Method and kit for in vitro diagnosis of infection due to enterobacteria with a hypervirulent genotype

Abstract

The present invention relates to a method and a kit for the in vitro diagnosis of infection by enterobacteria with a hypervirulent genotype, such as the enterobacterium Klebsiella pneumoniae (Kpn) with a hypervirulent genotype, by means of real-time PCR, end-point PCR, or real-time probe-based PCR.

Description

[0001] METHOD AND KIT FOR IN VITRO DIAGNOSIS OF INFECTION DUE TO ENTEROBACTERIA WITH A HYPERVIRULENT GENOTYPE

[0002] The present invention relates to a method and a kit for in vitro diagnosis of infection due to enterobacteria with a hypervirulent genotype.

[0003] In greater detail, the invention relates to a method and a kit for in vitro diagnosis of infection due to enterobacteria with a hypervirulent genotype, such as, for example, the enterobacterium Klebsiella pneumoniae (Kpn) with a hypervirulent genotype, for example by means of probe-based real-time PCR.

[0004] As is well known, starting from the first description of Kpn in the 19th century (Dai et al., 2022), both drug resistance and hypervirulence have been increasingly documented in this pathogen. Multidrug-resistant (MDR) Kpn is considered a global public health threat by the major international health organisations because of its rapid spread and high morbidity and mortality, as well as the economic costs associated with treating and controlling it (Russo et al., 2019). In recent decades, high-risk MDR clones have become endemic in various countries, including Italy, and, at the same time, the spread of highly virulent Kpn (designated as “hvKp”) has also been documented. These clones are capable of causing severe pyogenic infections, with metastatic dissemination, in immunodepressed individuals in hospital settings and in immunocompetent individuals in community settings (Kot et al., 2023). The infections ascribed to this pathogen are, in particular, pneumonia, urinary tract infections, endoabdominal infections, surgical wound infections and sepsis (Ballen et al., 2021 ; Fatima et al., 2021 ). HvKp clones produce different virulence factors, including a polysaccharide capsule which, by protecting the bacteria from phagocytosis and bactericidal serum factors, contribute to increasing the virulence. Furthermore, the capsular serotypes of hvKp are implicated in the formation of liver abscesses in both healthy and immunocompromised individuals. The liver abscesses caused by hvKp can spread far, causing pneumonia and lung abscesses, meningitis, necrotising fasciitis and endophthalmitis. In Australia a case of multifocal osteomyelitis was reported as a rare complication of hvKp in a young 20-year-old previously in good health (Sohrabi et al., 2022). The genes implicated in capsule production and the consequent mucosal phenotype and defined as the main virulence factors (Yu et al., 2006), are magA and rmpA the former, associated with mucoviscosity, codes for a structural protein of the outer membrane, whilst the latter is a gene regulating the gene responsible for the mucoid phenotype, and is located on a virulence plasmid (Holt et al., 2015). A recent Italian study demonstrated that the hvKp isolates most greatly represented and circulating in Italy show a convergence of the characteristics / determ inants of virulence, including yersinobactin and / or salmochelin and / or aerobactin, as well as the genes responsible for the mucoid phenotype and the antimicrobial resistance markers (in particular extended-spectrum beta-lactamase) (Arena et al., 2022).

[0005] Phenotype and molecular analyses are currently available for the purpose of detecting hvKp. The phenotype method provides for the use of an easy-to-perform assay, the string test. The test is performed by touching a colony with an inoculating loop and gently lifting it: if a mucous “string” 5 mm long or longer forms, the result is considered positive. Notwithstanding the advantages given by the extreme simplicity of execution and low costs, the string test has low sensitivity (Eisenmenger et al., 2021 ) and a negative result is not indicative of an absence of hypervirulence. In fact, Kpn may not possess or not express magA, despite possessing the remaining genes responsible for hypervirulence, whose presence is in any case a prognostic indicator of an unfavourable outcome of the infection. Conversely, a positive result does not necessarily indicate hypervirulence and requires further confirmation.

[0006] The molecular analysis provides for the use of next-generation sequencing (NGS) techniques, in particular whole genome sequencing (WGS). The results are analysed using various online platforms, including Pathogenwatch (https: / / pathogen. watch / ), managed by the Centre for Genomic Pathogen Surveillance (https: / / www.pathogensurveillance.net / ). The platform automatically analyses the assembled genomes according to the Kleborate and Inctyper pipelines. Kleborate software provides in-depth information on the species and sequence type (ST) according to the multilocus sequence typing (MLST) scheme based on 7 genes which include: virulence genes, capsule and locus typing genes and antimicrobial resistance markers (Lam et al., 2021 ). As regards the genes associated with virulence, Kleborate examines 5 key loci: the hypermucoid locus rmpADC, the siderophores yersiniabactin (ybt), aerobactin (iuc) and salmochelin ( / ro) and the genotoxin colibactin (clb). The isolates are then classified according to a virulence score that goes from 0 to 5:

[0007] • 0= negative • 1 = positive only for ybt

[0008] • 2= positive for ybt and clb (or only for clb)

[0009] • 3= positive for iuc

[0010] • 4= positive for iuc and ybt (without clb)

[0011] • 5= positive for ybt, clb and iuc

[0012] Although WGS allows an in-depth analysis of the hvKp isolates to be obtained, this technique is costly, is not applicable in all laboratories, and requires time and specific competences, as well as the availability of high-throughput sequencers. Such difficulties are the reason for the possible underestimation of this phenotype in the population of strains circulating in Italy.

[0013] In contrast, the assays that use real-time PCR technology are highly sensitive and specific, entail lower costs compared to WGS and are relatively easy to perform and interpret. In order for a real-time PCR assay to be able to determine hypervirulence, it must provide for a simultaneous search for all the genes implicated in this phenotype. At present, however, there exist no real-time PCR assays designed to detect the previously mentioned genes.

[0014] In this regard, patent application CN116574820 concerning a quantitative fluorescent PCR and an associated kit for detecting Kpn with high toxicity is known. However, the quantitative PCR method disclosed in the Chinese patent application is based on the simultaneous detection only of the / ucA, iroB, peg-344 and rmp / genes in Klebsiella pneumoniae. Therefore, the method does not take account of other genes and toxins correlated with hypervirulence.

[0015] In the light of the above, it appears evident that there is a need to be able to have available new methods and kits for detecting the hypervirulent genotype in enterobacteria such as, for example, Kpn, which are capable of overcoming the disadvantages of the known methods and kits.

[0016] The solution according to the present invention fits into this context; it aims to provide a method and a kit for detecting the hypervirulent genotype in enterobacteria, in particular in Kpn, by means of a probe-based real-time PCR technique that allows the above-described problems to be resolved. The present invention provides, in fact, a low-cost, easily implementable method and kit which enable an overall result as to the presence of genes associated with the profile of hypervirulence to be obtained in a short time. Given the major emergency due to the increase in the cases of hvKp, as stated by a recent warning of the ECDC (ECDC, 2024), it is of fundamental importance to develop a method allowing results to be obtained which are as exhaustive as possible. The panel of genes detected according to the present invention is, at present, the most complete, capable of detecting the presence of a profile of hypervirulence and simultaneously providing results with high sensitivity and specificity thanks to the selection of the targets.

[0017] According to the present invention the detection of the hypervirulent genotype in enterobacteria, such as, for example, Kpn, can be achieved starting from a bacterial suspension and positive blood culture, by means of a diagnostic kit for probe-based real-time PCR.

[0018] The method and the kit according to the present invention are based on the detection of specific genes correlated with the hypervirulence of enterobacteria and the use of specific primers and probes for the genes associated with hypervirulence, selected and designed after a thorough study of the currently available literature. Specifically, the method and kit enable the simultaneous detection of the following genes: ybtA, ybtS, iucA, iroB, iroN, clbA, rmpA, rmpA2, rmpC, rmpD, magA, hlyA, fimH, mrkD, cnf-1 and peg-344.

[0019] Compared to the string test, the assay according to the present invention shows to be sensitive and specific. According to the present invention, the detection of even only one gene among the ones indicated allows the presence of hypervirulence to be identified without further confirmatory tests.

[0020] Compared to the method of Chinese patent application CN116574820, the method of the present invention takes account of a larger number of targets and above all takes account of toxins and the genes coding for yersinobactin, colibactin, adhesin and fimbriae, which have a fundamental role in imparting hypervirulence.

[0021] It is therefore a specific object of the present invention an in vitro method for detecting, in a biological sample, an enterobacterium with a hypervirulent genotype, said method comprising or consisting of detecting the following genes (i.e. a set of genes comprising the following genes): rmpA, rmpA2, rmpC, rmpD, magA, cnf-1, ybtA, ybtS, iucA, iroB, iroN and clbA through the detection of the following nucleotide sequences:

[0022] ATGGGGAGGGTACAAAATGTTAAGGGGATCATTAAATATGATAAGCCA ATGGATGTGGCTTGACGTTTCGGGGGGGGGGCGGTTTTATCCTAAAGGGTG T SEQ ID NO:1 for the rmpA gene,

[0023] GTTAGCCGGATTGGAAATCATTACCCACAACTAACAAAAAAAGAACAAA TCATTCTACAATGCTTACTCTCCAGGAGGGAGGGCATCCATGAATTAAAAAGC CGTCTAAAAATTGAAGAGAAAACACTATCGTGTCACAGATG SEQ ID NO:2 for the rmpA2 gene,

[0024] AAGACTCCTTCTTTGTCAAATTTATTGGTTTATAATCAAGCTGCCCCCC CCCATTAAAGAGAGAAATCATTCTATCTTTTAAAATAGTGATAGCCTCGCGTG CGTTAATGATGTTAATTCCGATGAGGGTGGAATTA SEQ ID N0:3 for the rmpC gene,

[0025] GCTTTGTTGTAAGTGGACCAGTTGGTGATTCTGAACTTTTGCTTTGCCA CGGAATGGTCTGAGTTGTCGGGAGGATGCGTGATCTGATCCTTCAACTCAGC AAAAGTTCGATTTATTCAACAAAGCCGCGTCTATTCATCGCTTTTC

[0026] SEQ ID N0:4 for the rmpD gene,

[0027] AACGAATTGATGCTTGGCATCATGCAATAGCCACGTTTTATGAGCATCC AATACTTGGAGAGGGAGTGGGAATATTCATTCAAAGATTCAACATGTACCCTC ATAATATATTCTTCGAATCTGCAGCAGAAA SEQ ID N0:5 for the magA gene,

[0028] AACGTCGTTCTGCTATACCTGGTTTGGCGACAAATGCAGTACTGCTTG GTCTGTCTTTTGCAATTCCACTCTTGATTAGTAAGGCAGGAATAAACCAGGA SEQ ID N0:6 for the cnf-1 gene,

[0029] TTCGAGAGCATTACCCTCTCCTTCAGCCCTGAAACCCTCGCGCTGTGG GTGCCGGATATTAGCGCGGTCATTAAGAATAAGATCGACTCCCACTGCTGTT GCCAGCAGCAGCGTTGTAATGCTGAAACCCA SEQ ID N0:7 for the ybtA gene,

[0030] CTGGCGAACCGACTTTAAATCTTCAACCACCACGCTGCCCGGCAGGC AAACGGCCTCCAGTTCAGCAATCGCTTCTTTGAC SEQ ID N0:8 for the ybtS gene,

[0031] GTCCGTACTCTGTCTGTGAAAGAGGTGGAGCGAGGAATGCGCCTGGC ACGTCTGGCGCAAACCGACGGCTGGCAGATGCTACAGGCCCGCTTCCCTAC TTTCCGGGTAATG SEQ ID N0:9 for the iucA gene,

[0032] GGCATATCAAAGGCGTAACGCGTTCGCTTGCGGACACGTATCATCGC CACGGCGTCGGGGCCGCACCGCGCGATATGGCCTGGATCGACGTA SEQ ID NO: 10 for the iroB gene,

[0033] GTATCTTCACTGCGGGTTTCCGCATGAGTACGCGGTTTTTGTCTGCCA TATAACGTCCAGTTGACGCTGGCAGAAAACGCCTGGGTGATGGTCCAGTTAA GCGAGTTATTGATAGTATATTTCGGGATGACCGACAGAGGATTACC SEQ ID N0:11 for the iroN gene, and AGATACAGTAGCCGTTGCTATTAGTTCTCACTGCGAGCTTGGTGTCGA TATTGAACAAATAAAAGATTTAAACAACTCTTATCTGAATATCAGTCAGCATTTT TTTACTCCACAGGAAGCTACTAACA SEQ ID NO: 12 for the clbA gene.

[0034] According to the present invention, the above-described method can further comprise detecting one or more or all of the following genes: peg-344, hlyA, fimH and mrkD through the detection of the following nucleotide sequences:

[0035] TCGCATTAGCTTCACTTTCGTTCATCCTCATCACGGAGGCGCGGGAAA GGACAGAAAGCCAGTGGACGGGAGTTATTGCCCTTTCTTCTGCAGTA SEQ ID NO: 13 for the peg-344 gene,

[0036] GAGAATGGCTATGACGCAAGACATGCTGCGTTTTTAGAAGACTCTCTG TCTTTGCTTGCTGATCTTTCTCGTCAACATGGAGTAGAAAGAGCAGTCGCAAT AACCCAGCAACATTGGGATGAGAAGATCGGTGAACTTGC SEQ ID NO: 14 for the hlyA gene,

[0037] TGTGCTGTCGAGTTTTTCAGGCACCGTGAAATATAACGGCACCTCTTA CCCGTTCCCGACCACCACGGAAACCGCGCGGGTGATTTATGATTCACGGAC CGATAAACC SEQ ID NO: 15 for the fimH gene and

[0038] GGGTAAGTACACCTCCTACGACTGGGAGAGCGGCGGTAACCCGATCC TCGAAACCTATCTGAGCGCCAACGCCATCACCGTGGTCTCGCCCTCCTGTTC GGTGCTGAGCGGGAAAAATATG SEQ ID NO: 16 for the mrkD gene.

[0039] Therefore, according to the present invention the method aims to detect the entire set of genes which are indicators of hypervirulence, consisting of the rmpA, rmpA2, rmpC, rmpD, magA, cnf-1, ybtA, ybtS, iucA, iroB, iroN and clbA genes, in order to obtain a profile of hypervirulence. Moreover, the method can also provide for the detection of further genes such as peg-344, hlyA, fimH and mrkD with the aim of completing the profile of hypervirulence (where a positive result has already been obtained for the previous genes) or ascertaining the presence thereof in the event of a previous negative result. The method can provide for the detection of one, two, three or four of said further genes. When the method provides for the detection of more than one gene of said further genes, the detection can regard different combinations of said further genes such as, for example, peg-344, hlyA, fimH and mrkD', peg-344, hlyA, fimH', hlyA, fimH and mrkD', peg-344, mrkD, hlyA', peg-344, mrkD, fimH', peg-344, hlyA', hlyA, fimH', fimH and mrkD; peg-344 and fimH', peg-344 and mrkD; hlyA and mrkD.

[0040] According to one embodiment, so-called “panels” can be created, each one consisting of a set of genes. For example, the following panels can be created.

[0041] Panel 1 Hypermucoid: rmpA rmpA2 rmpC

[0042] HBB

[0043] Panel 2 Hypermucoid: magA rmpD cnf-1

[0044] HBB

[0045] Panel 3 Hypervirulence: ybtA ybtS iucA

[0046] HBB

[0047] Panel 4 Hypervirulence: iroB iroN clbA

[0048] HBB

[0049] Panel 5 Hypervirulence: peg-344 hlyA

[0050] HBB

[0051] Panel 6 Hypervirulence: fimH mrkD

[0052] HBB

[0053] The panels that are essential for the purpose of obtaining a profile of hypervirulence, thus the first ones on which a positive blood culture or the bacterial colony must be tested, are panels 1 , 2, 3 and 4. Panels 5 and 6 can complete the profile of hypervirulence (where a positive result has already been obtained from the previous panels) or ascertain its presence in the event of a previous negative result. It is well known that the methods of detecting genes also comprise internal controls. In the method according to the present invention, HBB, for example, can be used as the internal reference gene.

[0054] According to a preferred embodiment of the method according to the present invention, said nucleotide sequences are detected by using the following forward and reverse primers:

[0055] ATGGGGAGGG TACAAAATGT SEQ ID NO: 17

[0056] ACACCCTTTA GGATAAAACC G SEQ ID NO: 18 for the rmpA gene,

[0057] GTTAGCCGGA TTGGAAATCA SEQ ID NO: 19

[0058] CATCTGTGAC ACGATAGTGT SEQ ID NQ:20 for the rmpA2 gene,

[0059] >

[0060] AAGACTCCTT CTTTGTCAAA TTT SEQ ID NO:21

[0061] TAATTCCACC CTCATCGGAA SEQ ID NO:22 for the rmpC gene,

[0062] GCTTTGTTGTAAGTGGACCA SEQ ID NO:23

[0063] GAAAAGCGAT GAATAGACGC SEQ ID NO:24 for the rmpD gene,

[0064] AACGAATTGA TGCTTGGCAT SEQ ID NO:25

[0065] TTTCTGCTGC AGATTCGAAG SEQ ID NO:26 for the magA gene,

[0066] AACGTCGTTC TGCTATACCT SEQ ID NO:27

[0067] TCCTGGTTTA TTCCTGCCTT SEQ ID NO:28 for the cnf-1 gene,

[0068] TTCGAGAGCA TTACCCTCTC SEQ ID NO:29

[0069] TGGGTTTCAG CATTACAACG SEQ ID NQ:30 for the ybtA gene,

[0070] CTGGCGAACC GACTTTAAA SEQ ID NO:31

[0071] GTCAAAGAAG CGATTGCTGA SEQ ID NO:32 for the ybtS gene,

[0072] GTCCGTACTC TGTCTGTGAA SEQ ID NO:33

[0073] CATTACCCGG AAAGTAGGGA SEQ ID NO:34 for the iucA gene,

[0074] GGCATATCAA AGGCGTAACG SEQ ID NO:35 TACGTCGATC CAGGCCATA SEQ ID NO:36 for the iroB gene,

[0075] GTATCTTCACTGCGGGTTTC SEQ ID NO:37

[0076] GGTAATCCTCTGTCGGTCAT SEQ ID NO:38 for the iroN gene,

[0077] AGATACAGTA GCCGTTGCTA T SEQ ID NO:39

[0078] TGTTAGTAGC TTCCTGTGGA G SEQ ID NQ:40 for the clbA gene,

[0079] TCGCATTAGC TTCACTTTCG SEQ ID N0:41

[0080] TACTGCAGAA GAAAGGGCAA SEQ ID NO:42 for the peg-344 gene,

[0081] GAGAATGGCT ATGACGCAAG SEQ ID NO:43

[0082] GCAAGTTCAC CGATCTTCTC SEQ ID NO:44 for the hlyA gene,

[0083] >

[0084] TGTGCTGTCG AGTTTTTCAG SEQ ID NO:45

[0085] GGTTTATCGG TCCGTGAATC SEQ ID NO:46 for the fimH gene and

[0086] GGGTAAGTAC ACCTCCTACG SEQ ID NO:47

[0087] CATATTTTTC CCGCTCAGCA SEQ ID NO:48 for the mrkD gene

[0088] Furthermore, according to a further preferred embodiment, the abovedescribed method can further comprise the use of probes in combination with the respective primers used (see above) for each of said genes, wherein said probes can be for example the following:

[0089] AGC CAA TGG ATG TGG CTT GAC GTT TC SEQ ID NO:49 for the rmpA gene,

[0090] TCT CCA GGA GGG AGG GCA TCC A SEQ ID NQ:50 for the rmpA2 gene,

[0091] AGT GAT AGC CTC GCG TGC GTT AAT GA SEQ ID NO:51 for the rmpC gene,

[0092] GCC ACG GAA TGG TCT GAG TTG TCG SEQ ID NO:52 for the rmpD gene,

[0093] GCA TCC AAT ACT TGG AGA GGG AGT GGG SEQ ID NO:53 for the magA gene, TGG CGA CAA ATG CAG TAC TGC TTG G SEQ ID NO:54 for the cnf-1 gene,

[0094] GCC CTG AAA CCC TCG CGC TG SEQ ID NO:55 for the ybtA gene,

[0095] TTC AAC CAC CAC GCT GCC CG SEQ ID NO:56 for the ybtS gene,

[0096] AAC CGA CGG CTG GCA GAT GC SEQ ID NO:57 for the iucA gene,

[0097] GTA TCA TCG CCA CGG CGT CGG SEQ ID NO:58 for the iroB gene,

[0098] GCT GGC AGA AAA CGC CTG GGT SEQ ID NO:59 for the iroN gene,

[0099] TCT CAC TGC GAG CTT GGT GTC GA SEQ ID NQ:60 for the clbA gene,

[0100] TCA TCA CGG AGG CGC GGG AA SEQ ID N0:61 for the peg-344 gene,

[0101] GAG CAG TCG CAA TAA CCC AGC AAC A SEQ ID NO:62 for the hlyA gene,

[0102] CCG ACC ACC ACG GAA ACC GC SEQ ID NO:63 for the fimH gene and

[0103] AGC GGC GGT AAC CCG ATC CT SEQ ID NO:64 for the mrkD gene

[0104] Furthermore, according to the present invention, said probes can be bound at the 5’ end to a fluorophore and at the 3’ end to a quencher.

[0105] According to the present invention, the fluorophore can be selected in the group consisting of FAM, HEX, ROX and CY5. Furthermore, the quencher can be selected in the group consisting of BHQ1 , BHQ2 and BBQ 650.

[0106] Preferably, use can be made of a fluorophore and quencher combination selected from FAM-BHQ1 , HEX-BHQ1 , ROX-BHQ2 or CY5-BBQ 650.

[0107] According to specific embodiments of the method of the present invention, the FAM-BHQ1 combination is bound to the following probes:

[0108] AGT GAT AGC CTC GCG TGC GTT AAT GA (SEQ ID NO:51 ),

[0109] TGG CGA CAA ATG CAG TAC TGC TTG G (SEQ ID NO:54),

[0110] AAC CGA CGG CTG GCA GAT GC (SEQ ID NO:57), TCT CAC TGC GAG CTT GGT GTC GA (SEQ ID NO:60),

[0111] GAG CAG TCG CAA TAA CCC AGC AAC A (SEQ ID NO:62) and

[0112] AGC GGC GGT AAC CCG ATC CT (SEQ ID NO:64); the ROX-BHQ2 combination is bound to the following probes:

[0113] TCT CCA GGA GGG AGG GCA TCC A (SEQ ID NQ:50),

[0114] GCC ACG GAA TGG TCT GAG TTG TCG (SEQ ID NO:52),

[0115] TTC AAC CAC CAC GCT GCC CG (SEQ ID NO:56),

[0116] GCT GGC AGA AAA CGC CTG GGT (SEQ ID NO:59),

[0117] TCA TCA CGG AGG CGC GGG AA (SEQ ID NO:61 ) and

[0118] CCG ACC ACC ACG GAA ACC GC (SEQ ID NO:63); the CY5-BBQ650 combination is bound to the following probes:

[0119] AGC CAA TGG ATG TGG CTT GAC GTT T (SEQ ID NO:49),

[0120] GCA TCC AAT ACT TGG AGA GGG AGT GGG (SEQ ID NO:53),

[0121] GCC CTG AAA CCC TCG CGC TG (SEQ ID NO:55) and

[0122] GTA TCA TCG CCA CGG CGT CGG (SEQ ID NO:58).

[0123] When HBB is used as the internal reference gene, the latter can be detected, for example, by means of the following primers, forward and reverse, and probe respectively:

[0124] AGTGAGCTGCACTGTGACAA SEQ ID NO:66

[0125] CATCAAGCGTCCCATAGACTCA SEQ ID NO:67

[0126] CTGCACGTGGATCCTGAGAACTT SEQ ID NO:65. The probe can be bound to a fluorophore and quencher combination such as, for example, HEX- BHQ1.

[0127] The method according to the present invention can be applied to a biological sample such as, for example, a bacterial colony or a blood culture positive for enterobacteria, including Kpn.

[0128] It is well known that enterobacteria may be present in different human biological samples, such as, for example, rectal swabs, urine, blood, respiratory tract samples, wound swabs and drainage specimens. Therefore, the term bacterial colony means a biological sample comprising bacteria deriving from one of the abovementioned human biological samples.

[0129] According to the present invention, the enterobacterium with a hypervirulent genotype can be selected in the group consisting of Klebsiella pneumoniae, Escherichia coli, Yersinia spp and Enterobacter hormachei, preferably Klebsiella pneumoniae.

[0130] According to the method of the present invention, the detection of the enterobacterium with a hypervirulent genotype can be carried out by means of a technique selected from real-time PCR, end-point PCR or probe-based real-time PCR.

[0131] A further object of the present invention is a kit for detecting, in a biological sample, an enterobacterium with a hypervirulent genotype, said kit comprising or consisting of suitable primers and optionally also probes for detecting the following genes (i.e. a set of genes comprising the following genes): rmpA, rmpA2, rmpC, rmpD, magA, cnf-1, ybtA, ybtS, iucA, iroB, iroN and clbA, through the detection of the following nucleotide sequences:

[0132] ATGGGGAGGGTACAAAATGTTAAGGGGATCATTAAATATGATAAGCCA ATGGATGTGGCTTGACGTTTCGGGGGGGGGGCGGTTTTATCCTAAAGGGTG T SEQ ID NO:1 for the rmpA gene,

[0133] GTTAGCCGGATTGGAAATCATTACCCACAACTAACAAAAAAAGAACAAA TCATTCTACAATGCTTACTCTCCAGGAGGGAGGGCATCCATGAATTAAAAAGC CGTCTAAAAATTGAAGAGAAAACACTATCGTGTCACAGATG SEQ ID NO:2 for the rmpA2 gene,

[0134] AAGACTCCTTCTTTGTCAAATTTATTGGTTTATAATCAAGCTGCCCCCC CCCATTAAAGAGAGAAATCATTCTATCTTTTAAAATAGTGATAGCCTCGCGTG CGTTAATGATGTTAATTCCGATGAGGGTGGAATTA SEQ ID NO:3 for the rmpC gene,

[0135] GCTTTGTTGTAAGTGGACCAGTTGGTGATTCTGAACTTTTGCTTTGCCA CGGAATGGTCTGAGTTGTCGGGAGGATGCGTGATCTGATCCTTCAACTCAGC AAAAGTTCGATTTATTCAACAAAGCCGCGTCTATTCATCGCTTTTC SEQ ID NO:4 for the rmpD gene,

[0136] AACGAATTGATGCTTGGCATCATGCAATAGCCACGTTTTATGAGCATCC AATACTTGGAGAGGGAGTGGGAATATTCATTCAAAGATTCAACATGTACCCTC ATAATATATTCTTCGAATCTGCAGCAGAAA SEQ ID NO:5 for the magA gene,

[0137] AACGTCGTTCTGCTATACCTGGTTTGGCGACAAATGCAGTACTGCTTG GTCTGTCTTTTGCAATTCCACTCTTGATTAGTAAGGCAGGAATAAACCAGGA SEQ ID NO:6 for the cnf-1 gene,

[0138] TTCGAGAGCATTACCCTCTCCTTCAGCCCTGAAACCCTCGCGCTGTGG

[0139] GTGCCGGATATTAGCGCGGTCATTAAGAATAAGATCGACTCCCACTGCTGTT GCCAGCAGCAGCGTTGTAATGCTGAAACCCA SEQ ID NO:7 for the ybtA gene, CTGGCGAACCGACTTTAAATCTTCAACCACCACGCTGCCCGGCAGGC

[0140] AAACGGCCTCCAGTTCAGCAATCGCTTCTTTGAC SEQ ID N0:8 for the ybtS gene,

[0141] GTCCGTACTCTGTCTGTGAAAGAGGTGGAGCGAGGAATGCGCCTGGC ACGTCTGGCGCAAACCGACGGCTGGCAGATGCTACAGGCCCGCTTCCCTAC TTTCCGGGTAATG SEQ ID N0:9 for the iucA gene,

[0142] GGCATATCAAAGGCGTAACGCGTTCGCTTGCGGACACGTATCATCGC CACGGCGTCGGGGCCGCACCGCGCGATATGGCCTGGATCGACGTA SEQ ID NO: 10 for the iroB gene,

[0143] GTATCTTCACTGCGGGTTTCCGCATGAGTACGCGGTTTTTGTCTGCCA TATAACGTCCAGTTGACGCTGGCAGAAAACGCCTGGGTGATGGTCCAGTTAA GCGAGTTATTGATAGTATATTTCGGGATGACCGACAGAGGATTACC SEQ ID NO:11 for the iroN gene, and

[0144] AGATACAGTAGCCGTTGCTATTAGTTCTCACTGCGAGCTTGGTGTCGA TATTGAACAAATAAAAGATTTAAACAACTCTTATCTGAATATCAGTCAGCATTTT TTTACTCCACAGGAAGCTACTAACA SEQ ID NO: 12 for the clbA gene.

[0145] According to the present invention, the kit can further comprise primers and optionally also probes for detecting one or more or all of the following genes: peg- 344, hlyA, fimH and mrkD, through the detection of the following nucleotide sequences:

[0146] TCGCATTAGCTTCACTTTCGTTCATCCTCATCACGGAGGCGCGGGAAA GGACAGAAAGCCAGTGGACGGGAGTTATTGCCCTTTCTTCTGCAGTA SEQ ID NO: 13 for the peg-344 gene,

[0147] GAGAATGGCTATGACGCAAGACATGCTGCGTTTTTAGAAGACTCTCTG TCTTTGCTTGCTGATCTTTCTCGTCAACATGGAGTAGAAAGAGCAGTCGCAAT AACCCAGCAACATTGGGATGAGAAGATCGGTGAACTTGC SEQ ID NO: 14 for the hlyA gene,

[0148] TGTGCTGTCGAGTTTTTCAGGCACCGTGAAATATAACGGCACCTCTTA CCCGTTCCCGACCACCACGGAAACCGCGCGGGTGATTTATGATTCACGGAC CGATAAACC SEQ ID NO: 15 for the fimH gene and

[0149] GGGTAAGTACACCTCCTACGACTGGGAGAGCGGCGGTAACCCGATCC TCGAAACCTATCTGAGCGCCAACGCCATCACCGTGGTCTCGCCCTCCTGTTC GGTGCTGAGCGGGAAAAATATG SEQ ID NO: 16 for the mrkD gene. Therefore, according to the present invention the kit is adapted to detect the entire set of genes which are indicators of hypervirulence, consisting of the rmpA, rmpA2, rmpC, rmpD, magA, cnf-1, ybtA, ybtS, iucA, iroB, iroN and clbA genes, in order to obtain a profile of hypervirulence. Moreover, the kit can also provide for the detection of further genes such as peg-344, hlyA, fimH and mrkD with the aim of completing the profile of hypervirulence (where a positive result has already been obtained for the previous genes) or ascertaining the presence thereof in the event of a previous negative result. The kit can provide for the detection of one, two, three or four of said further genes. When the kit provides for the detection of more than one gene of said further genes, the detection can regard different combinations of said further genes come, for example peg-344, hlyA, fimH and mrkD', peg-344, hlyA, fimH', hlyA, fimH and mrkD', peg-344, mrkD, hlyA, peg-344, mrkD, fimH', peg-344, hlyA, hlyA, fimH', fimH and mrkD; peg-344 and fimH', peg-344 and mrkD; hlyA and mrkD.

[0150] According to one embodiment, kits with different “panels” can be created, each one for the detection of a set of genes. For example, the following panels can be created.

[0151] Panel 1 Hypermucoid: rmpA rmpA2 rmpC HBB

[0152] Panel 2 Hypermucoid: magA rmpD cnf-1 HBB

[0153] Panel 3 Hypervirulence: ybtA ybtS iucA HBB

[0154] Panel 4 Hypervirulence: iroB iroN clbA

[0155] HBB

[0156] Panel 5 Hypervirulence: peg-344 hlyA

[0157] HBB

[0158] Panel 6 Hypervirulence: fimH mrkD

[0159] HBB

[0160] The panels that are essential for the purpose of obtaining a profile of hypervirulence, thus the first ones on which a positive blood culture or the bacterial colony must be tested, are panels 1 , 2, 3 and 4. Panels 5 and 6 can complete the profile of hypervirulence (where a positive result has already been obtained from the previous panels) or ascertain its presence in the event of a previous negative result.

[0161] It is well known that gene detection kits also comprise internal controls. In the kit according to the present invention, HBB, for example, can be used as the internal control when the analysis is performed on the biological sample.

[0162] According to a preferred embodiment of the kit of the present invention, said kit comprises the following forward and reverse primers for detecting said nucleotide sequences:

[0163] ATGGGGAGGG TACAAAATGT SEQ ID NO: 17

[0164] ACACCCTTTA GGATAAAACC G SEQ ID NO: 18 for the rmpA gene,

[0165] GTTAGCCGGA TTGGAAATCA SEQ ID NO: 19

[0166] CATCTGTGAC ACGATAGTGT SEQ ID NQ:20 for the rmpA2 gene,

[0167] >

[0168] AAGACTCCTT CTTTGTCAAA TTT SEQ ID NO:21

[0169] TAATTCCACC CTCATCGGAA SEQ ID NO:22 for the rmpC gene,

[0170] GCTTTGTTGTAAGTGGACCA SEQ ID NO:23

[0171] GAAAAGCGAT GAATAGACGC SEQ ID NO:24 for the rmpD gene, AACGAATTGA TGCTTGGCAT SEQ ID NO:25

[0172] TTTCTGCTGC AGATTCGAAG SEQ ID NO:26 for the magA gene,

[0173] AACGTCGTTC TGCTATACCT SEQ ID NO:27

[0174] TCCTGGTTTA TTCCTGCCTT SEQ ID NO:28 for the cnf-1 gene,

[0175] TTCGAGAGCA TTACCCTCTC SEQ ID NO:29

[0176] TGGGTTTCAG CATTACAACG SEQ ID NQ:30 for the ybtA gene,

[0177] CTGGCGAACC GACTTTAAA SEQ ID N0:31

[0178] GTCAAAGAAG CGATTGCTGA SEQ ID NO:32 for the ybtS gene,

[0179] GTCCGTACTC TGTCTGTGAA SEQ ID NO:33

[0180] CATTACCCGG AAAGTAGGGA SEQ ID NO:34 for the iucA gene,

[0181] GGCATATCAA AGGCGTAACG SEQ ID NO:35

[0182] TACGTCGATC CAGGCCATA SEQ ID NO:36 for the iroB gene, GTATCTTCACTGCGGGTTTC SEQ ID NO:37

[0183] GGTAATCCTCTGTCGGTCAT SEQ ID NO:38 for the iroN gene,

[0184] AGATACAGTA GCCGTTGCTA T SEQ ID NO:39

[0185] TGTTAGTAGC TTCCTGTGGA G SEQ ID NQ:40 for the clbA gene,

[0186] TCGCATTAGC TTCACTTTCG SEQ ID N0:41

[0187] TACTGCAGAA GAAAGGGCAA SEQ ID NO:42 for the peg-344 gene,

[0188] GAGAATGGCT ATGACGCAAG SEQ ID NO:43

[0189] GCAAGTTCAC CGATCTTCTC SEQ ID NO:44 for the hlyA gene,

[0190] TGTGCTGTCG AGTTTTTCAG SEQ ID NO:45

[0191] GGTTTATCGG TCCGTGAATC SEQ ID NO:46 for the fimH gene and

[0192] GGGTAAGTAC ACCTCCTACG SEQ ID NO:47 CATATTTTTC CCGCTCAGCA SEQ ID NO:48 for the mrkD gene.

[0193] According to a further preferred embodiment, the kit further comprises probes in combination with the respective primers used for each of said genes, wherein said probes can be, for example, the following:

[0194] AGC CAA TGG ATG TGG CTT GAC GTT TC SEQ ID NO:49 for the rmpA gene,

[0195] TCT CCA GGA GGG AGG GCA TCC A SEQ ID NQ:50 for the rmpA2 gene,

[0196] AGT GAT AGC CTC GCG TGC GTT AAT GA SEQ ID NO:51 for the rmpC gene,

[0197] GCC ACG GAA TGG TCT GAG TTG TCG SEQ ID NO:52 for the rmpD gene,

[0198] GCA TCC AAT ACT TGG AGA GGG AGT GGG SEQ ID NO:53 for the magA gene,

[0199] TGG CGA CAA ATG CAG TAC TGC TTG G SEQ ID NO:54 for the cnf-1 gene,

[0200] GCC CTG AAA CCC TCG CGC TG SEQ ID NO:55 for the ybtA gene,

[0201] TTC AAC CAC CAC GCT GCC CG SEQ ID NO:56 for the ybtS gene,

[0202] AAC CGA CGG CTG GCA GAT GC SEQ ID NO:57 for the iucA gene,

[0203] GTA TCA TCG CCA CGG CGT CGG SEQ ID NO:58 for the iroB gene,

[0204] GCT GGC AGA AAA CGC CTG GGT SEQ ID NO:59 for the iroN gene,

[0205] TCT CAC TGC GAG CTT GGT GTC GA SEQ ID NQ:60 for the clbA gene,

[0206] TCA TCA CGG AGG CGC GGG AA SEQ ID NO:61 for the peg-344 gene,

[0207] GAG CAG TCG CAA TAA CCC AGC AAC A SEQ ID NO:62 for the hlyA gene,

[0208] CCG ACC ACC ACG GAA ACC GC SEQ ID NO:63 for the fimH gene and

[0209] AGC GGC GGT AAC CCG ATC CT SEQ ID NO:64 for the mrkD gene.

[0210] According to one embodiment of the present invention, the kit does not comprise primers and optionally also probes for detecting further genes other than the ones listed above, with the exception of the genes whose detection is necessary for implementing the detection technique applied, such as the internal control genes.

[0211] Furthermore, according to the present invention, the probes of the kit can be bound at the 5’ end to a fluorophore and at the 3’ end to a quencher. According to the present invention the fluorophore can be selected in the group consisting of FAM, HEX, ROX and CY5. Furthermore, the quencher can be selected in the group consisting of BHQ1 , BHQ2 and BBQ 650. Preferably, use can be made of a fluorophore and quencher combination selected from FAM-BHQ1 , HEX-BHQ1 , ROX-BHQ2 or CY5-BBQ 650.

[0212] According to specific embodiments of the kit of the present invention, the FAM-BHQ1 combination is bound to the following probes:

[0213] AGT GAT AGC CTC GCG TGC GTT AAT GA (SEQ ID NO:51 ), TGG CGA CAA ATG CAG TAC TGC TTG G (SEQ ID NO:54), AAC CGA CGG CTG GCA GAT GC (SEQ ID NO:57), TCT CAC TGC GAG CTT GGT GTC GA (SEQ ID NQ:60), GAG CAG TCG CAA TAA CCC AGC AAC A (SEQ ID NO:62) and AGC GGC GGT AAC CCG ATC CT (SEQ ID NO:64); the ROX-BHQ2 combination is bound to the following probes:

[0214] TCT CCA GGA GGG AGG GCA TCC A (SEQ ID NQ:50),

[0215] GCC ACG GAA TGG TCT GAG TTG TCG (SEQ ID NO:52),

[0216] TTC AAC CAC CAC GCT GCC CG (SEQ ID NO:56),

[0217] GCT GGC AGA AAA CGC CTG GGT (SEQ ID NO:59), TCA TCA CGG AGG CGC GGG AA (SEQ ID NO:61 ) and CCG ACC ACC ACG GAA ACC GC (SEQ ID NO:63); the CY5-BBQ650 combination is bound to the following probes:

[0218] AGC CAA TGG ATG TGG CTT GAC GTT T (SEQ ID NO:49),

[0219] GCA TCC AAT ACT TGG AGA GGG AGT GGG (SEQ ID NO:53), GCC CTG AAA CCC TCG CGC TG (SEQ ID NO:55) and GTA TCA TCG CCA CGG CGT CGG (SEQ ID NO:58). When HBB is used as the internal reference gene, the kit comprises primers and optionally also probes for detecting this gene, for example the following primers, forward and reverse, and probe respectively:

[0220] AGTGAGCTGCACTGTGACAA SEQ ID NO:66

[0221] CATCAAGCGTCCCATAGACTCA SEQ ID NO:67

[0222] CTGCACGTGGATCCTGAGAACTT SEQ ID NO:65. The probe can be bound to a fluorophore and quencher combination such as, for example, HEX- BHQ1.

[0223] According to the present invention, the biological sample can be selected from a bacterial colony or a blood culture positive for enterobacteria, including Kpn.

[0224] It is well known that enterobacteria can be present in different human biological samples, such as, for example, rectal swabs, urine, blood, respiratory tract samples, wound swabs and drainage specimens. Therefore, the term bacterial colony means a biological sample comprising bacteria deriving from one of the abovementioned human biological samples.

[0225] The enterobacterium with a hypervirulent genotype can be selected in the group consisting of Klebsiella pneumoniae, Escherichia coli, Yersinia spp and Enterobacter hormachei, preferably Klebsiella pneumoniae.

[0226] The detection of the enterobacterium with a hypervirulent genotype can be carried out by means of a technique selected from real-time PCR, end-point PCR or probe-based real-time PCR.

[0227] The present invention will now be described by way of non-limiting illustration with particular reference to some illustrative examples and to the figures of the appended drawings, wherein:

[0228] - figure 1 shows the amplification curves obtained for a strain positive for the ybtA (CY5), ybtS (ROX) and iucA (FAM) genes;

[0229] - figure 2 shows the amplification curve of the internal control HBB (haemoglobin beta) (HEX);

[0230] - figure 3 shows the amplification curves obtained for a strain positive for the rmpA2 (ROX) and rmpC (FAM) genes;

[0231] - figure 4 shows the amplification curves obtained for a strain positive for the fimH (ROX) and mrkD (FAM) genes;

[0232] - figure 5 shows a sensitivity test performed using the synthetic ybtS gene;

[0233] - figure 6 shows a sensitivity test performed using the synthetic ybtA gene; - figure 7 shows a sensitivity test performed using the synthetic iucA gene;

[0234] - figure 8 shows a sensitivity test performed using the synthetic fimH gene;

[0235] - figure 9 shows a sensitivity test performed using the synthetic mrkD gene;

[0236] - figure 10 shows a sensitivity test performed using the synthetic peg-344 gene;

[0237] - figure 11 shows a sensitivity test performed using the synthetic hlyA gene.

[0238] EXAMPLE 1: Study for the development of the method and kit according to the present invention

[0239] Materials and Methods

[0240] After a thorough study of the available literature present on PubMed® (https: / / pubmed.ncbi.nlm.nih.gov / ), the genes of interest associated with the hypervirulence of enterobacteria were selected. The nucleotide sequence of every gene was obtained via the online platform of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ).

[0241] For the purposes of the amplification reaction, no previous extraction of the bacterial DNA is necessary. This results in a shortening of the overall response times.

[0242] The genes selected to create the panels for probe-based real-time PCR are the following: rmpA, rmpA2, rmpC, rmpD, mag A, cnf-1, ybtA, ybtS, iucA, iroB, iroN, clbA, peg-344, hlyA, fimH and mrkD.

[0243] Each of these genes is responsible for the profile of hypervirulence in Kpn; the determinants of virulence of each gene present in the panels of the invention are listed below:

[0244] Determinant of virulence: Hypermucoviscosity :

[0245] • rmpA, rmpA2, rmpC, rmpD (regulators of the mucoid phenotype via increased capsule production)

[0246] • mag A (mucoviscosity -associated gene A)

[0247] Determinant of virulence: Siderophore production:

[0248] • iucA (aerobactin siderophore biosynthesis)

[0249] • iroB, iroN (salmochelin siderophore biosynthesis)

[0250] • ybtA, ybtS (yersiniabactin)

[0251] • peg-344 (putative transporter of metabolites)

[0252] Determinant of virulence: Colibactin: • clbA (colibactin A)

[0253] Determinant of virulence: Adhesin and Fimbriae:

[0254] • mrkD (gene encoding adhesin MrkD)

[0255] • fimH (gene encoding the minor subunit FimH)

[0256] Determinant of virulence: Toxins:

[0257] • hlyA (haemolysin A)

[0258] • cnf-1 (cytotoxic necrotizing factor-1)

[0259] The nucleotide sequences, obtained in the FASTA format, were subsequently analysed with the specific online primer design program, Primer- BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ).

[0260] Once the primers and probes of interest had been obtained, it was checked whether any nonspecific pairings were present which would prejudice their subsequent quality, using the specific online program for verifying pairings, Clustal Omega Multiple Sequence Alignment (MSA)

[0261] (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo).

[0262] The forward and reverse primers and the respective probes selected are shown in table 1 for each gene. Each probe is bound to a fluorophore at the 5’ end and to a quencher at the 3’ end.

[0263] Table 1

[0264] As shown above, the present invention provides for the probes to be bound to a fluorophore and a quencher. Different fluorophore-quencher combinations are possible; the ones selected as the preferred combination are the following: FAM- BHQ1 , HEX-BHQ1 , R0X-BHQ2, and CY5-BBQ650.

[0265] The selected fluorophores have the QY (quantum yield) value closest to 1 : this value indicates the energy efficiency of the fluorophore when it is subjected to light energy: the higher the QY value of the fluorescence, the more luminous the fluorophore and the stronger the intensity of the fluorescence signal emitted. This means a better sensitivity of the test, i.e. the possibility of detecting smaller quantities of the target.

[0266] The selected BHQ quenchers, specific for the fluorophores used, possess the highest capacity to absorb the energy emitted by the fluorophore and offer the best guarantee of eliminating background noise.

[0267] Table 2 below indicates the probes with the respective selected fluorophores and quenchers bound at the 5‘ and 3‘ ends, respectively, of the probe.

[0268] Table 2

[0269] Once the absence of nonspecific pairing was ascertained, the primers and probes were tested on bacterial suspensions and on a positive inoculated blood culture The Taq polymerase used possesses its own predefined standard buffer, while the primers and probe were brought to the working concentration by adding specific H2O (nuclease-free H2O).

[0270] Various experimental tests were carried out to establish the correct working concentration both of the primers and of the probes. Table 3 below shows the selected working concentrations.

[0271] Table 3

[0272]

[0273] A ready-to-use master mix containing dNTPs and a buffer containing DNAse and MgCl2 inhibitors to stabilise the reaction, in the optimal concentrations, was chosen for the amplification reaction.

[0274] The designed panels were tested on bacterial suspensions and on a positive inoculated blood culture. It is a future objective to perform tests on a larger number of positive blood cultures in order to increase the case records.

[0275] The bacterial suspensions were obtained from colonies cultured on plates containing solid culture media, identified by MALDI-TOF mass spectrometry. In order to standardise the quantity of colony-forming units (CFU) per ml of H2O, the bacterial suspensions were prepared starting from a 0.5 McFarland standard.

[0276] A 1 ml aliquot was taken from the positive inoculated blood culture and centrifuged twice: the supernatant was collected after the first centrifugation at 500 g and centrifuged a second time at 5000 g to obtain the pellet. The pellet was resuspended in nuclease-free H2O.

[0277] The following reaction scheme was used for each panel:

[0278] Taq polymerase: 5 pl

[0279] Primer-Probe mix (PPmix): 2 pl

[0280] Sample: 1 pl

[0281] H2O: 2 pl

[0282] Various experimental tests were carried out to obtain the most suitable thermal profile for the reaction. The thermal profile chosen is the following:

[0283] Denaturation: 95 °C for 1 minute and 30 seconds

[0284] Primer pairing: 95 °C for 10 seconds

[0285] Polymerisation: 60°C for 30 seconds

[0286] The reaction was set at 35 cycles, with a response time of about 1 hour.

[0287] The result is considered positive when even only one curve corresponding to the genes present in the panels is amplified, whereas it is considered negative if there is either an absence of amplification curves (starting from bacterial suspensions) or amplification solely of the curve corresponding to haemoglobin beta (starting from a positive blood culture).

[0288] The amplification reaction was designed so as to favour the detection of the genes that were the object of study rather than the internal control.

[0289] The sensitivity tests were carried out using synthetic genes obtained for every gene forming the object of our invention. Details relating to the preparation of the genes and sensitivity tests are given below.

[0290] The synthetic genes were received lyophilised with a weight of 100 ng. They were resuspended in 1 ml of nuclease-free H2O so as to have a concentration of 0.1 ng / pl.

[0291] Serial dilutions were made starting from this initial concentration in order to prepare the standard curve and test both the quality of the gene fragments and the yield of the primers on the new sequence; the latter was then used as our standard.

[0292] Dilutions:

[0293] - take 10 pl from the stock solution (total 1 ng)

[0294] - make a 1 :2 dilution to obtain 0.5 ng / pl,

[0295] - from this first dilution, make the 1 : 10 serial dilutions (the first dilution will be 0.05 ng / pl).

[0296] The quantity per microlitre was chosen so that 2 pl of every dilution could be introduced into the PCR mix. The dilutions made are shown below (only the quantity in ng is shown, since every gene has a specific number of copies per pl, which varies based on the length of the nucleotide sequence):

[0297] 1 ) 1 ng

[0298] 2) 0.1 ng

[0299] 3) 0.01 ng

[0300] 4) 0.001 ng

[0301] 5) 0.0001 ng

[0302] 6) 0.00001 ng

[0303] 7) 0.000001 ng

[0304] 8) 0.0000001 ng

[0305] 9) 0.00000001 ng

[0306] Results The results obtained from the tests carried out are given in Table 4 below. The table shows the genes detected by the test, the type of samples analysed, with the respective identification code and the tested microorganism.

[0307] Table 4

[0308] References

[0309] Arena, F., Menchinelli, G., of Pilato, V., Torelli, R., Antonelli, A., Henrici De Angelis, L., . . Rossolini, G. M. (2022). Resistance and virulence features of hyperm ucoviscous. Front Microbiol, 13, 983294. https: / / doi.org / 10.3389 / fmicb.2022.983294

[0310] Ballen, V., Gabasa, Y., Ratia, C., Ortega, R., Tejero, M., & Soto, S. (2021 ). Antibiotic Resistance and Virulence Profiles of. Front Cell Infect Microbiol, 11 , 738223. https: / / doi.Org / 10.3389 / fcimb.2021 .738223

[0311] Eisenmenger, E. F., Guajardo, E., Finch, N., Atmar, R. L., & Sargsyan, Z.

[0312] (2021 ). 'String Test' for Hyperm ucoviscous Klebsiella pneumoniae. Am J Med, 134(10), e520-e521 . https: / / doi.Org / 10.1016 / j.amjmed.2O21 .03.047

[0313] Dai, P., Hu, D. (2022). The making of hypervirulent Klebsiella pneumoniae. J Clin Lab Anal, 2022 Dec; 36(12):e24743. doi: 10.1002 / jcla.24743. Epub 2022 Nov 8. PMID: 36347819; PMCID: PMC9757020. doi: 10.1002 / jcla.24743.

[0314] Fatima, S., Liaqat, F., Akbar, A., Sahfee, M., Samad, A., Anwar, M., . . . Khan, A. (2021 ). Virulent and multidrug-resistant Klebsiella pneumoniae from clinical samples in Balochistan. Int Wound J, 18(4), 510-518. https: / / doi.Org / 10.1111 / iwj.13550

[0315] Holt, K. E., Wertheim, H., Zadoks, R. N., Baker, S., Whitehouse, C. A., Dance, D., . . . Thomson, N. R. (2015). Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci U S A, 112(27), E3574-3581. https: / / doi.Org / 10.1073 / pnas.1501049112

[0316] Kot, B., Piechota, M., Szweda, P., Mitrus, J., Wicha, J., Gruzewska, A., & Witeska, M. (2023). Virulence analysis and antibiotic resistance of Klebsiella pneumoniae isolates from hospitalised patients in Poland. Sci Rep, 13(1 ), 4448. https: / / doi.Org / 10.1038 / s41598-023-31086-w

[0317] Lam, M. M. C., Wick, R. R., Watts, S. C., Cerdeira, L. T., Wyres, K. L., & Holt, K. E. (2021 ). A genomic surveillance framework and genotyping tool for Klebsiella pneumoniae and its related species complex. Nat Commun, 12(1 ), 4188. https: / / doi.Org / 10.1038 / s41467-021 -24448-3

[0318] Russo, T. A., & Marr, C. M. (2019). Hypervirulent Klebsiella pneumoniae. Clin Microbiol Rev, 32(3). https: / / doi.org / 10.1128 / CMR.00001 -19

[0319] Sohrabi, M., Alizade Naini, M., Rasekhi, A., Oloomi, M., Moradhaseli, F., Ayoub, A., Badmasti, F. (2022). Emergence of K1 ST23 and K2 ST65 hypervirulent. Front Cell Infect Microbiol, 12, 964290. https: / / doi.org / 10.3389 / fcimb.2022.964290

[0320] Yu, W. L., Ko, W. C., Cheng, K. C., Lee, H. C., Ke, D. S., Lee, C. C., Chuang, Y. C. (2006). Association between rmpA and magA genes and clinical syndromes caused by Klebsiella pneumoniae in Taiwan. Clin Infect Dis, 42(10), 1351 -1358. https: / / www.ecdc.europa.eu / en / news-events / increase-hypervirulent-carbapenem- resistant-klebsiella-pneumoniae-eueea

[0321] The present invention has been described by way of non-limiting illustration according to the preferred embodiments thereof, but it is understood that variations and / or modifications may be introduced by the person skilled in the art without going outside the scope of protection hereof, as defined by the appended claims.

Claims

CLAIMS1 ) An in vitro method for detecting, in a biological sample, an enterobacterium with a hypervirulent genotype, said method comprising detecting the following genes: rmpA, rmpA2, rmpC, rmpD, magA, cnf-1, ybtA, ybtS, iucA, iroB, iroN and clbA through the detection of the following nucleotide sequences:ATGGGGAGGGTACAAAATGTTAAGGGGATCATTAAATATGATAAGCCA ATGGATGTGGCTTGACGTTTCGGGGGGGGGGCGGTTTTATCCTAAAGGGTG T SEQ ID NO:1 for the rmpA gene,GTTAGCCGGATTGGAAATCATTACCCACAACTAACAAAAAAAGAACAAA TCATTCTACAATGCTTACTCTCCAGGAGGGAGGGCATCCATGAATTAAAAAGC CGTCTAAAAATTGAAGAGAAAACACTATCGTGTCACAGATG SEQ ID NO:2 for the rmpA2 gene,AAGACTCCTTCTTTGTCAAATTTATTGGTTTATAATCAAGCTGCCCCCC CCCATTAAAGAGAGAAATCATTCTATCTTTTAAAATAGTGATAGCCTCGCGTG CGTTAATGATGTTAATTCCGATGAGGGTGGAATTA SEQ ID NO:3 for the rmpC gene,GCTTTGTTGTAAGTGGACCAGTTGGTGATTCTGAACTTTTGCTTTGCCA CGGAATGGTCTGAGTTGTCGGGAGGATGCGTGATCTGATCCTTCAACTCAGC AAAAGTTCGATTTATTCAACAAAGCCGCGTCTATTCATCGCTTTTCSEQ ID NO:4 for the rmpD gene,AACGAATTGATGCTTGGCATCATGCAATAGCCACGTTTTATGAGCATCC AATACTTGGAGAGGGAGTGGGAATATTCATTCAAAGATTCAACATGTACCCTC ATAATATATTCTTCGAATCTGCAGCAGAAA SEQ ID NO:5 for the magA gene,AACGTCGTTCTGCTATACCTGGTTTGGCGACAAATGCAGTACTGCTTG GTCTGTCTTTTGCAATTCCACTCTTGATTAGTAAGGCAGGAATAAACCAGGA SEQ ID NO:6 for the cnf-1 gene,TTCGAGAGCATTACCCTCTCCTTCAGCCCTGAAACCCTCGCGCTGTGG GTGCCGGATATTAGCGCGGTCATTAAGAATAAGATCGACTCCCACTGCTGTT GCCAGCAGCAGCGTTGTAATGCTGAAACCCA SEQ ID NO:7 for the ybtA gene, CTGGCGAACCGACTTTAAATCTTCAACCACCACGCTGCCCGGCAGGC AAACGGCCTCCAGTTCAGCAATCGCTTCTTTGAC SEQ ID NO:8 for the ybtS gene,GTCCGTACTCTGTCTGTGAAAGAGGTGGAGCGAGGAATGCGCCTGGCACGTCTGGCGCAAACCGACGGCTGGCAGATGCTACAGGCCCGCTTCCCTAC TTTCCGGGTAATG SEQ ID NO:9 for the iucA gene,GGCATATCAAAGGCGTAACGCGTTCGCTTGCGGACACGTATCATCGC CACGGCGTCGGGGCCGCACCGCGCGATATGGCCTGGATCGACGTA SEQ ID NO: 10 for the iroB gene,GTATCTTCACTGCGGGTTTCCGCATGAGTACGCGGTTTTTGTCTGCCA TATAACGTCCAGTTGACGCTGGCAGAAAACGCCTGGGTGATGGTCCAGTTAA GCGAGTTATTGATAGTATATTTCGGGATGACCGACAGAGGATTACC SEQ ID NO:11 for the iroN gene, andAGATACAGTAGCCGTTGCTATTAGTTCTCACTGCGAGCTTGGTGTCGA TATTGAACAAATAAAAGATTTAAACAACTCTTATCTGAATATCAGTCAGCATTTT TTTACTCCACAGGAAGCTACTAACA SEQ ID NO: 12 for the clbA gene.2) The method according to claim 1 , wherein said method further comprises detecting one or more or all of the following genes: peg-344, hlyA, fimH and mrkD through the detection of the following nucleotide sequences:TCGCATTAGCTTCACTTTCGTTCATCCTCATCACGGAGGCGCGGGAAA GGACAGAAAGCCAGTGGACGGGAGTTATTGCCCTTTCTTCTGCAGTA SEQ ID NO: 13 for the peg-344 gene,GAGAATGGCTATGACGCAAGACATGCTGCGTTTTTAGAAGACTCTCTG TCTTTGCTTGCTGATCTTTCTCGTCAACATGGAGTAGAAAGAGCAGTCGCAAT AACCCAGCAACATTGGGATGAGAAGATCGGTGAACTTGC SEQ ID NO: 14 for the hlyA gene,TGTGCTGTCGAGTTTTTCAGGCACCGTGAAATATAACGGCACCTCTTA CCCGTTCCCGACCACCACGGAAACCGCGCGGGTGATTTATGATTCACGGAC CGATAAACC SEQ ID NO: 15 for the fimH gene andGGGTAAGTACACCTCCTACGACTGGGAGAGCGGCGGTAACCCGATCC TCGAAACCTATCTGAGCGCCAACGCCATCACCGTGGTCTCGCCCTCCTGTTC GGTGCTGAGCGGGAAAAATATG SEQ ID NO: 16 for the mrkD gene.3) The method according to any one of the preceding claims, wherein said nucleotide sequences are detected by using the following primers:ATGGGGAGGG TACAAAATGT SEQ ID NO: 17ACACCCTTTA GGATAAAACC G SEQ ID NO: 18 for the rmpA gene,GTTAGCCGGA TTGGAAATCA SEQ ID NO: 19CATCTGTGAC ACGATAGTGT SEQ ID NO:20 for the rmpA2 gene,AAGACTCCTT CTTTGTCAAA TTT SEQ ID N0:21TAATTCCACC CTCATCGGAA SEQ ID NO:22 for the rmpC gene,GCTTTGTTGTAAGTGGACCA SEQ ID NO:23GAAAAGCGAT GAATAGACGC SEQ ID NO:24 for the rmpD gene,AACGAATTGA TGCTTGGCAT SEQ ID NO:25TTTCTGCTGC AGATTCGAAG SEQ ID NO:26 for the magA gene,AACGTCGTTC TGCTATACCT SEQ ID NO:27TCCTGGTTTA TTCCTGCCTT SEQ ID NO:28 for the cnf-1 gene,TTCGAGAGCA TTACCCTCTC SEQ ID NO:29TGGGTTTCAG CATTACAACG SEQ ID NQ:30 for the ybtA gene,CTGGCGAACC GACTTTAAA SEQ ID N0:31GTCAAAGAAG CGATTGCTGA SEQ ID NO:32 for the ybtS gene,GTCCGTACTC TGTCTGTGAA SEQ ID NO:33CATTACCCGG AAAGTAGGGA SEQ ID NO:34 for the iucA gene,GGCATATCAA AGGCGTAACG SEQ ID NO:35TACGTCGATC CAGGCCATA SEQ ID NO:36 for the iroB gene,GTATCTTCACTGCGGGTTTC SEQ ID NO:37GGTAATCCTCTGTCGGTCAT SEQ ID NO:38 for the iroN gene,AGATACAGTA GCCGTTGCTA T SEQ ID NO:39TGTTAGTAGC TTCCTGTGGA G SEQ ID NQ:40 for the clbA gene,TCGCATTAGC TTCACTTTCG SEQ ID N0:41 TACTGCAGAA GAAAGGGCAA SEQ ID NO:42for the peg-344 gene,GAGAATGGCT ATGACGCAAG SEQ ID NO:43GCAAGTTCAC CGATCTTCTC SEQ ID NO:44 for the hlyA gene,TGTGCTGTCG AGTTTTTCAG SEQ ID NO:45GGTTTATCGG TCCGTGAATC SEQ ID NO:46 for the fimH gene andGGGTAAGTAC ACCTCCTACG SEQ ID NO:47CATATTTTTC CCGCTCAGCA SEQ ID NO:48 for the mrkD gene4) The method according to claim 3, said method further comprising the use of probes in combination with the respective primers used for each of said genes, wherein said probes are the following:AGC CAA TGG ATG TGG CTT GAC GTT TC SEQ ID NO:49 for the rmpA gene,TCT CCA GGA GGG AGG GCA TCC A SEQ ID NQ:50 for the rmpA2 gene,AGT GAT AGC CTC GCG TGC GTT AAT GA SEQ ID NO:51 for the rmpC gene,GCC ACG GAA TGG TCT GAG TTG TCG SEQ ID NO:52 for the rmpD gene,GCA TCC AAT ACT TGG AGA GGG AGT GGG SEQ ID NO:53 for the magA gene,TGG CGA CAA ATG CAG TAC TGC TTG G SEQ ID NO:54 for the cnf-1 gene,GCC CTG AAA CCC TCG CGC TG SEQ ID NO:55 for the ybtA gene,TTC AAC CAC CAC GCT GCC CG SEQ ID NO:56 for the ybtS gene,AAC CGA CGG CTG GCA GAT GC SEQ ID NO:57 for the iucA gene,GTA TCA TCG CCA CGG CGT CGG SEQ ID NO:58 for the iroB gene,GCT GGC AGA AAA CGC CTG GGT SEQ ID NO:59for the iroN gene,TCT CAC TGC GAG CTT GGT GTC GA SEQ ID NO:60 for the clbA gene,TCA TCA CGG AGG CGC GGG AA SEQ ID N0:61 for the peg-344 gene,GAG CAG TCG CAA TAA CCC AGC AAC A SEQ ID NO:62 for the hlyA gene,CCG ACC ACC ACG GAA ACC GC SEQ ID NO:63 for the fimH gene andAGC GGC GGT AAC CCG ATC CT SEQ ID NO:64 for the mrkD gene5) The method according to the preceding claim, wherein said probes are bound to a fluorophore and to a quencher.6) The method according to the preceding claim, wherein the fluorophore is selected in the group consisting of FAM, HEX, ROX and CY5.7) The method according to claim 5, wherein the quencher is selected in the group consisting of BHQ1 , BHQ2 and BBQ 650.8) The method according to any one of claims 5-7, wherein the fluorophore and quencher combination is selected from FAM-BHQ1 , HEX-BHQ1 , ROX-BHQ2 or CY5-BBQ 650.9) The method according to claim 7, wherein the FAM-BHQ1 combination is bound to the following probes:AGT GAT AGC CTC GCG TGC GTT AAT GA (SEQ ID NO:51 ),TGG CGA CAA ATG CAG TAC TGC TTG G (SEQ ID NO:54),AAC CGA CGG CTG GCA GAT GC (SEQ ID NO:57),TCT CAC TGC GAG CTT GGT GTC GA (SEQ ID NQ:60),GAG CAG TCG CAA TAA CCC AGC AAC A (SEQ ID NO:62) andAGC GGC GGT AAC CCG ATC CT (SEQ ID NO:64); the ROX-BHQ2 combination is bound to the following probes:TCT CCA GGA GGG AGG GCA TCC A (SEQ ID NQ:50),GCC ACG GAA TGG TCT GAG TTG TCG (SEQ ID NO:52),TTC AAC CAC CAC GCT GCC CG (SEQ ID NO:56),GCT GGC AGA AAA CGC CTG GGT (SEQ ID NO:59),TCA TCA CGG AGG CGC GGG AA (SEQ ID NO:61 ) andCCG ACC ACC ACG GAA ACC GC (SEQ ID NO:63); the CY5-BBQ650 combination is bound to the following probes:AGC CAA TGG ATG TGG CTT GAC GTT T (SEQ ID NO:49),GCA TCC AAT ACT TGG AGA GGG AGT GGG (SEQ ID NO:53),GCC CTG AAA CCC TCG CGC TG (SEQ ID NO:55) andGTA TCA TCG CCA CGG CGT CGG (SEQ ID NO:58).10) The method according to any one of the preceding claims, wherein the biological sample is selected from a bacterial colony or a blood culture positive for enterobacteria, including Kpn.11 ) The method according to any one of the preceding claims, wherein the enterobacterium with a hypervirulent genotype is selected in the group consisting of Klebsiella pneumoniae, Escherichia coli, Yersinia spp and Enterobacter hormaechei, preferably Klebsiella pneumoniae.12) The method according to any one of the preceding claims, wherein the detection of the enterobacterium with a hypervirulent genotype is carried out by means of a technique selected from real-time PCR, end-point PCR or probe-based real-time PCR.13) A kit for detecting, in a biological sample, an enterobacterium with a hypervirulent genotype, said kit comprising suitable primers and optionally probes for detecting the following genes: rmpA, rmpA2, rmpC, rmpD, magA, cnf-1, ybtA, ybtS, iucA, iroB, iroN and clbA, through the detection of the following nucleotide sequences:ATGGGGAGGGTACAAAATGTTAAGGGGATCATTAAATATGATAAGCCA ATGGATGTGGCTTGACGTTTCGGGGGGGGGGCGGTTTTATCCTAAAGGGTG T SEQ ID NO:1 for the rmpA gene,GTTAGCCGGATTGGAAATCATTACCCACAACTAACAAAAAAAGAACAAA TCATTCTACAATGCTTACTCTCCAGGAGGGAGGGCATCCATGAATTAAAAAGC CGTCTAAAAATTGAAGAGAAAACACTATCGTGTCACAGATG SEQ ID NO:2 for the rmpA2 gene,AAGACTCCTTCTTTGTCAAATTTATTGGTTTATAATCAAGCTGCCCCCC CCCATTAAAGAGAGAAATCATTCTATCTTTTAAAATAGTGATAGCCTCGCGTG CGTTAATGATGTTAATTCCGATGAGGGTGGAATTA SEQ ID NO:3 for the rmpC gene,GCTTTGTTGTAAGTGGACCAGTTGGTGATTCTGAACTTTTGCTTTGCCACGGAATGGTCTGAGTTGTCGGGAGGATGCGTGATCTGATCCTTCAACTCAGC AAAAGTTCGATTTATTCAACAAAGCCGCGTCTATTCATCGCTTTTC SEQ ID NO:4 for the rmpD gene,AACGAATTGATGCTTGGCATCATGCAATAGCCACGTTTTATGAGCATCC AATACTTGGAGAGGGAGTGGGAATATTCATTCAAAGATTCAACATGTACCCTC ATAATATATTCTTCGAATCTGCAGCAGAAA SEQ ID N0:5 for the magA gene,AACGTCGTTCTGCTATACCTGGTTTGGCGACAAATGCAGTACTGCTTG GTCTGTCTTTTGCAATTCCACTCTTGATTAGTAAGGCAGGAATAAACCAGGA SEQ ID N0:6 for the cnf-1 gene,TTCGAGAGCATTACCCTCTCCTTCAGCCCTGAAACCCTCGCGCTGTGG GTGCCGGATATTAGCGCGGTCATTAAGAATAAGATCGACTCCCACTGCTGTT GCCAGCAGCAGCGTTGTAATGCTGAAACCCA SEQ ID N0:7 for the ybtA gene,CTGGCGAACCGACTTTAAATCTTCAACCACCACGCTGCCCGGCAGGC AAACGGCCTCCAGTTCAGCAATCGCTTCTTTGAC SEQ ID N0:8 for the ybtS gene,GTCCGTACTCTGTCTGTGAAAGAGGTGGAGCGAGGAATGCGCCTGGC ACGTCTGGCGCAAACCGACGGCTGGCAGATGCTACAGGCCCGCTTCCCTAC TTTCCGGGTAATG SEQ ID N0:9 for the iucA gene,GGCATATCAAAGGCGTAACGCGTTCGCTTGCGGACACGTATCATCGC CACGGCGTCGGGGCCGCACCGCGCGATATGGCCTGGATCGACGTA SEQ ID NO: 10 for the iroB gene,GTATCTTCACTGCGGGTTTCCGCATGAGTACGCGGTTTTTGTCTGCCA TATAACGTCCAGTTGACGCTGGCAGAAAACGCCTGGGTGATGGTCCAGTTAA GCGAGTTATTGATAGTATATTTCGGGATGACCGACAGAGGATTACC SEQ ID N0:11 for the iroN gene, andAGATACAGTAGCCGTTGCTATTAGTTCTCACTGCGAGCTTGGTGTCGA TATTGAACAAATAAAAGATTTAAACAACTCTTATCTGAATATCAGTCAGCATTTT TTTACTCCACAGGAAGCTACTAACA SEQ ID NO: 12 for the clbA gene.14) The kit according to claim 13, wherein said kit further comprises primers and optionally also probes for detecting one or more or all of the following genes: peg-344, hlyA, fimH and mrkD, through the detection of the following nucleotide sequences:TCGCATTAGCTTCACTTTCGTTCATCCTCATCACGGAGGCGCGGGAAAGGACAGAAAGCCAGTGGACGGGAGTTATTGCCCTTTCTTCTGCAGTA SEQ IDNO: 13 for the peg-344 gene,GAGAATGGCTATGACGCAAGACATGCTGCGTTTTTAGAAGACTCTCTG TCTTTGCTTGCTGATCTTTCTCGTCAACATGGAGTAGAAAGAGCAGTCGCAAT AACCCAGCAACATTGGGATGAGAAGATCGGTGAACTTGC SEQ ID NO: 14 for the hlyA gene,TGTGCTGTCGAGTTTTTCAGGCACCGTGAAATATAACGGCACCTCTTA CCCGTTCCCGACCACCACGGAAACCGCGCGGGTGATTTATGATTCACGGAC CGATAAACC SEQ ID NO: 15 for the fimH gene andGGGTAAGTACACCTCCTACGACTGGGAGAGCGGCGGTAACCCGATCC TCGAAACCTATCTGAGCGCCAACGCCATCACCGTGGTCTCGCCCTCCTGTTC GGTGCTGAGCGGGAAAAATATG SEQ ID NO: 16 for the mrkD gene.15) The kit according to claims 13-14, wherein said kit comprises the following primers for detecting said nucleotide sequences:ATGGGGAGGG TACAAAATGT SEQ ID NO: 17ACACCCTTTA GGATAAAACC G SEQ ID NO: 18 for the rmpA gene,GTTAGCCGGA TTGGAAATCA SEQ ID NO: 19CATCTGTGAC ACGATAGTGT SEQ ID NQ:20 for the rmpA2 gene,>AAGACTCCTT CTTTGTCAAA TTT SEQ ID NO:21TAATTCCACC CTCATCGGAA SEQ ID NO:22 for the rmpC gene,GCTTTGTTGTAAGTGGACCA SEQ ID NO:23GAAAAGCGAT GAATAGACGC SEQ ID NO:24 for the rmpD gene,AACGAATTGA TGCTTGGCAT SEQ ID NO:25TTTCTGCTGC AGATTCGAAG SEQ ID NO:26 for the magA gene,AACGTCGTTC TGCTATACCT SEQ ID NO:27TCCTGGTTTA TTCCTGCCTT SEQ ID NO:28 for the cnf-1 gene,TTCGAGAGCA TTACCCTCTC SEQ ID NO:29TGGGTTTCAG CATTACAACG SEQ ID NQ:30 for the ybtA gene,CTGGCGAACC GACTTTAAA SEQ ID NO:31GTCAAAGAAG CGATTGCTGA SEQ ID NO:32 for the ybtS gene,GTCCGTACTC TGTCTGTGAA SEQ ID NO:33CATTACCCGG AAAGTAGGGA SEQ ID NO:34 for the iucA gene,GGCATATCAA AGGCGTAACG SEQ ID NO:35TACGTCGATC CAGGCCATA SEQ ID NO:36 for the iroB gene,GTATCTTCACTGCGGGTTTC SEQ ID NO:37GGTAATCCTCTGTCGGTCAT SEQ ID NO:38 for the iroN gene,AGATACAGTA GCCGTTGCTA T SEQ ID NO:39TGTTAGTAGC TTCCTGTGGA G SEQ ID NQ:40 for the clbA gene,TCGCATTAGC TTCACTTTCG SEQ ID N0:41TACTGCAGAA GAAAGGGCAA SEQ ID NO:42 for the peg-344 gene,GAGAATGGCT ATGACGCAAG SEQ ID NO:43GCAAGTTCAC CGATCTTCTC SEQ ID NO:44 for the hlyA gene,>TGTGCTGTCG AGTTTTTCAG SEQ ID NO:45GGTTTATCGG TCCGTGAATC SEQ ID NO:46 for the fimH gene andGGGTAAGTAC ACCTCCTACG SEQ ID NO:47CATATTTTTC CCGCTCAGCA SEQ ID NO:48 for the mrkD gene.16) The kit according to claims 14-15, said kit further comprising probes in combination with the respective primers used for each of said genes, wherein said probes are the following:AGC CAA TGG ATG TGG CTT GAC GTT TC SEQ ID NO:49 for the rmpA gene,TCT CCA GGA GGG AGG GCA TCC A SEQ ID NQ:50 for the rmpA2 gene,AGT GAT AGC CTC GCG TGC GTT AAT GA SEQ ID NO:51 for the rmpC gene,GCC ACG GAA TGG TCT GAG TTG TCG SEQ ID NO:52 for the rmpD gene,GCA TCC AAT ACT TGG AGA GGG AGT GGG SEQ ID NO:53 for the magA gene,TGG CGA CAA ATG CAG TAC TGC TTG G SEQ ID NO:54 for the cnf-1 gene,GCC CTG AAA CCC TCG CGC TG SEQ ID NO:55 for the ybtA gene,TTC AAC CAC CAC GCT GCC CG SEQ ID NO:56 for the ybtS gene,AAC CGA CGG CTG GCA GAT GC SEQ ID NO:57 for the iucA gene,GTA TCA TCG CCA CGG CGT CGG SEQ ID NO:58 for the iroB gene,GCT GGC AGA AAA CGC CTG GGT SEQ ID NO:59 for the iroN gene,TCT CAC TGC GAG CTT GGT GTC GA SEQ ID NQ:60 for the clbA gene,TCA TCA CGG AGG CGC GGG AA SEQ ID N0:61 for the peg-344 gene,GAG CAG TCG CAA TAA CCC AGC AAC A SEQ ID NO:62 for the hlyA gene,CCG ACC ACC ACG GAA ACC GC SEQ ID NO:63 for the fimH gene andAGC GGC GGT AAC CCG ATC CT SEQ ID NO:64 for the mrkD gene.17) The kit according to the preceding claim, wherein said probes are bound to a fluorophore and to a quencher.18) The kit according to the preceding claim wherein the fluorophore is selected in the group consisting of FAM, HEX, ROX and CY5.19) The kit according to claim 17, wherein the quencher is selected in the group consisting of BHQ1 , BHQ2 and BBQ 650.20) The kit according to any one of claims 17-19, wherein the fluorophore and quencher combination is selected from FAM-BHQ1 , HEX-BHQ1 , ROX-BHQ2 or CY5-BBQ 650.21 ) The kit according to claim 20, wherein the FAM-BHQ1 combination is bound to the following probes:AGT GAT AGC CTC GCG TGC GTT AAT GA (SEQ ID NO:51 ),TGG CGA CAA ATG CAG TAC TGC TTG G (SEQ ID NO:54),AAC CGA CGG CTG GCA GAT GC (SEQ ID NO:57),TCT CAC TGC GAG CTT GGT GTC GA (SEQ ID NQ:60),GAG CAG TCG CAA TAA CCC AGC AAC A (SEQ ID NO:62) andAGC GGC GGT AAC CCG ATC CT (SEQ ID NO:64); the ROX-BHQ2 combination is bound to the following probes:TCT CCA GGA GGG AGG GCA TCC A (SEQ ID NQ:50),GCC ACG GAA TGG TCT GAG TTG TCG (SEQ ID NO:52),TTC AAC CAC CAC GCT GCC CG (SEQ ID NO:56),GCT GGC AGA AAA CGC CTG GGT (SEQ ID NO:59),TCA TCA CGG AGG CGC GGG AA (SEQ ID NO:61 ) andCCG ACC ACC ACG GAA ACC GC (SEQ ID NO:63); the CY5-BBQ650 combination is bound to the following probes:AGC CAA TGG ATG TGG CTT GAC GTT T (SEQ ID NO:49),GCA TCC AAT ACT TGG AGA GGG AGT GGG (SEQ ID NO:53),GCC CTG AAA CCC TCG CGC TG (SEQ ID NO:55) andGTA TCA TCG CCA CGG CGT CGG (SEQ ID NO:58).22) The kit according to any one of claims 13-21 , wherein the biological sample is selected from a bacterial colony or a blood culture positive for enterobacteria, including Kpn.23) The kit according to any one of claims 13-22, wherein the enterobacterium with a hypervirulent genotype is selected in the group consisting of Klebsiella pneumoniae, Escherichia coli, Yersinia spp and Enterobacter hormaechei, preferably Klebsiella pneumoniae.24) The kit according to any one of claims 13-23, wherein the detection of the enterobacterium with a hypervirulent genotype is carried out by means of a technique selected from real-time PCR, end-point PCR or probe-based real-time PCR.

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