ES2817896A1 - METHOD FOR THE DETECTION OF PSEUDOMONAS AERUGINOSA ST175 (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Method for the detection of Pseudomonas aeruginosa ST175. The present invention relates to an in vitro method of diagnosis of an infection caused by Pseudomonas aeruginosa ST175 characterized by comprising the identification of the nucleotide sequence SEQ ID NO: 1 or, alternatively, of a fragment of it or, alternatively, of a sequence with at least 99% identity with the nucleotide sequence SEQ ID NO: 1 or, alternatively, of an expression product thereof. It also refers to an in vitro method of decision or recommendation of an antibiotic treatment that includes the detection of said sequences, as well as their use and a kit that includes the specific tools to be identified. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Method for the detection of Pseudomonas aerugínosa ST175

The present invention relates to an in vitro method of diagnosis of Pseudomonas aeruginosa ST175 in a biological sample. It also refers to an in vitro method of recommending an antibiotic treatment in a patient with P. aerugínosa infection. Said method is useful in treating an infection caused by said bacteria, making it useful in the field of medicine.

STATE OF THE ART

Pseudomonas aeruginosa is a nosocomial opportunistic Gram-negative pathogenic bacterium that causes infections in humans. It is frequently isolated in bacteremia, wound infections, pneumonia, intra-abdominal sepsis, urogenital sepsis, in patients with indwelling catheterization or who have undergone surgery. P. aerugínosa is the main cause of chronic lung colonization in patients with cystic fibrosis, in bronchiectasis and in other chronic obstructive pulmonary diseases. It is of special relevance in immunosuppressed patients, especially neutropenic.

The prevalence of nosocomial infections caused by multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of P. aerugínosa is increasing.

P. aerugínosa ST175 ( P. aerugínosa "sequence type" 175) is part of the so-called high-risk clones of P. aerugínosa together with clones ST111 and ST235 (Woodford N et al. 2011; Oliver A, et al 2015). In multicenter studies of bacteremia isolates in Spain, it has been found that almost 85% of multiresistant and extensive resistance isolates (MDR / XDR) belong to clone ST175 (Peña C, et al. 2015; Cabot G, et al. 2012 ; and Mulet X, et al. 2013) P. aerugínosa ST175, associated with serotype O4, is widely distributed in multiple European countries, especially in Spain and France.

Infections with P. aerugínosa are frequently fatal and often difficult to treat due to the susceptibility of P. aerugínosa to a limited number of antimicrobial agents, and the frequent development of resistance during therapy (Carmeli Y, et al. 1999 (a) ).

P. aerugínosa ST175 presents a defined phenotypic resistance profile, characterized by the sensitivity only to a few antibiotics, for example, colistin and ceftolozane / tazobactam (Mensa J, et al.

2018). The study of the mechanisms responsible for the phenotype in multiresistant isolates belonging to clone ST175 have determined a series of specific mutations of said clone, for example, the mutation in AmpR (G154R) that leads to the hyperexpression of AmpC, the mutational inactivation of porin membrane OprD leading to carbapenem resistance and mutations in the Quinolone Resistance Determinant Regions (QRDR) of GyrA (T83I and D87N) (Cabot G, et al. 2012).

When there is a P. aeruginosa infection, the patient's treatment should begin as soon as possible, since suboptimal treatments can result in the development of antibiotic resistance and a negative clinical result, which occurs frequently (Carmeli Y, et al. 1999 (a); Carmeli Y, et al.

1999 (b)). Therefore, a rapid identification of P. aeruginosa isolates is of great importance, especially those belonging to high-risk clones, mainly ST175 (Oliver A, et al. 2015), at the time of selection and administration. of an adequate treatment to combat said infection and one of the key factors to increase the rates of therapeutic success in the treatment and control of the infection.

Several approaches have been developed that have tried to solve this problem, among which we can find serotyping (based on the agglutination reaction of various sera with the O antigen), identification by MALDI-TOF MS analysis (desorption / ionization matrix-assisted laser - Time of flight) and the combination of both (Cabrolier N, et al. 2015; and Mulet X, et al.

2019). However, these techniques require the culture and growth of the bacteria for subsequent analysis, which delays the possibility of selecting the optimal treatment for this type of infection and the early identification of those isolates belonging to clone ST175. On the other hand, genetic markers of resistance specific to clone ST175 have been reported to try to use them as a biomarker for the identification of isolates belonging to clone ST175 (Cabot G, et al.

2012), however, the need for sequencing hinders and slows down the identification of isolates.

Therefore, there is a need to find a rapid and robust identification method of the P. aeruginosa clone ST175 in clinical samples in order to provide the patient with an adequate rapid treatment, thus also avoiding the appearance of resistance against antibiotics.

EXPLANATION OF THE INVENTION

Researchers have developed a tool to identify P. aeruginosa ST175 from quickly and reliably both in isolates of the bacteria and in clinical samples, which allows a rapid diagnosis of infection by said pathogen.

Researchers have found specific polynucleotides and proteins of the ST175 clone genome that allow rapid, sensitive and specific identification of said pathogen. Among them, the genomic region SEQ ID NO: 1 has been found. In the present invention, the identification of the region SEQ ID NO: 1 has been exemplified by detecting the polynucleotide sequence called SEQ ID NO: 2. In the region SEQ ID NO: 1 the specific genomic DNAs of clone ST175 called SEQ ID NO: 3 and SEQ ID NO: 5 have been found, which encode the proteins called SEQ ID NO: 4 and SEQ ID NO: 6, respectively, which have been found which are also specific to clone ST175. The polynucleotides of sequence SEQ ID NO: 7 and SEQ ID NO: 8 have also been found to be specific for clone ST175, where the latter encodes the protein SEQ ID NO: 9, also specific for said clone. In addition, the polynucleotides SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14 that encode, respectively, for the specific proteins of clone ST175 SEQ ID NO: 11, SEQ ID have also been found to be specific for clone ST175 NO: 13 and SEQ ID NO: 15.

By detecting the sequence SEQ ID NO: 1, the researchers have been able to identify clone ST175 with a specificity and a sensitivity of 100%, in clinical samples such as urine, respiratory samples and blood cultures (see example 3). Furthermore, the identification of said pathogen is more sensitive and specific compared to the use of specific biomarkers of the clone ST175 already known and compared to pathogen detection techniques, such as agglutination and MALDI-TOF (see examples 2 and 3).

Therefore, a first aspect of the invention refers to an in vitro method of diagnosis of an infection caused by Pseudomonas aeruginosa ST175 in an isolated sample from a subject, characterized by comprising the identification of the nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, of an expression product of any of them.

A particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided below, refers to an in vitro method of diagnosis of an infection caused by Pseudomonas aeruginosa ST175 in an isolated sample from a subject, characterized by comprising the identification of the nucleotide sequence SEQ ID NO: 1 or, alternatively, of a fragment thereof or, alternatively, of a sequence with at least 99% identity with the nucleotide sequence SEQ ID NO: 1; or, alternatively, of an expression product thereof.

Pseudomonas aeruginosa ST175 ("ST175" or "clone ST175" in the present invention), is defined as that strain of P. aeruginosa that presents the following alleles defined by the "multilocus sequence typing" (MLST) scheme developed by Curran B, et al 2004: acsA_28; aroE_22; guaA_5; mutL_3; nuoD_3; ppsA_14 and trpE_19. Various isolates belonging to this clone have been extensively characterized previously by the person skilled in the art, for example, in Cabot G, et al. 2012 and with sequences complete data published in Viedma E, et al. 2013 (NCBI Reference Sequence NZ_AOIH00000000.1) or Cabot G, et al. 2016 ( European Nucleotide Archive Study Number ERP016726 and European Nucleotide Archive Accession Number ERS1280254 to ERS1280271 and ERS1280273 to ERS1280276.)

In the present invention, the term "identity" refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. If, in optimal alignment, a position in a first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, the sequences exhibit identity with respect to that position. The level of identity between two sequences (or "percent sequence identity") is measured as a ratio of the number of identical positions shared by the sequences to the size of the sequences (that is, percent sequence identity = (number of identical positions / total number of positions) x 100).

A series of mathematical algorithms to quickly obtain the optimal alignment and calculate the identity between two or more sequences are known and incorporated in a number of available software programs. Examples of such programs include the MATCH-BOX, MULTAIN, GCG, FASTA, and ROBUST programs, among others. Preferred software analysis programs include the ALIGN, CLUSTAL W, and BLAST programs (eg, BLAST 2.1, BL2SEQ, and later versions thereof).

BLAST programs provide analysis of at least two sequences, either by aligning a selected sequence with multiple sequences in a database (eg, GenSeq), or, with BL2SEQ, between two selected sequences. BLAST programs are preferably modified by low complexity filter programs, such as DUST or SEG programs, which are preferably integrated into the operations of the BLAST program. BLAST programs and principles are described in more detail in, for example, Altschul ST, et al., "Basic local alignment search tool", 1990, J. Mol. Biol, 215: 403-410.

For multiple sequence analysis, the CLUSTAL W program can be used. Preferably, the CLUSTAL W program is run using the "dynamic" (rather than "fast") settings. Sequences can be evaluated using a variable set of BLOSUM matrices depending on the level of identity between the sequences. The CLUSTAL W program and the underlying operating principles are described in more detail, for example, in Higgins DG, et al., "CLUSTAL V: improved software for multiple sequence alignment", 1992, CABIOS, 8 (2): 189- 191.

In the present invention, the terms "nucleotide sequence", "polynucleotide", "nucleic acid sequence" and "nucleic acid" are used interchangeably.

A particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided above or below, the in vitro method further comprises the identification of one or more nucleotide sequences that are selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of said sequences; or, alternatively, of an expression product of any of them; or, alternatively, one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; or, alternatively, of any combinations of them.

In a particular embodiment of the first aspect of the invention, optionally in combination with any particular embodiment provided above or below, the expression product of SEQ ID NO: 1 is SEQ ID NO: 4 or SEQ ID NO: 6.

When the sequence (s), fragment or expression product defined in the first aspect of the invention, for example, of SEQ ID NO: 1, is identified in an isolated sample from the subject, it is inferred that the bacterium is resistant to most commonly used conventional anti-pseudomonics (antibiotics), eg, ceftazidime, piperacillin in combination with tazobactam (piperacillin / tazobactam), cefepime, imipenem, meropenem, gentamicin, tobramycin, and ciprofloxacin; but which is sensitive to colistin, ceftolozane in combination with tazobactam (ceftolozane / tazobactam), ceftazidime in combination with avibactam (ceftazidime / avibactam) or amikacin. Therefore, this information can be used in making decisions for the selection of the most appropriate treatment.

For this reason, a second aspect of the invention refers to an in vitro method of decision or recommendation of an antibiotic treatment in a subject with an infection by Pseudomonas aeruginosa, characterized in that the method comprises the identification of Pseudomonas aeruginosa ST175 by identifying of the nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, of an expression product of any of them; where when the sequence (s), fragment or expression product is identified, this is indicative that the antibiotic to be administered is colistin, ceftolozane in combination with tazobactam, ceftazidime in combination with avibactam, amikacin, or any combination of them.

A particular embodiment of the second aspect of the invention, optionally in combination with any particular embodiment provided above or below, refers to an in vitro method of deciding or recommending an antibiotic treatment in a subject with an infection by Pseudomonas aeruginosa, characterized in that the The method comprises the identification of Pseudomonas aeruginosa ST175 by identifying the nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, of an expression product of any of them; where when the sequence (s), fragment or expression product is not identified, this is indicative that the antibiotic to be administered is, for example, ceftazidime, piperacillin / tazobactam, cefepime, imipenem, meropenem, gentamicin, tobramycin, or ciprofloxacin, or any combination of them, or any combination of them.

A particular embodiment of the second aspect of the invention, optionally in combination with any particular embodiment provided above or below, refers to an in vitro method of deciding or recommending an antibiotic treatment in a subject with an infection by Pseudomonas aeruginosa, characterized in that The method comprises the identification of Pseudomonas aeruginosa ST175 by identifying the nucleotide sequence SEQ ID NO: 1 in an isolated sample from the subject; or, alternatively, of a fragment thereof, or, alternatively, of a sequence with at least 99% identity with the nucleotide sequence SEQ ID NO: 1 or, alternatively, of an expression product thereof; where, when the sequence (s), the fragment or the expression product of SEQ ID NO: 1 the antibiotic to be administered is colistin, ceftolozane in combination with tazobactam, ceftazidime in combination with avibactam, amikacin, or any combination of them (Mensa J, et al. 2018)

The amounts to be administered of each antibiotic are those known to those skilled in the art, for example, those described in Mensa J, et al. 2018. For example, when piperacillin is used in combination with tazobactam, 2g of piperacillin and 0.25g of tazobactam can be used. For example, when ceftazidime is used in combination with avibactam, 2g of ceftazidime and 0.25g of avibactam can be used.

In a particular embodiment of the second aspect of the invention, optionally in combination with any particular embodiment provided above or below, the in vitro method further comprises the identification of one or more nucleotide sequences that are selected from the group consisting of: SEQ ID NO : 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of said sequences; or, alternatively, of an expression product of any of them; or, alternatively, one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; or, alternatively, of any combinations of them; where, when the sequence (s), fragment or expression product of SEQ ID NO: 7 is identified, the antibiotic to be administered is colistin, ceftolozane in combination with tazobactam, ceftazidime in combination with avibactam, amikacin or any combination from them.

In a particular embodiment of the second aspect of the invention, optionally in combination with any particular embodiment provided above or below, when the sequence (s), the fragment or the expression product defined in the first are not identified Aspect of the invention, the antibiotic to be administered is, for example, ceftazidime, piperacillin / tazobactam, cefepime, imipenem, meropenem, gentamicin, tobramycin, or ciprofloxacin, or any combination thereof.

In a particular embodiment of the second aspect of the invention, optionally in combination with any particular embodiment provided above or below, when the sequence (s), the fragment or the expression product of SEQ is (are) not identified ID NO: 1, the antibiotic to be administered is, for example, ceftazidime, piperacillin / tazobactam, cefepime, imipenem, meropenem, gentamicin, tobramycin, or ciprofloxacin, or any combination of them.

In a particular embodiment of the first or second aspect of the invention, optionally in combination with any particular embodiment provided above or below, the fragment of the nucleotide sequence SEQ ID NO: 1 comprises, in one example, consists of, the sequence of nucleotides SEQ ID NO: 2. In another particular embodiment, the fragment of the nucleotide sequence SEQ ID NO: 1 comprises, in one example, consists of the nucleotide sequence SEQ ID NO: 3 or SEQ ID NO: 5.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the identification is made of the genomic DNA of the microorganism, for example SEQ ID NO: 1, SEQ ID NO : 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any combination thereof.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the expression product is messenger RNA (mRNA).

When the expression product is mRNA, the detection can be carried out by any method known to the person skilled in the art, provided that said method allows the detection or quantification of the mRNA in the biological sample. Examples of these procedures include polymerase chain reaction (PCR), quantitative real-time PCR (QPCR), multiplex PCR, Nucleic Acid Sequence Based Amplification (NASBA), ligase chain reaction (LCR ), reverse transcriptase polymerase chain reaction (RT-PCR), RNA sequencing, Northern blotting, hybridization (eg, by nucleic acid chips, mircoarrays), or combinations thereof. For example, mRNA determination is done by using specific primers and / or probes to detect and / or quantify the mRNA of interest.

In most of the RNA detection and quantification methods mentioned above, before performing this procedure it is necessary to convert the RNA into complementary DNA (cDNA). This conversion is carried out by techniques known to those skilled in the art, such as reverse transcription, among others. For this reason, in a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the expression product is messenger RNA and is identified by detecting its complementary DNA .

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the expression product is a protein.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any embodiment provided above or below, the expression product of the nucleotide sequence SEQ ID NO: 1 is a protein, in one example, where the protein is SEQ ID NO: 4 and / or, SEQ ID NO: 6.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the identification of the nucleotide sequence (s) or their fragments is carried out in the Pseudomonas aeruginosa ST175 DNA comprised in the isolated sample from the subject.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the identification of the nucleotide sequence (s) or their fragments or the product of When this expression is mRNA, it is carried out using a technique based on an amplification reaction, for example by polymerase chain reaction (PCR), Nucleic Acid Sequence Based Amplification (NASBA) or ligase chain reaction (LCR). Detection can also be carried out by hybridization, for example by means of DNA chips ( microarrays) comprising specific probes.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the PCR is selected from the group consisting of: PCR, quantitative real-time PCR (qPCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR and multiplex PCR.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the PCR comprises the use of at least one pair of primers that are selected from the list consisting of : SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, and SEQ ID NO: 26 and SEQ ID NO: 27, or any combination thereof.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the PCR comprises the use of at least the pair of primers SEQ ID NO: 16 and SEQ ID NO : 17; or alternatively, the primer pair SEQ ID NO: 24 and SEQ ID NO: 25; or, alternatively, the primer pair SEQ ID NO: 26 and SEQ ID NO: 27.

The term "biological sample" includes, but is not limited to, biological tissues and / or fluids of an individual, obtained by any method designed for that purpose known to those skilled in the art. The biological sample comprises a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO : 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, of an expression product of any of them; In one example, the sample comprises SEQ ID NO: 1; or, alternatively, a fragment of it; or, alternatively, a sequence with at least 99% identity with the sequence SEQ ID NO: 1 or, alternatively, of an expression product of said sequence.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the isolated sample is an isolated biological sample that is selected from the list consisting of: tissue, blood , plasma, serum, lymph, bronchoalveolar lavage, saliva, urine, or ascitic fluid.

In the present invention, the terms "subject", "patient" and "individual" are used interchangeably. The subject can be of any age, gender or race.

In the present invention the subject is a mammal, such as a mouse, rat, guinea pig, rabbit, dog, cat, bovine, horse, goat, sheep, primate or human being, preferably it is a human being.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the subject is an immunosuppressed subject, for example, is an immunosuppressed subject presenting neutropenia.

In a particular embodiment of the in vitro method of the first and second aspects of the invention, optionally in combination with any particular embodiment provided above or below, the subject is a subject with cystic fibrosis.

A third aspect of the invention relates to a use of a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, of an expression product of any of them; as a diagnostic marker of an infection caused by Pseudomonas aeruginosa ST175; or to decide or recommend an antibiotic treatment in a subject with an infection caused by Pseudomonas aeruginosa.

A particular embodiment of the third aspect of the invention, optionally in combination with any particular embodiment provided above or below, refers to a use of the nucleotide sequence SEQ ID NO: 1 or, alternatively, of a fragment thereof; or, alternatively, of a sequence with at least 99% identity with the sequence SEQ ID NO: 1; or, alternatively, of an expression product thereof; as a diagnostic marker of an infection caused by Pseudomonas aeruginosa ST175; or to decide or recommend an antibiotic treatment in a subject with an infection caused by Pseudomonas aeruginosa.

In a particular embodiment of the third aspect of the invention, optionally in combination with any particular embodiment provided above or below, in addition one or more nucleotide sequences are used that are selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO : 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of said sequences; or, alternatively, of an expression product of any of them; or, alternatively, one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; or, alternatively, of any combinations of them; as a diagnostic marker of an infection caused by Pseudomonas aeruginosa ST175; or to decide or recommend an antibiotic treatment in a subject with an infection caused by Pseudomonas aeruginosa.

All the particular embodiments of the first and second aspects of the invention form part of the third aspect of the invention.

A fourth aspect of the invention refers to a use of specific tools to identify a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, of an expression product of any of them, in any of the in vitro methods of the first and second aspects of the invention

A particular embodiment of the fourth aspect of the invention, optionally in combination with any particular embodiment provided above or below, refers to the use of specific tools to identify the sequence SEQ ID NO: 1; or, alternatively, a fragment of it; or, alternatively, a sequence with at least 99% identity to the sequence SEQ ID NO: 1; or, alternatively, an expression product thereof, in any of the in vitro methods of the first and second aspects of the invention.

In a particular embodiment of the fourth aspect of the invention, optionally in combination with any embodiment provided above or below, one or more nucleotide sequences are further identified that are selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of said sequences; or, alternatively, of an expression product of any of them; or, alternatively, one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; or, alternatively, of any combinations of them.

In a particular embodiment of the fourth aspect of the invention, optionally in combination with any embodiment provided above or below, the specific tools are part of a kit.

A fifth aspect of the invention refers to a kit to identify Pseudomonas aeruginosa ST175, which comprises the specific tools to identify a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, an expression product of any of them.

A particular embodiment of the fifth aspect of the invention, optionally in combination with any embodiment provided above or below, refers to a kit to identify Pseudomonas aeruginosa ST175, which comprises the specific tools to identify the sequence SEQ ID NO: 1; or, alternatively, a fragment of it; or, alternatively, a sequence with at least 99% identity to the sequence SEQ ID NO: 1; or, alternatively, an expression product thereof.

In a particular embodiment of the fifth aspect of the invention, optionally in combination with any embodiment provided above or below, the kit further comprises the specific tools to identify one or more nucleotide sequences that are selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of said sequences; or, alternatively, of an expression product of any of them; or, alternatively, one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; or, alternatively, of any combinations of them.

All the particular embodiments of the first, second and third aspects of the invention form part of the fourth and fifth aspects of the invention.

In a particular embodiment of the fourth and fifth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the specific tools for identifying the sequences are primers, probes or antibodies that specifically bind to the sequences or to the products. expression described in the present invention.

In a particular embodiment of the fourth and fifth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the specific tools to identify the nucleotide sequence SEQ ID NO: 1 is at least one pair of primers that are selected from the list consisting of: SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, and SEQ ID NO: 26 and SEQ ID NO: 27, or any combination thereof.

In a particular embodiment of the fourth and fifth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the specific tools to identify the nucleotide sequence SEQ ID NO: 1 are the primer pair SEQ ID NO: 16 and SEQ ID NO: 17; or, alternatively, the primer pair SEQ ID NO: 18 and SEQ ID NO: 19; or, alternatively, the primer pair SEQ ID NO: 20 and SEQ ID NO: 21; or, alternatively, the primer pair SEQ ID NO: 22 and SEQ ID NO: 23.

Antibodies can be monoclonal or polyclonal. Antibodies can also be nanobodies.

There are well known means in the current state of the art to prepare and characterize antibodies. Methods for generating polyclonal antibodies are well known in the current state of the art. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition and collecting serum from the immunized animal. A wide range of animal species can be used to produce the antiserum. Animals typically used to produce antiserum can be rabbits, mice, rats, hamsters, guinea pigs, or goats.

On the other hand, monoclonal antibodies (MAbs) can be easily prepared using well-known techniques, such as those specified in US patent US 4,196,265. This technique usually consists of immunizing a suitable animal with a protein composition as an immunogen. The immunization composition is administered in an amount effective to stimulate the antibody-producing cells.

Monoclonal antibody preparation procedures are generally started in the same manner as polyclonal antibody preparation. Animals are injected with antigen as above. The antigen can be mixed with adjuvants, such as Freund's complete or incomplete adjuvant. Vaccination with the same antigen is repeated approximately every two weeks. After immunization, somatic cells with potential for antibody production, specifically B lymphocytes (B cells), are chosen for use in the MAb generation protocol. These cells can be obtained from a biopsy of the spleen, lymph nodes, or tonsils, or from peripheral blood samples. The antibody-producing B lymphocytes of the immunized animal are fused with cells of an immortal myeloma cell line, generally of the same species as the immunized animal. For example, myeloma cell lines that are suitable for use in fusion procedures for the production of hybridomas have high fusion efficiency and enzyme deficiencies that make them unable to grow in certain culture media that only support the growth of cells. desired fused cells (hybridomas).

A nanobody or single domain antibody (sdAb) is an antibody fragment consisting of a single domain of variable monomeric antibodies. It can be obtained by immunizing camelids or cartilaginous fish.

In a particular embodiment of the fourth and fifth aspects of the invention, optionally in combination with any particular embodiment provided above or below, the antibody is a humanized antibody, to prevent immunological reactions of a human organism against the antibody.

In a particular embodiment of the fourth and fifth aspects of the invention, optionally in combination with any of the embodiments provided above or below, the detection tools, for example, primers, probes or antibodies, can be located on a solid support for example, but not limited to, glass, plastic, tubes, multiwell plates, membranes, or any other known support.

In a particular embodiment of the fourth and fifth aspects of the invention, optionally in combination with any of the embodiments provided above or below, the specific tools to identify the sequence (s) comprise at least 10%, at least 20 %, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents to analyze sequences that make up the kit. Therefore, in the particular case of kits that comprise reagents to identify SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and / or SEQ ID NO: 15, the specific reagents of said sequences (for example, primers, probes and / or antibodies) comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50% , at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the specific tools present in the kit. These kits are therefore simplified kits that mainly include the reagent means for detecting the sequences of the present invention.

In a particular embodiment of the fifth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit can further comprise at least one DNA polymerase, a reverse transcriptase, an RNA polymerase, a fluorophore, a mixture of tri-deoxynucleotides -phosphate (dNTPs), a mixture of nucleotide tri-phosphate (NTPs), deoxyribonuclease (DNase), ribonuclease inhibitors (RNase), dithiothreitol (DTT), inorganic pyrophosphatase (PPi) and / or the necessary buffers for the enzymes provided in the kit.

In a particular embodiment of the fifth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit further comprises instructions (such as a brochure) with the indications for the use of the specific tools.

In a particular embodiment of the fifth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit further comprises at least one antibiotic, for example: ceftazidime, piperacillin, tazobactam, cefepime, imipenem, meropenem, gentamicin, Tobramycin, Ciprofloxacin, Colistin, Ceftolozane, Avibactam, Amikacin, or any of their combinations. For example, the kit comprises: colistin, amikacin, ceftolozane / tazobactam and / or ceftazidime / avibactam; or, alternatively, the kit comprises ceftazidime, piperacillin / tazobactam, cefepime, imipenem, meropenem, gentamicin, tobramycin, and / or ciprofloxacin.

A sixth aspect of the invention relates to a polynucleotide isolated from Pseudomonas aeruginosa ST175 comprising a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them (for example, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5); or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, of an expression product (mRNA) of any of them.

All the particular embodiments of the first, second, third, fourth or fifth aspects of the invention form part of the sixth aspect of the invention.

A seventh aspect of the present invention relates to an expression cassette comprising the isolated polynucleotide of the sixth aspect of the invention.

All the particular embodiments of the first, second, third, fourth, fifth or sixth aspects of the invention form part of the seventh aspect of the invention.

In the present invention the expression cassette is a gene construct comprising the isolated DNA of the seventh aspect of the invention operably linked to a promoter that controls its expression. Being "operably linked" should be understood as that the DNA sequence is arranged following the promoter sequence or relatively closely in the case that restriction fragments or elements that provide stability to the construct are included. The gene construct can also comprise small fragments with sequences useful to adapt it to the expression systems desired and known to the person skilled in the art.

An eighth aspect of the present invention relates to a vector comprising the isolated DNA of the seventh aspect of the invention.

The vector is a DNA molecule refers to a DNA molecule into which the isolated DNA of the seventh aspect of the invention can be integrated without losing its capacity for self-replication. The vector can be a plasmid, cosmid, DNA phage, or an artificial chromosome (eg, yeast). If the vector is an expression vector, the protein encoded by the gene of interest is produced by the host cell's transcription and translation machinery, once the vector is within the host cell.

All the particular embodiments of the first, second, third, fourth, fifth, sixth or seventh aspects of the invention form part of the eighth aspect of the invention.

A ninth aspect of the invention relates to a host cell comprising the isolated DNA of the seventh aspect of the invention, the expression cassette of the eighth aspect of the invention, or the vector of the ninth aspect of the invention. The host cell can be prokaryotic or eukaryotic.

All the particular embodiments of the first, second, third, fourth, fifth, sixth, seventh or eighth aspects of the invention are part of the ninth aspect of the invention.

A tenth aspect of the invention refers to the use of the isolated DNA of the sixth aspect of the invention for the elaboration of specific tools, for example, primers or probes, for the identification of Pseudomonas aeruginosa ST175 as defined in the first and second aspects of the present invention.

All particular embodiments of the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspects of the invention form part of the tenth aspect of the invention.

An eleventh aspect of the invention relates to the use of an expression product protein of the nucleotide sequence SEQ ID NO: 1; or, alternatively, of a sequence with at least 99% identity with the sequence SEQ ID NO: 1; or, alternatively, of a fragment thereof, or, alternatively, of a protein product of expression of a nucleotide sequence that is selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them (SEQ ID NO: 3 and SEQ ID NO: 5); or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, from a protein that is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15, for the development of specific tools for the identification of Pseudomonas aeruginosa ST175.

In a particular embodiment of the eleventh aspect of the invention, optionally in combination with any particular embodiment provided above or below, the specific tool is an antibody that specifically binds to the proteins described in the eleventh aspect of the invention. For this reason, any of the proteins described in the eleventh aspect of the invention can be used as an immunogen.

An immunogen comprises an epitope that is capable of eliciting a specific immune response. An "epitope" is understood as the part of a peptide (or an antigen) whose sequence and / or special configuration is recognized by the immune system (antibodies, T cells or B cells).

All particular embodiments of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth aspects of the invention form part of the eleventh aspect of the invention.

Therefore, a twelfth aspect of the invention relates to an antibody that specifically binds to an expression product of one or more nucleotide sequences that are selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them (for example, SEQ ID NO: 3 and SEQ ID NO: 5); or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, it binds to a protein that is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15.

All the particular embodiments of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or eleventh aspects of the invention form part of the twelfth aspect of the invention.

The antibodies of the twelfth aspect of the invention can be used for the methods described in the present invention and can also be used as therapeutic tools in individuals with infection by the ST175 clone.

The present invention also relates to a method of prophylactic treatment of an individual, to protect against an infection by Pseudomonas aeruginosa ST175, which comprises the use of a protein product of expression of a nucleotide sequence that is selected from the group consisting of : SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them (for example, SEQ ID NO: 3 and SEQ ID NO: 5); or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, from a protein that is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: fifteen.

The present invention also relates to a method of treating an individual in need where the individual has a P. aeruginosa infection, comprising the identification of one or more nucleotide sequences that are selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them (for example, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 5); or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, the identification of a protein that is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; where, when the sequence (s) is identified, treatment comprises the administration of one or more antibiotics selected from the group consisting of: colistin, ceftolozane in combination with tazobactam, ceftazidime in combination with avibactam, amikacin, or a combination of them. In a particular embodiment, SEQ ID NO: 1 is identified. In another particular embodiment, when SEQ ID NO: 1; or, alternatively, one or more nucleotide sequences selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them (for example, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 5); or, alternatively, a sequence with at least 99% identity to any of them; or, alternatively, a protein that is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15 ; the antibiotic to be administered is, for example, one or more antibiotics that are selected from the group consisting of: ceftazidime, piperacillin / tazobactam, cefepime, imipenem, meropenem, gentamicin, tobramycin, or ciprofloxacin, or any combination of them.

The present invention also relates to a method of identification of P. aeruginosa ST175 that comprises contacting an isolated biological sample with a specific tool of one or more nucleotide sequences that are selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them (for example, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 5); or, alternatively, of a sequence with at least 99% identity with any of them; or, alternatively, the identification of a protein that is selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; where when the tool binds in a specific way (for example, it is captured) said specific binding is indicative of the presence of P. aeruginosa ST175 in the sample.

The in vitro methods of the invention provide diagnostic or recommendation information for an antibiotic treatment. In one embodiment, the methods of the invention further comprise the steps of (i) collecting the diagnostic information and (ii) saving the information on a data carrier.

In the sense of the invention, a "data carrier" should be understood as any medium that contains information data significant for the diagnosis of P. aeruginosa ST175 or the recommendation of an antibiotic treatment for infection by P. aeruginosa ST175, such as paper. The carrier can also be any entity or device capable of carrying the diagnostic data. For example, the carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disk or a hard disk. Furthermore, the carrier can be a transmissible carrier such as an electrical or optical signal, which can be carried by electrical or optical cable or by radio or other means. When data is incorporated into a signal that can be transmitted directly over a cable or other device or medium, the operator may be constituted by said cable or other device or medium. Other operators are related to USB devices and computer files. Examples of suitable data carriers are paper, CD, USB, computer files on PC or sound record with the same information.

All terms used in this application, unless otherwise indicated, will be understood in their ordinary meaning as known in the art. Other more specific definitions of certain terms used in the present application are those set forth herein and are intended to be applied uniformly throughout the description and in the claims, unless an expressly stated definition otherwise provides a broader definition. The definitions given herein are included for the purpose of understanding and are expected to apply throughout the description, claims, and drawings.

For the purposes of the present invention, any given range includes both the lowest value and the highest value of that range.

Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. Furthermore, the word "comprises" includes the case "consists of". For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limitative of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments indicated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Gel of a PCR of the biological marker (SEQ ID NO: 2) in a biological sample (blood culture) for the identification of the Pseudomonas aeruginosa clone ST175. The lanes correspond to (in this order from the left): molecular weight marker; ST175, PCR of clone ST175; PAO1, PCR of PAO1; C-, negative control (sterile distilled water).

EXAMPLES

1. Identification of specific regions of the P. aeruginosa clone ST175:

Material and methods:

Strains and clinical samples:

The reference strain of P. aeruginosa PAO1 (NC_002516.2, of January 24, 2019, corresponded to P. aeruginosa (Schroeter) Migula (ATCC®BAA-47 ™). The clinical strains of clone ST175 from various previous studies (Cabot G, et al. 2012; del Barrio-Tofiño E, et al. 2017; del Barrio-Tofiño E, et al. 2019) for the analysis of the sequences and the evaluation of the PCR.

A total of 69 samples were analyzed, of which 17 were blood cultures, 27 urine samples and 25 samples of respiratory origin. Both samples from patients diagnosed with infection by P. aeruginosa (n = 38) and negative for this pathogen (n = 31) were analyzed. The clinical samples were prepared for the extraction of genomic DNA, once routinely processed following the conventional microbiological procedures used in the Recommendations of the Spanish Society of Infectious Diseases and Clinical Microbiology of 2019, for the management of urinary infections (Andreu A, et al. 2010), blood cultures (Rodríguez JC, et al. 2017) and respiratory infections (Batista N, et al. 2006; Cacho JB, et al. 2007 and anonymized so as not to influence the hospital routine.

Genomic DNA extraction:

A total of 25 isolates were analyzed (12 of them belonging to clone ST175) randomly selected from the study collection of del Barrio-Tofiño E, et al. 2019 to obtain the genomic DNA of the isolates, a commercial capture system ("High Pure PCR Template Preparation Kit"; Roche Diagnostics) was used.

To obtain genomic DNA from clinical samples, a commercial capture system was used using the "Quiagen Biorobot EZ1" (Quiagen).

Library preparation and massive sequencing:

From the genomic DNA of the isolates, obtained using a commercial capture system ( High Pure PCR Template Preparation Kit; Roche Diagnostics), paired-end libraries were generated using a commercial library preparation kit ( Nextera XT DNA Library Preparation Kit; Illumina). The libraries were subsequently sequenced on a MiSeq System bench sequencer, using a MiSeq Reagent Kit (version 3; Illumina), generating 300 base pair (bp) paired-end reads, following the manufacturer's instructions.

PacBio sequencing and de novo assembly:

One of the clinical isolates from the work of Cabot G, et al. 2016 belonging to the P. aeruginosa clone ST175 was sequenced in a PacBio RSII sequencer (Pacific Bioscience of California Inc.) and a de novo assembly was obtained through the commercial services of the company Macrogen Inc. The de novo assembly of the P aeruginosa obtained by PacBio sequencing was annotated using Prokka v.1.12 software (Seeman T, 2014).

Variant calling:

22 isolates belonging to clone ST175 that appear in Cabot G, et al.

2016, also 44 XDR isolates, of which 18 belonged to the ST175 included in the document of del Barrio-Tofiño E, et al. 2017, and, in addition, the sequences of 185 isolates belonging to the Barrio-Tofiño E, et al. Study were also included in the analysis. 2019; of which 55 were ST175; where all of them had been sequenced previously (see their corresponding publications). The readings of each of the isolates were initially mapped against the genome of the reference strain of P. aeruginosa PAO1 (RefSeq NC_002516.2, "taxon: 208964") using Bowtie2 software v.2.2.4 (Langmead B, et al. 2009). Complete listings of mapped readings were obtained using SAMtools v.0.1.16 (Li H, et al. 2009) and PicardTools v.1.140 (developed by the Broad Institute). The alignments around the possible indels were realigned using the Genome Analysis Toolkit v.3.4-46 (GATK; McKeena A, 2010). For massive sequencing data analysis, single nucleotide polymorphism (SNPs) analysis was used as described in Cabot G et al., 2016e. In addition, those positions in which at least one of the isolates differed from the P. aeruginosa strain PAO1 in the rest of the unfiltered listings were cured (the sequences were checked) manually. The indels were extracted from the total listings when they met the criteria also described above (Cabot G et al., 2016).

The readings of the reference strain of P. aeruginosa PAO1 and of all isolates of the clone ST175 of Cabot G, et al. Were also mapped. 2016, against the de novo assembly of the isolate of clone ST175 obtained by PacBio sequencing.

Visualization and analysis of the maps:

The data of the mapping of the isolates against the de novo assembly of clone ST175 were visualized using the SeqMonk v.1.45.2 software (developed by the Bioinformatics Group, Babraham Institute , United Kingdom) for the visualization, identification and subsequent analysis of possible specific regions belonging to clone ST175. These regions were delimited in the de novo assembly of clone ST175 for the extraction of their sequence for subsequent comparison in the available databases, using the online blast tool (Altschul SF, et al. 1990).

Results:

Once specific areas present in all the isolates of clone ST175 and that were not present in the reference strain of P. aeruginosa PAO1 had been identified, the corresponding sequences were analyzed in depth to assess their suitability with the use of the BLASTN online tool. ( Basic Local Alignment Search Tool Nucleotide), which allowed further narrowing down those sub-regions that were clearly specific to the isolates of clone ST175 and also those that were not present in the available genomic databases. In addition, with the annotation of the genome, the different proteins annotated in each of the regions were searched in the databases of the National Center for Biotechnology Information (NCBI). As a result, the following regions of the genome of P. aeruginosa clone ST175 that were specific to it were identified: SEQ ID NO: 1, in which the sequences SEQ ID NO: 3 and SEQ ID NO: 5 that code for proteins were found. SEQ ID NO: 4 and SEQ ID NO: 6, respectively; and SEQ ID NO: 7, and the sequence SEQ ID NO: 8 (which included the sequence SEQ ID NO: 7) that codified for the protein SEQ ID NO: 9. In addition, other specific sequences of the clone ST175 of P. aeruginosa were found, such as SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, encoding the proteins SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15, respectively.

Finally, one of the regions (SEQ ID NO: 1) was selected as a marker for the identification of the isolates of the P. aeruginosa clone ST175, as it is the largest completely specific fragment, for the performance of the PCR indicated in the following examples.

2. Comparison between the amplified region for the detection of the ST175 clones with specific genetic markers of the ST175 clone and other techniques previously described:

In order to verify whether the specificity and sensitivity of the tool of the invention was advantageous compared to other tools and specific markers of the ST175 clone, the specificity and sensitivity of the same was verified.

Material and methods:

An analysis of the predictive values, sensitivity and specificity of specific genetic markers for clone ST175 identified in previous works (Cabot G, et al. 2012) was carried out in two collections of strains, one comprising 44 multi-resistant isolates (see table 1) and another 185 isolates from a multicenter study belonging to different resistance profiles (del Barrio-Tofiño E, et al 2017; del Barrio-Tofiño E, et al. 2019) (see table 2), in order to compare in the same I collect some of these markers and those of the present invention. Genetic markers were analyzed following the methodology described in Cabot G, et al. 2012 (by amplification and sequencing) and were the following: the presence of the aadB gene (responsible for resistance to gentamicin and tobramycin), the presence of the W142X mutation in the gene that codes for OprD, the presence of the T83I and D87N mutations in the gene that codes for GyrA, both individually and in combination, and the presence of the G154R mutation in the gene that codes for AmpR (as described in Cabot G, et al. 2012).

In addition, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) obtained were compared with those previously obtained using serotyping with the O antigen and MALDI-TOF (as described in Mulet X, et al. 2019).

In addition, the presence of the sequence SEQ ID NO: 1 in the genomes of the two collections was analyzed.

of P. aeruginosa isolates (see table 1 and table 2).

Results:

It was observed in silico (by sequence analysis using the indicated SeqMonk software

previously) that the region SEQ ID NO: 1 was present in all genomes of P. aeruginosa ST175

of the two collections analyzed, while it was not in any of the genomes that were not P.

aeruginosa ST175 (see results in tables 1 and 2 "" PCR "( in silico)". In addition, the

analysis using BLASTN between SEQ_ID NO: 1 and those of the de novo genomes, with a

also resulted in 100% specificity and sensitivity.

Table 1. Results of the typing using some of the genetic markers described

previously as specific for clone ST175 (Cabot G, et al. 2012) compared to the presence of the

Amplified region for the detection of the ST175 clones, in the collection of 44 multiresistant isolates

(MDR / XDR) from P. aeruginosa.

GyrA "PCR" OprD GyrA GyrA AmpR

aadB (T83I ( in silico) (W142X) (T83I) (D87N) (G154R)

n = 44 D87N)

Sensitivity (%) 100.0% 72.2% 100.0% 100.0% 100.0% 72.2% 100.0% Specificity (%) 96.2% 100.0% 19.5% 92.3% 92.3% 100.0% 100.0% PPV (%) 94.7% 100.0% 46.2% 90.0% 90.0% 100.0% 100.0% NPV (%) 100.0% 80.8% 100.0% 100.0% 100.0% 80.8% 100.0%

Table 2. Results of the typing using some of the genetic markers described

previously as specific for clone ST175 (Cabot G, et al. 2012) compared to the presence of the

region amplified for the detection of clones ST175, in the collection of 185 isolates of P.

aeruginosa from different Spanish regions.

OprD GyrA GyrA GyrA AmpR "PCR" aadB

n = 185 (W142X) (T83I) (D87N) (T83I D87N) (G154R) ( in silico) Sensitivity (%) 80.0% 54.5% 100.0% 89.1% 89.1% 54.5% 100.0% Specificity (%) 98.5% 100.0% 35.4 % 94.6% 95.4% 99.2% 100.0%

PPV (%) 95.7% 100.0% 39.6% 87.5% 89.1% 96.8% 100.0%

NPV (%) 91.4% 80.8% 100.0% 95.1% 95.2% 80.6% 100.0%

By detecting SEQ ID NO: 1, a clear improvement in sensitivity and specificity was achieved with respect to the

Genetic markers analyzed, allowing an unequivocal identification of the isolates

belonging to ST175.

In addition, it should be taken into account that for the detection of mutations, amplification and subsequent sequencing of the amplicon was required (as described in Cabot G, et al. 2012), which is less advantageous than the method described in the present invention ( identification of SEQ ID NO: 1) regarding the speed of identification of the pathogen.

On the other hand, in relation to the comparison using agglutination methods against the O4 antigen, MALDI-TOF or a combination of both for the identification of the ST175 clone, the sensitivity, specificity and predictive values did not reach 100% (as shown in table 2 in Mulet X, et al. 2019). Therefore, in comparison, the identification of P. aeruginosa ST175 by identifying the region SEQ ID NO: 1 was better than that achieved with these other techniques (comparing the sensitivity and specificity that was achieved in the two collections of Tables 1 and 2 by identifying SEQ ID NO: 1 and the sensitivity and specificity that was achieved in Mulet X, et al. 2019 by agglutination and / or MALDI-TOF).

3. Identification of the presence of P. aeruginosa clone ST175 in isolates and in clinical samples:

The identification of clone ST175 was carried out both in isolates and in clinical samples obtained from patients and the sensitivity and specificity of the method of the invention was assessed.

Material and methods:

Once the specific regions belonging to clone ST175 had been identified in example 1, primers were designed for certain subregions, which allowed a region belonging to these areas to be amplified by PCR and subsequent detection by means of an agarose gel.

Table 3. Primers for the identification of Pseudomonas aeruginosa isolates belonging to clone ST175.

"bp", base pairs

All PCR components, except primers and dimethyl sulfoxide (DMSO), belonged to the "AmpliTaq Gold ™ DNA Polymerase with Buffer II & MgCh" Kit, from Applied Biosystems (Ref. N8080247). DMSO (Ref. D5879) and primers were from Sigma Aldrich.

The PCR conditions for all primer pairs described in Table 3 were as follows:

Components and final concentration in the reaction: Taq Gold (1.25 U), Buffer 10x (1x in reaction), MgCl ² (6 pM), primer will turn (SEQ ID NO: 16) (1 pM), primer reverse ( SEQ ID NO: 17) (1 pM), dNTPs (200 pM each), dimethylsulfoxide (DMSO) (> 99.5%) (10%) and H ² O up to 50pl (final reaction volume of 50pl ).

Table 4: Amplification reaction:

By using the sequence primers SEQ ID NO: 16 and SEQ ID NO: 17, the specific amplicon SEQ ID NO: 2 was achieved. By using the sequence primers SEQ ID NO: 24 and SEQ ID NO: 25, the obtained the specific amplicon SEQ ID NO: 29 comprising a fragment of the sequence SEQ ID NO: 10 and a fragment of SEQ ID NO: 12. By using the sequence primers SEQ ID NO: 26 and SEQ ID NO: 27 the specific amplicon SEQ ID NO: 31 was obtained which comprised a fragment of the sequence SEQ ID NO: 14.

As a positive control, it was used for the PCR of a sample (ID: 179) from the work Cabot G, 2012.

to. Identification of clone ST175 in clinical isolates:

A test panel was carried out for the amplification of the specific region selected as a biomarker for the identification of 25 isolates (pure culture of the bacteria) where 12 of them were. strains belonging to clone ST175 (identified by MLST, which is the technique used to define the "ST" ( sequence type), Curran B, et al 2004). The isolates were obtained from the study by Barrio-Tofiño E, et al. 2019, preserved at -80 ° C and grown on Columbia Agar 5% sheep blood plates (Niomériuex) that were isolated from blood culture samples in different Spanish regions (Andalusia, Aragón, Cantabria, Castilla y León, Castilla-La Mancha , Catalonia, Valencian Community, Extremadura, Galicia, Balearic Islands, Canary Islands, Madrid, Murcia, Navarra, Basque Country and the Principality of Asturias)

The region SEQ ID NO: 1 was amplified with the primers (primers SEQ ID NO: 16 and SEQ ID NO: 17, which amplified SEQ ID NO: 2) and conditions indicated above.

b. Identification of clone ST175 in clinical samples:

The marker of the invention was tested for direct identification of the presence of clone ST175 by analyzing 69 clinical samples, comprising 17 blood culture samples, 27 urine samples, and 25 samples of respiratory origin. The clinical samples were obtained from the Clinical Microbiology Service of the Son Espases Hospital, once the samples had been routinely processed following conventional microbiological processes and anonymized to preserve the confidentiality of the patients and perform a test without any type of bias.

The region SEQ ID NO: 1 was amplified with the primers (primers SEQ ID NO: 16 and SEQ ID NO: 17, which amplified SEQ ID NO: 2) and conditions indicated above.

Results:

Figure 1 is an example of a PCR of a sample (blood culture) (ID: 179) from the work Cabot G, et al.

2012 for the detection of ST175 by amplification of the fragment SEQ ID NO: 2, with the pair of primers SEQ ID NO: 16 and SEQ ID NO: 17.

The amplification of the SEQ ID NO: 1 region allowed the unequivocal identification of 100% of the strains belonging to clone ST175 from the isolates and clinical samples (Table 5). As can be seen in Table 5, the sensitivity and specificity in the detection of the P. aeruginosa clone ST175 both in the isolates and in direct clinical samples was 100% in both cases.

Table 5. Result of the use of the specific region SEQ ID NO: 1 as a marker for the detection of the ST175 clones by PCR amplification

Clinical samples

Isolated

TOTAL H O R

(n = 25)

(n = 69) (n = 17) (n = 27) (n = 25)

ST175 12 5 1 3 1

Sensitivity (%) 100.0% 100.0% 100.0% 100.0% 100.0% Specificity (%) 100.0% 100.0% 100.0% 100.0% 100.0%

PPV (%) 100.0% 100.0% 100.0% 100.0% 100.0%

NPV (%) 100.0% 100.0% 100.0% 100.0% 100.0%

(H: blood culture, O: urine, R: respiratory sample).

Conclution:

The method of the present invention allowed the identification of the P. aeruginosa clone ST175 both in isolates and in clinical samples with a sensitivity, specificity and predictive values (PPV and NPV) of 100%.

Furthermore, as can be seen comparing with the results of examples 2 and 3, the amplification of the specific regions of the present invention improved the identification of the P. aeruginosa ST175 clone compared to the use of the known specific genetic markers that were evaluated (the resistance markers described previously). Furthermore, it should be noted that the identification of P. aeruginosa ST175 by detecting SEQ ID NO: 1 using PCR allowed a faster detection than by detecting resistance genetic marker mutations since it did not require a sequencing step.

In comparison with other known identification techniques of the clone ST175 (agglutination against the O4 antigen and MALDI-TOF) (as described in example 2), the method of the present invention allowed the identification of the clone ST175 of P. aeruginosa with higher sensitivity, specificity and predictive values (PPV and NPV).

The identification of the presence of the specific regions of the invention directly from the clinical sample represented a considerable reduction in the time necessary for the identification of isolates belonging to clone ST175, since it did not even require the growth of the strain in a culture pure ("isolated"), thus saving 24 hours compared to the fastest methods to date, being able to obtain the results in just a few hours after receiving the sample in the laboratory. This was an advantage in terms of selection and administration of adequate treatment and therefore an improvement in the treatment of infections due to isolates belonging to clone ST175 of P. aeruginosa.

LIST OF APPOINTMENTS

Patent literature:

US4.196.265

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Claims (23)

1. An in vitro method of diagnosis of an infection caused by Pseudomonas aeruginosa ST175 in an isolated sample from a subject, characterized by comprising the identification of the nucleotide sequence SEQ ID NO: 1; or, alternatively, a fragment of it; or, alternatively, of a sequence with at least 99% identity with the nucleotide sequence SEQ ID NO: 1; or, alternatively, of an expression product thereof. The in vitro method according to claim 1 further comprising identifying one or more nucleotide sequences that are selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of said sequences; or, alternatively, of an expression product of any of them; or, alternatively, one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; or, alternatively, of any combinations of them. 3. An in vitro method for deciding or recommending an antibiotic treatment in a subject with a Pseudomonas aeruginosa infection, characterized in that the method comprises the identification of Pseudomonas aeruginosa ST175 by identifying the nucleotide sequence SEQ ID NO: 1 in an isolated sample from the subject; or, alternatively, a fragment thereof; or, alternatively, of a sequence with at least 99% identity with the nucleotide sequence SEQ ID NO: 1; or, alternatively, of an expression product thereof; where, when the sequence (s), fragment or expression product of SEQ ID NO: 1 is identified, the antibiotic to be administered is colistin, ceftolozane in combination with tazobactam, ceftazidime in combination with avibactam, amikacin, or any combination of them. 4. The in vitro method of deciding or recommending an antibiotic treatment according to claim 3, characterized in that it also comprises the identification of one or more nucleotide sequences that are selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of said sequences; or, alternatively, a product of expression of any of them; or, alternatively, one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; or, alternatively, of any combinations of them; where, when the sequence (s), the fragment (s) or the expression product (s) are identified, the antibiotic to be administered is colistin, ceftolozane in combination with tazobactam, ceftazidime in combination with avibactam, amikacin, or any combination of them 5. The in vitro diagnostic method according to any of claims 1 or 2 or the in vitro method of deciding or recommending an antibiotic treatment according to any of claims 3 or 4, wherein the fragment of the nucleotide sequence SEQ ID NO: 1 comprises the nucleotide sequence SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 5. 6. The in vitro method according to any of claims 1 to 5, wherein the identification of the nucleotide sequence (s) or their fragments is carried out in the DNA of Pseudomonas aeruginosa ST175 comprised in the isolated sample from the subject. 7. The in vitro diagnostic method according to any of claims 1 or 2 or the in vitro method of deciding or recommending an antibiotic treatment according to any of claims 3 or 4, wherein the expression product of the nucleotide sequence is RNA delivery courier. 8. The in vitro method according to any of claims 1 to 7, wherein the identification of the nucleotide sequence (s) or their fragments or the expression product when this is mRNA, is carried out by means of a technique based on the polymerase chain reaction (PCR) or, alternatively, by hybridization. The in vitro method according to claim 8 wherein the PCR is selected from the group consisting of: PCR, real-time quantitative PCR, RT-PCR, real-time quantitative RT-PCR, multiplex PCR; and, where the hybridization is carried out on a chip ( microarray). 10. The in vitro method according to any of claims 8 or 9 wherein the in vitro method comprises the use of at least one pair of primers that are selected from the list consisting of: SEQ ID NO: 16 and SEQ ID NO: 17 , SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25 and SEQ ID NO: 26 and SEQ ID NO: 27, or any combination of them. The in vitro method according to any of claims 1 to 10 wherein the biological sample is selected from the list consisting of: tissue, blood, plasma, serum, lymph, bronchoalveolar lavage, saliva, urine or ascites fluid. 12. The in vitro method according to any of claims 1 to 11 wherein the subject is an immunosuppressed subject. 13. The in vitro method according to any of claims 1 to 12 wherein the subject is a subject with cystic fibrosis 14. Use of a polynucleotide of sequence SEQ ID NO: 1 or, alternatively, of a fragment thereof; or, alternatively, a polynucleotide with at least 99% identity with the sequence SEQ ID NO: 1; or, alternatively, an expression product thereof; as a diagnostic marker of an infection caused by Pseudomonas aeruginosa ST175; or to decide or recommend an antibiotic treatment in a subject with an infection caused by Pseudomonas aeruginosa. 15. Use according to claim 14, wherein one or more nucleotide sequences selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of said sequences; or, alternatively, of an expression product of any of them; or, alternatively, one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; or, alternatively, of any combinations of them; as a diagnostic marker of an infection caused by Pseudomonas aeruginosa ST175; or to decide or recommend an antibiotic treatment in a subject with an infection caused by Pseudomonas aeruginosa. 16. Use according to any of claims 14 or 15, wherein the fragment of SEQ ID NO: 1 is selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 5; or, alternatively, where the expression product of SEQ ID NO: 1 is SEQ ID NO: 4 or SEQ ID NO: 6. 17. Use of specific tools to identify the sequence SEQ ID NO: 1; or, alternatively, a fragment of it; or, alternatively, a sequence with at least 99% identity to the sequence SEQ ID NO: 1; or, alternatively, a product of expression thereof, as defined in claims 1 to 16 in any of the in vitro methods defined in claims 1 to 13. 18. Use according to claim 17 wherein one or more nucleotide sequences are also identified that are selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of said sequences; or, alternatively, of an expression product of any of them; or, alternatively, one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; or, alternatively, of any combinations of them. 19. Use according to any of claims 17 or 18, wherein the fragment of SEQ ID NO: 1 is selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 5; or, alternatively, where the expression product of SEQ ID NO: 1 is SEQ ID NO: 4 or SEQ ID NO: 6. 20. The use according to any one of claims 17 to 19 wherein the tools are part of a kit. 21. A kit to identify Pseudomonas aeruginosa ST175, comprising the specific tools to identify the sequence SEQ ID NO: 1; or, alternatively, a fragment of it; or, alternatively, a sequence with at least 99% identity to the sequence SEQ ID NO: 1; or, alternatively, an expression product thereof. 22. The kit according to claim 21, wherein it further comprises the specific tools to identify one or more isolated nucleotide sequences that are selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14; or, alternatively, a fragment of any of them; or, alternatively, of a sequence with at least 99% identity with any of said sequences; or, alternatively, of an expression product of any of them; or, alternatively, one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; or, alternatively, of any combinations of them. 23. The kit according to any of claims 21 or 22, wherein the fragment of SEQ ID NO: 1 is selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 5; or, alternatively, where the expression product of SEQ ID NO: 1 is SEQ ID NO: 4 or SEQ ID NO: 6.