

2229648, 2230617, 2230750, 2230836, 2230874, 2231569, 2231570, 2231571,

2231581, 2231794, 238100, 2243936, 2335800, 2340329, 2442677, 2442685,

2472332, 2473402, 2476250, 2476332, 2476597, 2476603, 2476615, 2476742,

2478782, 2478897, 2481987, 2482024, 2482224, 2482760, 2482761, 2482867,

2483098, 2483108, 2483232, 2483791, 2581759, 2590008, 2590377, 2592098,

2644614, 2644726, 2644737, 2769143, 2769146, 2769209, 2769248, 2769288,

2772170, 2772179, 2772279, 2772642, 2775062, 2775162, 2775272, 2786911,

2786926, 2787314, 2787433, 2787446, 2787447, 2787827, 2787951, 2788142,

2788300, 2788384, 2788451, 2788539, 2788541, 2788586, 2898978, 2904023,

2968996, 2969025, 2969694, 2969753, 2969784, 2969798, 2971794, 2971795,

2971796, 2971797, 2971798, 2971959, 2972346, 2972726, 2988736, 2988934,

3000293, 3000434, 3003519, 3003544, 3003546, 3004546, 3004697, 3006261,

3006611, 3006612, 3006613, 3006627, 3006658, 3006708, 3006709, 3006715, 3006791, 3006810, 3006832, 3006847, 3006902, 3006904, 3006910, 3007284,

3007321, 3007477, 3041804, 3041820, 3041823, 3042357, 3042468, 3055027,

3055162, 3055257, 3055258, 3055273, 3055564, 3058839, 3058882, 3059496,

3059509, 3059675, 3059676, 3059963, 3060607, 3060792, 3060994, 3061157,

3061158, 3061179, 3061620, 3062379, 3062488, 3062683, 3062701, 3062723,

3062866, 3062945, 3063015, 3063544, 3064080, 3064098, 3064099, 3064103,

3064277, 3064283, 3065011, 3065028, 3065306, 3065502, 3065511, 3065512,

3065580, 3067456, 3067463, 3067493, 3067563, 3067579, 3067693, 3067720,

3067731, 3067732, 3067739, 3067798, 3067942, 3068551, 3069985, 3070082,

3070525, 3070560, 3070598, 3070599, 3070610, 3070624, 3070666, 3070672,

3070699, 3075527, 3075770, 3075960, 3076139, 3076150, 3076157, 3076168,

3076170, 3078449, 3078450, 3140400, 3140993, 3141038, 3173976, 3173988,

3173997, 3174309, 3174402, 3174403, 3174429, 3174528, 3174529, 3174532,

3174594, 3174618, 3174619, 3174632, 3210333, 3210381, 3210582, 3210583,

3211688, 3211694, 3211774, 3220249, 3220312, 3220529, 3220580, 3220803,

3220811, 3220832, 3220880, 3220883, 3220965, 3221159, 3221172, 3221380,

3221587, 3221611, 3223265, 3225405, 3225451, 3225529, 3299712, 3299749,

3326024, 3358602, 3361509, 3361588, 3361606, 3361612, 3361614, 3361621,

3361871, 3361872, 3361937, 3361992, 3361994, 3362009, 3362011, 3362031,

3362078, 3362345, 3362393, 3363067, 3363185, 3363326, 3363663, 3363684,

3363730, 3363803, 3363969, 3364748, 3364941, 3364955, 3364959, 3364963,

3365950, 3365960, 3365994, 3366006, 3366059, 3366266, 3366827, 3366832,

3367976, 3367999, 3368000, 3368115, 3368631, 3421606, 3422011, 3422089,

3422272, 3422315, 3422593, 3422660, 3422746, 3422758, 3422800, 3422827,

3422947, 3422998, 3423002, 3423301, 3423314, 3423339, 3423347, 3423349,

3423479, 3423571, 3423572, 3423593, 3423641, 3442266, 3442411, 3442455,

3442482, 3442995, 3443468, 3443474, 3443514, 3443600, 3443627, 3443740,

3443741, 3443807, 3443816, 3443933, 3443937, 3443945, 3443948, 3443951,

3443952, 3443954, 3444678, 3444693, 3444702, 3446029, 3446261, 3446713,

3446933, 3448971, 3448972, 3448991, 3449040, 3449042, 3449052, 3449075,

3449081, 3449123, 3449280, 3449348, 3449349, 3449403, 3449612, 3449698,

3449885, 3449889, 3449925, 3449970, 3450028, 3450545, 3450640, 3470490,

3470647, 3470648, 3496360, 3496871, 3496900, 3497196, 3497557, 3497558,

3497560, 3530618, 3530628, 3530903, 3531614, 3531625, 3531774, 3531804,

3535639, 3557063, 3557092, 3557151, 3568751, 3568760, 3568840, 3568944,

3568978, 3578816, 3578860, 3578891, 3581355, 3581400, 3581673, 3581980,

3582010, 3582844, 3583192, 3583558, 3583833, 3585451, 3585537, 3585546,

3586561, 3586562, 3586567, 3586831, 3586849, 3587263, 3587297, 3587299,

3588101, 3588942, 3589709, 3589828, 3589840, 3589862, 3589930, 3590344,

3590345, 3590700, 3590900, 3591026, 3591624, 3591647, 3593035, 3593310, 3593315 3595160, 3595163, 3595208, 3612449, 3612512, 3612769, 3612829, 3612890 3612907, 3612935, 3613063, 3613446, 3613509, 3615780, 3615990, 3632585 3632588, 3632597, 3632608, 3644924, 3645043, 3647693, 3647816, 3647822 3647885, 3647911, 3652470, 3658169, 3658352, 3692300, 3692462, 3692463 3692464, 3692465, 3692477, 3694213, 3694436, 3696887, 3696968, 3696971 3697375, 3703133, 3703141, 3703214, 3703248, 3703310, 3703384, 3703388 3704011, 3704123, 3727857, 3727858, 3730091, 3731610, 3732055, 3732064 3732455, 3732867, 3736431, 3736432, 3737164, 3737782, 3741525, 3741540 3741553, 3741562, 3741571, 3742055, 3742803, 3742837, 3742916, 3742967 3743001, 3743006, 3743365, 3743369, 3743419, 3743430, 3743434, 3743435 3743436, 3743453, 3743622, 3743713, 3743794, 3743802, 3744367, 3744368 3744390, 3744402, 3744517, 3744612, 3744870, 3744951, 3745026, 3745062 3745264, 3745279, 3745282, 3745433, 3745437, 3745460, 3749482, 3749941 3749942, 3752300, 3752306, 3752575, 3752583, 3752605, 3753216, 3793195 3838549, 3838757, 3838877, 3838879, 3838939, 3899203, 3917376, 3917431 3918057, 3918248, 3918273, 3918304, 3918459, 3918870, 3921420, 3921481 3942358, 3943893, 3944031, 3960166, 3960289, 3960435, 3960436, 3960447 3960448, 3960566, 3960569, 3960577, 3960778, 3965450, 3965463, 3965465 3965787, 3965944, 3966152, 3966202, 3966203, 3966226, 3977953, 3978005 3980725, 3980737, 3980791, 3980810, 3980884, 3981160, 3981182, 3984702 3984759, 3984780, 4005175, 4005457, 4029846, 4030230, 4030296, 4030298 4030314, 4030336, 4030378, 4030434, 4030446, 4030541, 4032086, 4032192 4032215, 4032242, 4032245, 4032266, 4032293, 4032332, 4032335, 4032343 4032365, 4032713, 4032870, 4033003, 4033006, 4033170, 4033178, 4033231 4033240, 4033241, 4033242, 4033245, 4033249, 4033255, 4033359, 4033382 4033392, 4033958, 4034032, 4034039, 4034044, 4034054, 4034064, 4034069 4034095, 4034111, 4034132, 4034134, 4034139, 4034147, 4034167, 4034192 4034231, 4034237, 4034261, 4060077, 4060130, 4060144, 4060163, 4060171 4060243, 132508, 1875294, 1963153, 2590425, 2766897, 3995229.

Examples

The present invention will now be described in detail with reference to several examples thereof. However, these exam ^¬ ples are illustrative and do not limit the scope of the in ^¬ vention . Example 1

Whole genome sequencing was carried out in addition to clas ^¬ sical antimicrobial susceptibility testing of the same iso ^¬ lates for a cohort of 583 specimens of Proteus species, par- ticularly Proteus mirabilis, Proteus penneri and Proteus vul ^¬ garis. This allowed performing genome wide correlation stud ^¬ ies to find genetic variants (e.g. point mutations, small in ^¬ sertions and deletion, larger structural variants, plasmid copy number gains, gene dosage effects) in the genome and plasmids that are significantly correlated to the resistance against one or several drugs. The approach also allows for comparing the relevant sites in the genome to each other.

In the approach the different sources of genetic resistance as well as the different ways of how bacteria can become re ^¬ sistant were covered. By measuring clinical isolates collect ^¬ ed in a broad geographical area and across a broad time span of three decades a complete picture going far beyond the ra ^¬ ther artificial step of laboratory generated resistance mech- anisms was tried to be generated.

To this end, a set of 21 clinically relevant antimicrobial agents with 5 different modes of action was put together, and the minimally inhibitory concentration (MIC) of the 21 drugs for the Proteus isolates was measured.

The detailed procedure is given in the following:

Bacterial Strains

The inventors selected 583 Proteus strains from the microbi ^¬ ology strain collection at Siemens Healthcare Diagnostics (West Sacramento, CA) for susceptibility testing and whole genome sequencing. Antimicrobial Susceptibility Testing (AST) Panels Frozen reference AST panels were prepared following Clinical Laboratory Standards Institute (CLSI) recommendations. The following antimicrobial agents (with yg/ml concentrations shown in parentheses) were included in the panels: Amoxicil- lin/K Clavulanate (0.5/0.25-64/32), Ampicillin (0.25-128), Ampicillin/Sulbactam (0.5/0.25-64/32), Aztreonam (0.25-64), Cefazolin (0.5-32), Cefepime (0.25-64), Cefotaxime (0.25- 128), Ceftazidime (0.25-64), Ceftriaxone (0.25-128), Cefurox- ime (1-64), Cephalothin (1-64), Ciprofloxacin (0.015-8),

Ertepenem (0.12-32), Gentamicin (0.12-32), Imipenem (0.25- 32), Levofloxacin (0.25-16), Meropenem (0.12-32),

Piperacillin/Tazobactam (0.25/4-256/4), Tetracycline (0.5- 64), Tobramycin (0.12-32), and Trimethoprim/Sulfamethoxazole (0.25/4.7-32/608). Prior to use with clinical isolates, AST panels were tested with QC strains. AST panels were consid ^¬ ered acceptable for testing with clinical isolates when the QC results met QC ranges described by CLSI16. Inoculum Preparation

Isolates were cultured on trypticase soy agar with 5% sheep blood (BBL, Cockeysville, Md.) and incubated in ambient air at 35±1 ^° C for 18-24 h. Isolated colonies (4-5 large colonies or 5-10 small colonies) were transferred to a 3 ml Sterile Inoculum Water (Siemens) and emulsified to a final turbidity of a 0.5 McFarland standard. 2 ml of this suspension was add ^¬ ed to 25 ml Inoculum Water with Pluronic-F (Siemens) . Using the Inoculator (Siemens) specific for frozen AST panels, 5 μΐ of the cell suspension was transferred to each well of the AST panel. The inoculated AST panels were incubated in ambi ^¬ ent air at 35±1 ^° C for 16-20 h. Panel results were read visu ^¬ ally, and minimal inhibitory concentrations (MIC) were deter ^¬ mined . DNA extraction

Four streaks of each Gram-negative bacterial isolate cultured on trypticase soy agar containing 5% sheep blood and cell suspensions were made in sterile 1.5 ml collection tubes con- taining 50 μΐ Nuclease-Free Water (AM9930, Life Technolo ^¬ gies) . Bacterial isolate samples were stored at -20 °C until nucleic acid extraction. The Tissue Preparation System (TPS) (096D0382-02_01_B, Siemens) and the VERSANT® Tissue Prepara ^¬ tion Reagents (TPR) kit (10632404B, Siemens) were used to ex- tract DNA from these bacterial isolates. Prior to extraction, the bacterial isolates were thawed at room temperature and were pelleted at 2000 G for 5 seconds. The DNA extraction protocol DNAext was used for complete total nucleic acid ex ^¬ traction of 48 isolate samples and eluates, 50 μΐ each, in 4 hours. The total nucleic acid eluates were then transferred into 96-Well qPCR Detection Plates (401341, Agilent Technolo ^¬ gies) for RNase A digestion, DNA quantitation, and plate DNA concentration standardization processes. RNase A (AM2271, Life Technologies) which was diluted in nuclease-free water following manufacturer's instructions was added to 50 μΐ of the total nucleic acid eluate for a final working concentra ^¬ tion of 20 μg/ml. Digestion enzyme and eluate mixture were incubated at 37 °C for 30 minutes using Siemens VERSANT® Am ^¬ plification and Detection instrument. DNA from the RNase di- gested eluate was quantitated using the Quant-iT™ PicoGreen dsDNA Assay (P11496, Life Technologies) following the assay kit instruction, and fluorescence was determined on the Sie ^¬ mens VERSANT® Amplification and Detection instrument. Data analysis was performed using Microsoft® Excel 2007. 25 μΐ of the quantitated DNA eluates were transferred into a new 96- Well PCR plate for plate DNA concentration standardization prior to library preparation. Elution buffer from the TPR kit was used to adjust DNA concentration. The standardized DNA eluate plate was then stored at -80°C until library prepara ^¬ tion .

Next Generation Sequencing

Prior to library preparation, quality control of isolated bacterial DNA was conducted using a Qubit 2.0 Fluorometer (Qubit dsDNA BR Assay Kit, Life Technologies) and an Agilent 2200 TapeStation (Genomic DNA ScreenTape, Agilent Technolo ^¬ gies) . NGS libraries were prepared in 96 well format using NexteraXT DNA Sample Preparation Kit and NexteraXT Index Kit for 96 Indexes (Illumina) according to the manufacturer's protocol. The resulting sequencing libraries were quantified in a qPCR-based approach using the KAPA SYBR FAST qPCR

MasterMix Kit (Peqlab) on a ViiA 7 real time PCR system (Life Technologies) . 96 samples were pooled per lane for paired-end sequencing (2x lOObp) on Illumina Hiseq2000 or Hiseq2500 se ^¬ quencers using TruSeq PE Cluster v3 and TruSeq SBS v3

sequncing chemistry (Illumina). Basic sequencing quality parameters were determined using the FastQC quality control tool for high throughput sequence data (Babraham Bioinformat- ics Institute) .

Data analysis

Raw paired-end sequencing data for the 583 Proteus samples were mapped against the Proteus reference (NC_010554) with

BWA 0.6.1.20. The resulting SAM files were sorted, converted to BAM files, and PCR duplicates were marked using the Picard tools package 1.104 (http://picard.sourceforge.net/). The Ge ^¬ nome Analysis Toolkit 3.1.1 (GATK) 21 was used to call SNPs and indels for blocks of 200 Proteus samples (parameters: - ploidy 1 -glm BOTH -stand_call_conf 30 -stand_emit_conf 10) . VCF files were combined into a single file and quality fil ^¬ tering for SNPs was carried out (QD < 2.0 | | FS > 60.0 I I MQ < 40.0) and indels (QD < 2.0 | | FS > 200.0). Detected vari ^¬ ants were annotated with SnpEff22 to predict coding effects. For each annotated position, genotypes of all Proteus samples were considered. Proteus samples were split into two groups, low resistance group (having lower MIC concentration for the considered drug) , and high resistance group (having higher MIC concentrations) with respect to a certain MIC concentra ^¬ tion (breakpoint) . To find the best breakpoint all thresholds were evaluated and p-values were computed with Fisher' s exact test relying on a 2x2 contingency table (number of Proteus samples having the reference or variant genotype vs. number of samples belonging to the low and high resistance group) . The best computed breakpoint was the threshold yielding the lowest p-value for a certain genomic position and drug. For further analyses positions with non-synonymous alterations and p-value < 10 ^~10 were considered.

Since a potential reason for drug resistance is gene duplica ^¬ tion, gene dose dependency was evaluated. For each sample the genomic coverage for each position was determined using BED

Tools. Gene ranges were extracted from the reference assembly NC_010554. gff and the normalized median coverage per gene was calculated. To compare low- and high-resistance isolates the best area under the curve (AUC) value was computed. Groups of at least 20% of all samples having a median coverage larger than zero for that gene and containing more than 15 samples per group were considered in order to exclude artifacts and cases with AUC > 0.75 were further evaluated. To include data on the different ways how resistance mecha ^¬ nisms are acquired Proteus isolates collected over more than three decades were analyzed such that also horizontal gene transfer could potentially be discovered. In detail, the following steps were carried out:

Proteus strains to be tested were seeded on agar plates and incubated under growth conditions for 24 hours. Then, colo ^¬ nies were picked and incubated in growth medium in the pres- ence of a given antibiotic drug in dilution series under growth conditions for 16-20 hours. Bacterial growth was de ^¬ termined by observing turbidity.

Next mutations were searched that are highly correlated with the results of the phenotypic resistance test.

For sequencing, samples were prepared using a Nextera library preparation, followed by multiplexed sequencing using the Illuminat HiSeq 2500 system, paired end sequencing. Data were mapped with BWA (Li H. and Durbin R. (2010) Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinfor- matics, Epub . [PMID: 20080505] ) and SNP were called using samtools (Li H.*, Handsaker B.*, Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. and 1000 Ge- nome Project Data Processing Subgroup (2009) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics , 25, 2078-9. [PMID: 19505943] ) .

As reference genome, NC_010554 as annotated at the NCBI was determined as best suited.

The mutations were matched to the genes and the amino acid changes were calculated. Using different algorithms (SVM, ho ^¬ mology modeling) mutations leading to amino acid changes with likely pathogenicity / resistance were calculated.

In total, whole genomes and plasmids of 583 different clini ^¬ cal isolates of Proteus species were sequenced, and classical antimicrobial susceptibility testing (AST) against 21 therapy forms as described above was performed for all organisms. From the classical AST a table with 583 rows (isolates) and 21 columns (MIC values for 21 drugs) was obtained. Each table entry contained the MIC for the respective isolate and the respective drug. The genetic data were mapped to different reference genomes of Proteus that have been annotated at the NCBI (http://www.ncbi.nlm.nih.gov/), and the best reference was chosen as template for the alignment - NC_010554 as anno- tated at the NCBI. Additionally, assemblies were carried out and it was verified that the sequenced genomes fulfil all quality criteria to become reference genomes.

Next, genetic variants were evaluated. This approach resulted in a table with the genetic sites in columns and the same isolates in 583 rows. Each table entry contained the genetic determinant at the respective site (A, C, T, G, small inser ^¬ tions and deletions, ...) for the respective isolate. In a next step different statistical tests were carried out

1) For comparing resistance / susceptibility to genetic

sites we calculated contingency tables and determined the significance using Fishers test

2) For comparing different sites to each other we calculat- ed the correlation between different genetic sites

3) For detecting gene dosage effects, e.g. loss or gain of genes (in the genome or on plasmids) we calculated the coverage (i.e. how many read map to the current posi ^¬ tion) at each site for resistant and not resistant iso- lates.

From the data, first the 21 genes with the best p-value were chosen for the list of mutations as well as the list of cor- related antibiotic resistance, representing Tables 1 and 2. As for a lot of genes the p-values were very low, also the next p-values up to l,04565E-62 were considered, leading to the genes in Table 13, respectively the gene positions dis- closed with regard to the 22 ^nd , 23 ^rd and/or 24 ^th aspect.

A full list of all genetic sites, drugs, drug classes, af ^¬ fected genes etc. is provided in Tables 3 and 4a, 4b and 4c, wherein Table 3 corresponds to Table 1 and represents the genes having the lowest p-values after determining mutations in the genes, and Table 4, respectively Tables 4a, 4b and 4c correspond to Table 2 and represent the genes having the low ^¬ est p-values after correlating the mutations with antibiotic resistance .

In addition, the data with the best p-values for each antibi ^¬ otic class with the most antibiotic drugs, as well as each antibiotic, respectively, were evaluated, being disclosed in Tables 5 - 10.

In Tables 3 - 10 the columns are designated as follows:

Gene name: affected gene;

POS : genomic position of the SNP / variant in the Proteus reference genome (see above) ;

p-value: significance value calculated using Fishers exact test (determined according to FDR (Benjamini Hochberg) method (Benjamini Hochberg, 1995));

genbank protein accession number: (NCBI) Accession number of the corresponding protein of the genes Table 3: Detailed results for the genes in Example 1 (corresponding to Table

*: (tetracycline)

Table 4a: Detailed results for the genes in Example 1 (corresponding to Table

*: (tetracycline)

Table 4b: Detailed results for the genes in Example 1 (corresponding to Table 2, continued)

POS best #significant #significant #significant #significant #significant drug Lactams fluoroquinolones aminoglycosides polyketide other (benzene

(tetracycline) derived) / sulfonamide

2562578 CP 9 2 1 0 1

3741905 CFZ 8 2 0 1 0

131826 CFZ 8 2 0 1 0

1482764 CFZ 8 2 0 1 0

1771087 CFZ 8 2 0 1 0

1771119 CFZ 8 2 0 1 0

1918241 CFZ 8 2 0 1 0

1968294 CFZ 8 2 0 1 0

2238063 CFZ 8 2 0 1 0

2238072 CFZ 8 2 0 1 0

2238088 CFZ 8 2 0 1 0

2238090 CFZ 8 2 0 1 0

2454709 TE 7 2 0 1 0

3039125 TE 7 2 0 1 0

3221491 CFZ 8 2 0 1 0

3221494 CFZ 8 2 0 1 0

3422635 TE 7 2 0 1 1

4059624 CFZ 8 2 0 1 0

4059634 CFZ 8 2 0 1 0

4060202 CFZ 8 2 0 1 0

131835 CFZ 8 2 0 1 0

Table 4c: Detailed results for the genes in Example 1 (corresponding to Table 2, continued)

Also the antibiotic/drug classes, the number of significant antibiotics correlated to the mutations (over all antibiotics or over certain classes) , as well as the correlated antibiot ^¬ ics are denoted in the Tables.

The p-value was calculated using the Fisher exact test based on contingency table with 4 fields: #samples Resistant / wild type; #samples Resistant / mutant; #samples not Resistant / wild type; #samples not Resistant / mutant

The test is based on the distribution of the samples in the 4 fields. Even distribution indicates no significance, while clustering into two fields indicates significance. The following results were obtained

- A total of 27.140 different correlations between genetic sites and anti-microbial agents were detected (p-value < 10 ^{~ 10} ).

- The biggest part of these were point mutations (i.e. single base exchanges)

- The highest significance (10 ^~71 ) was reached for a non- synonymous coding in YP_002152062.1, particular in position 2562578 with regard to reference genome NC_010554 as annotat ^¬ ed at the NCBI, which is a non-synonymous coding, particular- ly a codon change aGc/aTc

- Besides these, insertions or deletions of up to four bases were discovered

- Further, potential genetic tests for five different drug classes relating to resistances were discovered

· β-lactams (includes Penicillins, Cephalosporins,

Carbapenems, Monobactams )

• Quinolones, particularly Fluoroquinolones

• Aminoglycosides • Polyketides, particularly Tetracyclines

• Folate synthesis inhibitors

- Potential genetic tests for all tested drugs/drug combina ^¬ tions were discovered:

Amoxicillin/Clavulanate, Ampicillin, Ampicillin/Sulbactam, Aztreonam, Cefazolin, Cefepime, Ceftazidime, Cefuroxime, Cephalothin, Imipenem, Piperacillin/Tazobactam, Ciprofloxacin, Levofloxacin, Gentamycin, Tobramycin, Tetracycline, Trimethoprim/Sulfamethoxazol

- Mutations were observed in 2.223 different genes

Although some strains of Proteus are sensitive to ampicillin and cephalosporins, we observed a high resistance against these and other anti-bacterial agents.

A genetic test for the combined pathogen identification and antimicrobial susceptibility testing direct from the patient sample can reduce the time-to actionable result significantly from several days to hours, thereby enabling targeted treat ^¬ ment. Furthermore, this approach will not be restricted to central labs, but point of care devices can be developed that allow for respective tests. Such technology along with the present methods and computer program products could revolu- tionize the care, e.g. in intense care units or for admis ^¬ sions to hospitals in general. Furthermore, even applications like real time outbreak monitoring can be achieved using the present methods. Instead of using only single variants, a combination of sev ^¬ eral variant positions can improve the prediction accuracy and further reduce false positive findings that are influ ^¬ enced by other factors . Compared to approaches using MALDI-TOF MS, the present ap ^¬ proach has the advantage that it covers almost the complete genome and thus enables us to identify the potential genomic sites that might be related to resistance. While MALDI-TOF MS can also be used to identify point mutations in bacterial proteins, this technology only detects a subset of proteins and of these not all are equally well covered. In addition, the identification and differentiation of certain related strains is not always feasible.

The present method allows computing a best breakpoint for the separation of isolates into resistant and susceptible groups. The inventors designed a flexible software tool that allows to consider - besides the best breakpoints - also values de- fined by different guidelines (e.g. European and US guide ^¬ lines) , preparing for an application of the GAST in different countries .

The inventors demonstrate that the present approach is capa- ble of identifying mutations in genes that are already known as drug targets, as well as detecting potential new target sites .

The current approach enables

a. Identification and validation of markers for genetic

identification and susceptibility/resistance testing within one diagnostic test

b. validation of known drug targets and modes of action c. detection of potentially novel resistance mechanisms

leading to putative novel target / secondary target genes for new therapies