WO2017081709A1 - Bacteriophage able to lyse klebsiella pneumoniae expressing a cpskkbo-4 capsular polysaccharide and related medical uses thereof - Google Patents

Abstract

The present invention concerns a bacteriophage able to lyse Klebsiella pneumoniae expressing a CPS_KKBO-4 capsular polysaccharide, including but not limited to, strains of sequence type ST258-2, the major genotype of Klebsiella pneumoniae carbapenemase (KPC) producing strains, and related uses thereof in surface sanitization, colonization prevention, decolonization of carriers, diagnosis and therapy.

Description

Bacteriophage able to lyse Klebsiella pneumoniae expressing a CPS capsular polysaccharide and related medical uses thereof

The present invention concerns a bacteriophage able to lyse Klebsiella pneumoniae expressing a CPS_KKBO-4 capsular polysaccharide and related medical uses thereof. Particularly, the present invention concerns a bacteriophage able to lyse Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide, including but not limited to, strains of sequence type ST258-2, the major genotype of Klebsiella pneumoniae carbapenemase (KPC) producing strains, and related uses thereof in surface sanitization, colonization prevention, decolonization of carriers, diagnosis and therapy.

The resistance to the antibiotics commonly used to treat nosocomial bacterial infections has now reached alarming rates and, sometimes, only a very few pharmacological options are left (1 ). The use and/or the abuse of such drugs, used to prevent and treat the infectious diseases, has triggered an evolutionary adaptation to the new conditions, facilitated by lateral gene transfer, particularly common among bacteria (2). This phenomenon is now so widespread and alarming that international organizations indicate the 21^th century as the posf-antibiotic era in which common infections, previously treated according to well-established protocols, may be difficult if not impossible to manage using the classical approach with antibiotics. Moreover, as a result of higher globalization and the frequent and numerous movements of individuals between different nations, outbreaks of antibiotic-resistant microorganisms expand more easily and rapidly affecting many countries and, sometimes, evolving into real pandemics.

The name "Klebsiella pneumoniae" is referred to the whole species which comprises several capsular types so that the cellular envelope of each strain belonging to a defined capsular type is different from those strains producing other capsular type. By using conventional serotyping techniques, 79 capsular types have been historically recognized and characterized, even though data from genome sequencing suggest that this number is likely underestimated given the very high genetic heterogeneity of capsular polysaccharide gene clusters observed within the species.

Antibiotic resistance of K. pneumoniae is a well known problem and depends mainly upon acquired genetic determinants. Some of them are the so-called ESBL (Extended spectrum beta-lactamases) genes transferred with mobile DNA so that, within the same capsular type, some strains may be endowed with this trait and some other may be not. ESBL were the main cause of resistance to extended spectrum beta-lactam antibiotics (not to carbapenems) before the appearance of carbapenemases in this species. Later, carbapenemases appeared. Carbapenemases are another group of beta-lactamases, which are active on carbapenems, considered as one of the last resort antibiotics. The strains (K pneumoniae so as other bacterial species) bearing them are even more dangerous than the ESBL producing ones, mostly because these isolates are resistant also to carbapenems with very few, if any, therapeutic options left. Among the most prevalent carbapenemases in K. pneumoniae strains are the KPC-type carbapenemases; the strains producing KPC are defined "KPC-producing K. pneumoniae".

Therefore, ESBL-producing Klebsiella pneumoniae and carbapenemase-producing Klebsiella pneumoniae are completely different types of resistant pathogens in terms of antibiotic resistance profiles and clinical relevance. The strains of Klebsiella pneumoniae producing the KPC-type carbapenemases (KPC-KP) frequently show a phenotype spanning from a multiple to a total resistance to the antimicrobials that can be used in therapy. These strains, which certainly represent the most worrying example of carbapenem-resistant enterobacteria (CRE), have spread since some years, in an epidemic way in many countries (eg: USA, Israel, Greece, Italy and Colombia) and cause infections associated with a high morbidity and mortality (3-5). In Italy, the dramatic increase of CRE strains has been documented by the European Antimicrobial Resistance Surveillance Network (EARS-Net), which showed that the percentage of invasive isolates of K. pneumoniae resistant to carbapenems, which until 2009 had remained <2%, increased to 15% in 2010 and reached 35% in 2013, and national surveillance studies showed that this phenomenon was mostly contributed by a clonal diffusion of KPC-KP largely sustained by ST258-2 isolates (6-7). With regard to mortality, a study conducted in a hospital in New York has shown, for example, that it was 48% in hospitalized patients infected by K. pneumoniae resistant to carbapenems, with a mortality rate directly attributable to infection of 38% (8). These rates are significantly higher than those recorded for patients infected with strains of K. pneumoniae susceptible to carbapenems (20% and 12%, respectively; p<0.001 ). Studies from the USA showed that in a hospital outbreak of KPC-KP, mortality attributable to infection with KPC-KP was 33%, while another more recent study from Italy, involving the largest sample of patients with infections caused by KPC-KP analysed to date, reported a 14 days mortality of 34.1 %(9,10). These rates, already very high and worrying, are even higher in special patient groups such as recipients of allogeneic stem cells, for whom a mortality of 70% has been reported (1 1 ). As previously mentioned, isolates of KPC-KP are susceptible to the action of a few antibiotic molecules, often only tigecycline, colistin, fosfomycin and gentamicin. The use of gentamicin and colistin is hampered by the nephrotoxic effects of both drugs; on the other hand the clinical efficacy of tigecycline, especially when used in monotherapy, is doubtful. Moreover, in many countries we are facing the appearance of isolates resistant to these molecules and, as in the case of colistin, the rate of resistance in some settings is dramatically high: 18.6% in isolates of a Greek hospital and 22.4% in isolates collected within the first national Italian study on CRE (6,12-14). Most recent Italian data show an overall rate of colistin resistance >40% (7). In this case the resistance is of particular concern as the colistin is often a fundamental component of combination therapies (3,15).

Genotyping analysis on KPC-KP strains have shown that isolates belonging to the Sequence Type (ST) 258 (5,16) or to similar STs, collectively named "isolates belonging to Clonal Complex (CC) 258" play an important role in the dissemination of this type of carbapenemases.

Outbreaks of KPC-KP belonging to CC258, which represents the dominant clone of KPC-KP in many cases have been registered in many countries such as, for example, Italy, USA, South America, Israel and Asia (16-17).

Klebsiella pneumoniae ST258 clones still persists in hospital environments and are often associated with high morbidity and mortality rates, mostly due to the production of carbapenemases and extremely multi-drug resistant phenotype. ST258 is a genetic hybrid arising from an homologous recombination of other K.pneumoniae clones. This event was followed by additional evolution leading to the appearance of two novel and distinct genetic lineages, that differ from each other mainly by a -215- kb region of divergence that includes genes involved in capsular polysaccharide (CPS) biosynthesis: ST258 clade I and ST258 clade II. The ST258 clade I (with a cps₂07-2 capsular polysaccharide gene cluster) has the already known capsular type K41 , while the ST258 clade II has the new cps«KBO-4 capsular polysaccharide gene cluster. ST258 clade II is in fact untypeable by classical serotyping method and presents an original genetic structure. Therefore, ST258 clade II is different from all the previously characterized clones. On the basis of the above, strains of ST258-II are novel hybrid clones, with new, unexpected genetic features that account for their success as high-risk clones and for their associated high-morbidity and mortality rates.

Most probably due to this new and different external envelope, ST258- II behaves differently from the other Klebsiella pneumoniae. Klebsiella pneumoniae, so as some other species of the genus Klebsiella, are enterobacteria, commonly present in the gut of man and animals. They are not regarded as pathogens, but can cause opportunistic infections, facilitated by the possible presence of antibiotic resistance in the bacteria, combined with an altered immune response in the host. Differently from other Klebsiella pneumoniae, the ST258-II CPS_KKBO-4 acts as a gut colonizer, entering the gut of the host, usually in a nosocomial environment, and expanding itself, often without any sign, by using their ability to evade the immune response. Within the colonized host, instead of being gradually eliminated, as it happens with most bacterial strains when they enter the gut, ST258-II CPS_KKBO4 keeps persisting and multiplying. At any time in the future, an unaware host could become infected by these strains, when entering in a weakness state. The ST258- II colonized status is an actual risk both for the patient and for all the other patients coming in contact with him/her. The colonization is often only detected during surveillance analysis, if the host is hospitalized again.

On the basis of the above, and considering the shortage of new molecules suitable for therapy and that can shortly enter the market (4), intervention strategies alternative to antibiotics are advisable such as the use of bacteriophages or bacteriophages-derived proteins.

Bacteriophages have been traditionally used in microbiology to type bacterial strains within bacterial species. This application is made possible by the narrow host specificity of each bacteriophage, that interacts in a specific manner with a group of strains, within a bacterial species. Bacteriophages interact with the external layers of the bacterial cell, targeting specific receptors. Therefore, each bacteriophage is specific for a subset of strains that share the same external asset i.e. each bacteriophage is specific only for a subset of strains sharing the same receptors.

The ability of some bacteriophages to specifically lyse particular bacterial clones, through the selective recognition of specific components exposed on the bacterial cell, means that they constitute a promising weapon to fight some of the above infections and, especially, to prevent the colonization that it is at basis. KPC-KP isolates of the ST258-II clade, in fact, although not particularly aggressive, are able of rapidly colonizing new hosts, especially in a hospital environment, and persist for a long time (18-20). The new host is then exposed to the risk of infections that cannot be treated with antibiotics as well as becoming in turn a source of spread. In a similar manner to that recorded for the mortality rate, the risk of infection in patients colonized with KPC-KP is particularly high in some cases as among patients subject to autologous bone marrow transplant, where 40% of the colonized subjects develop infection (11 ). The specificity of an approach based on bacteriophages has also, compared to the existing antibiotics, the undoubted advantage to act exclusively on the pathogen, either infecting or simply acting as a colonizer, without altering the normal resident microbiota. Moreover, since the spread of KPC-KP is predominantly given by oligoclonal diffusion, the use of phage or its derived proteins i.e. the protein or proteins of the bacteriophage which are responsible of the specificity against the CPSKKBO-4 capsular type of Klebsiella pneumoniae ST258-2, provides a strong rationale for the effectiveness of this approach.

Several bacteriophages are known which are able to lyse some strains of . pneumoniae such as ESBL-producing K. pneumoniae isolates or K. pneumoniae with capsular polysaccharide of the K2 type (21-24). However, no bacteriophage able to lyse isolates of K. pneumoniae having the new CPSKKBO-4 capsular type is known.

It is therefore evident the need to provide new strategies able to face with the emergency caused by these bacteria and to overcome the disadvantages of the known methods.

According to the present invention, a new bacteriophage has been isolated and characterized. The bacteriophage, named cpBOI E (also referred as phiBOI E) and deposited at IZSLER - - Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini" on June 17^TH ,2015 with deposit number VIR RE RSCIC 299, is able to specifically lyse isolates of K. pneumoniae having the new CPS«KBO-4 capsular type (otherwise said cps-2), recently described as the predominant capsular type within the KPC-KP belonging to the pandemic clone of KPC-KP ST258, and occasionally found in other STs (25,26).

This phage, in whole or through the use of its specific components, thus constitutes a promising weapon to fight infections caused by the above mentioned clone, for the decolonization of the carriers, and to limit the spreading of these strains among hospitalized patients. Moreover, the cpBOI E phage provides a very inexpensive diagnostic tool for the rapid identification of clinical isolates of K. pneumoniae, equipped with this capsule.

The bacteriophage according to the present invention is able to contrast these infections by fighting the colonization as the first goal. Differently from other phage-therapy approaches, the phage according to the present invention has a very strict specificity, so that the re-expansion of the naturally occurring gut microbiota is facilitated, and interacts only with the strains of the new capsular type. Rather than treat an "infection in act", the bacteriophage of the invention is able to decolonize the host lysing strains of the ST258-II clade, so to prevent both the possible future infections and the spreading of these clones in the hospital and in the community. Therefore, differently from other approaches, the bacteriophage according to the present invention is able to hamper the colonization success of ST258-II Klebsiella pneumoniae and/or to get a decolonization in the already colonized, asymptomatic patients, thus reducing the chanche of new colonizations of other individuals.

To do so, both the whole phage and/or components thereof such as protein/s that are responsible of the specificity against the new capsular type CPSKKBO-4 can be used.

It is therefore specific object of the present invention the bacteriophage VIR RE RSCIC 299 able to lyse Klebsiella pneumoniae expressing a CPSKKBO- capsular polysaccharide, variants of said bacteriophage and/or components of said bacteriophage or variants of said components, said variants or components being able to lyse Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide. The bacteriophage of the present invention is effective against any Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide such as for example strains of sequence type ST258-2.

According to the present invention, the term "variants" of VIR RE RSCIC 299 is understood as meaning bacteriophages having the same phenotypic characteristics as said bacteriophage and the same activity against the strains with the CPSKKBO-4 capsular type. The variants or homologous of VIR RE RSCIC 299 can have an identity of at least 60%, preferably 80%, at nucleotide level and/or an identity of at least 40%, preferably 60%, at aminoacid level, in comparison with the DNA sequence of VIR RE RSCIC 299 or the proteins encoded by the DNA sequence of VIR RE RSCIC 299, in particular concerning the comparison of the genes or proteins most typical of the bacteriophage VIR RE RSCIC 299, such as the ORFs 9; 13; 21 ; 34; 35;37 and their products (i.e. the proteins encoded by said genes), and/or linked to its specificity for the CPSKKBO-4 capsular type, such as the ORF59. The variants can be natural or artificial variants comprising a substitution, deletion, and/or insertion of one or more amino acids of the polypeptides encoded by the bacteriophage VIR RE RSCIC 299 DNA sequence and having a specificity against Klebsiella pneumoniae strains with capsular type CPSKKBO-4 or a homologous sequence thereof. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly- histidine tract, an antigenic epitope or a binding domain.

The term "components" of bacteriophage VIR RE RSCIC 299 is understood as meaning the components of the bacteriophage (i.e. complete genes or parts thereof, or products codified by said genes or parts thereof) having a lytic activity against Klebsiella pneumoniae strains with capsular type CPSKKBO-4- These components being, for example but not limited to, one or more proteins selected from the group consisting of depolymerases such as tail proteins, or tail proteins conferring the specificity of the bacteriophage VIR RE RSCIC 299, lytic enzymes such as spanines, holins, endolysins, isolated genes encoding said one or more proteins.

The term "variants of components" of VIR RE RSCIC 299 is understood as meaning proteins and genes which can have an identity of at least 60%, preferably 80%, at nucleotide level and/or an identity of at least 40%, preferably 60%, at aminoacid level in comparison to the genes or proteins of bacteriophage VIR RE RSCIC 299. In particular, said genes can be the ORFs 9; 13; 21 ; 34; 35; 37 and their products (i.e. the proteins encoded by said genes), and/or linked to the specificity of the bacteriophage for the CPSKKBO-4 capsular type, such as the ORF59. Therefore, according to the present invention, components of both bacteriophage VIR RE RSCIC 299 and/or variants thereof can be effectively used against Klebsiella pneumoniae strains with capsular type CPSKKBO-4. AS mentioned above, the variants can be natural or artificial variants comprising a substitution, deletion, and/or insertion of one or more amino acids of the polypeptides encoded by the bacteriophage VIR RE RSCIC 299 DNA sequence and having a specificity against Klebsiella pneumoniae strains with capsular type CPSKKBO-4 or a homologous sequence thereof. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly- histidine tract, an antigenic epitope or a binding domain.

The present invention concerns also a pharmaceutical composition comprising or consisting of the bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined above together with one or more excipient and/or adjuvant, said variants or components being able to lyse Klebsiella pneumoniae expressing a CPS_KKBO-4 capsular polysaccharide.

The pharmaceutical composition according to the present invention can further comprise one or more antimicrobial drugs, bacteriophages different from VIR RE RSCIC 299 or components thereof and/or probiotic microorganisms.

Antimicrobial drugs can be chemical compounds having bactericidal and/or bacteriostatic activity, such as, but not limited to, antiseptic compounds, such as active oxygen, able to reduce the bacterial load of infectious microbes and which can be used on living tissues or skin; natural and/or synthetic antimicrobial peptides or proteins of eukaryotic origin and/or their derivatives such as components of innate or adaptive eukaryotic immune system endowed of specific or general antimicrobial activity; antibiotic drugs such as beta-lactams, aminoglycosides, quinolones, tetracyclines, phenicols, polymyxins, sulfonamides and/or bactericidal peptides or proteins or combinations of the above drugs.

According to the present invention, bacteriophages different from VIR RE RSCIC 299 to be used in combination with the bacteriophage of the present invention can be chosen from whatever lytic or temperate phages for example bacteriophages having lytic activity against MRSA (Methicillin Resistant Staphylococcus aureus), Clostridium difficile, Pseudomonas aeruginosa or Acinetobacter baumannii. In addition, phage tail-like peptides or proteins of bacteriophages different from VIR RE RSCIC 299 and derived from induced or not induced bacterial cultures can be used according to the present invention in combination with VIR RE RSCIC 299.

In regards to probiotic microorganisms, bacteria belonging to the genus Bifidobacterium or Lactobacillus can advantageously be used according to the present invention.

In addition, the present invention concerns a pharmaceutical composition, as defined above, further comprising one or more disinfecting compounds. The term "disinfecting compounds" means a chemical, such as ammonia quaternary compounds, to be applied to non living objects to reduce the bacterial load of potentially infectious microorganisms.

Further aspect of the present invention is bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined above or composition as defined above for use as a medicament, said variants or components being able to lyse Klebsiella pneumoniae expressing a CPS_KKBO-4 capsular polysaccharide.

It is therefore an object of the present invention, bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined above or composition as defined above for use in prevention or treatment of the infection or colonization of tissues, such as mucosa of gut, urinary tract, oropharyngeal tract or skin, by Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide, such as strains of sequence type ST258-2, said variants or components being able to lyse Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide. The term "infection" means the invasion and multiplication of microorganisms in the hosts tissues. An infection may cause no symptoms and be subclinical, or it may cause symptoms and be clinically apparent. An infection may remain localized, or it may spread through the blood or lymphatic vessels to become systemic (bodywide). An infection generally involves a reaction of the immune system and is usually preceded by a colonization step.

According to the present invention, the term "colonization" means multiplication and persistence of microorganisms on surfaces of skin or mucosa without apparent tissue damage or clinical symptoms. Therefore, decolonization by topical or systemic agents consists of eliminating or reducing the colonization by one or more specific microorganisms.

The present invention concerns also the use of the pharmaceutical composition as defined above for the sanitization of objects' surfaces, such as the surface of surgical instruments.

A further embodiment of the present invention is a combination of the bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined above, said variants or components being able to lyse Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide, together with one or more antimicrobial drugs, bacteriophages different from VIR RE RSCIC 299 or components of said bacteriophages different from VIR RE RSCIC 299 and/or probiotic microorganisms, said combination being for separate or sequential use in prevention or treatment of the infection or colonization of tissues, such as mucosa of gut, urinary tract, oropharyngeal tract or skin, by Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide, such as strains of sequence type ST258-2. The antimicrobial drugs, bacteriophages different from VIR RE RSCIC 299 or components of said bacteriophages different from VIR RE RSCIC 299 and/or probiotic microorganisms to be used according to the present invention have been described above.

According to the present invention "separate use" is understood as meaning the administration, at the same time, of the two or more compounds of the combination according to the invention in distinct pharmaceutical forms. "Sequential use" is understood as meaning the successive administration of the two or more compounds of the composition according to the invention, each in a distinct pharmaceutical form.

The present invention concerns also the use of the bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined above, for in vitro diagnosis of the infection or colonization of tissues, such as mucosa of gut, urinary tract, oropharyngeal tract or skin, by Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide, such as strains of sequence type ST258-2, or for in vitro bacteriotyping of Klebsiella pneumoniae, said variants or components being able to lyse Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide.

Therefore the present invention concerns a method for in vitro diagnosis of the infection or colonization of tissues, such as skin, mucosa of gut, urinary tract, oropharyngeal tract, by Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide, such as strains of sequence type ST258-2, or for in vitro bacteriotyping of Klebsiella pneumoniae, said method comprising or consisting of a) contacting a bacteria culture with the bacteriophage, variants thereof and/or components of said bacteriophage or variants thereof as defined in claim 1 and b) observing the possible lysis of the cultured bacteria; the infection or colonization of tissues by Klebsiella pneumoniae expressing a CPSKKBO- 4 capsular polysaccharide being detected, or the bacteriotyping of the Klebsiella pneumoniae as Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide occurring, when a lysis of the culture is observed. The lysis of the culture can be observed in the form of lysis plaques on top-agar, by spot-test or as clearing of the liquid cultures or other methods apparent to those skilled of the art. According to the method of the present invention, said bacterial culture is obtained by a biological sample of a patient when in vitro diagnosis has to be carried out or it is a bacterial culture of Klebsiella pneumoniae isolated from environment, such as waste waters, expressing an unknown capsular polysaccharide and therefore to be subjected to bacteriotyping.

In addition, the present invention concerns a kit for the in vitro diagnosis of the infection or colonization of tissues, such as mucosa of gut, urinary tract, oropharyngeal tract or skin, by Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide, such as strains of sequence type ST258-2, or for in vitro bacteriotyping of Klebsiella pneumoniae, said kit comprising or consisting of the bacteriophage, variants thereof and/or components of said bacteriophage or variants of said components as defined above, said variants or components being able to lyse Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide.

The present invention now will be described by illustrative but not limitative way according to preferred embodiment thereof with particular reference to the enclosed drawings, wherein:

Figure 1 shows the plaques produced by the infection of the (pBOI E phage present in selected samples of nosocomial waste waters on the test strain KKBO-1. Plaques characterized by different diameters can be observed.

Figure 2 shows the restriction profile with Hindlll of the 3 selected phage preparations. Sizes of fragments of molecular weight standard, expressed in number of base pairs, are reported on the left. Lane 1 , Lambda/Hindlll; Lane 2, <pB01Y; Lane 3, <pB01 E; Lane 4, <pB01W.

Figure 3 shows the image of the φΒΟΙ Ε phage observed by TEM, after staining with uranyl acetate.

Figure 4 shows the effect of temperature on the infection ability of the φΒΟΙ Ε bacteriophage, that has been incubated at 40, 50, and 60°C for 10, 20, 40, and 60 minutes.

Figure 5 shows the influence of pH on the infection ability of the φΒ01 E bacteriophage incubated at the reported pH values for 1 hour.

Figure 6 shows the One-step growth curve of bacteriophage φΒΟΙ Ε. The ratio between PFU and infected bacterial cells at different time intervals is shown.

Figure 7 compares phages φΒΟΙ Ε and KP34, the closest homologous of <pB01 E as identified by a ClustalW alignment performed on the phages detected by a BLASTN search on the INSDC databases (http://blast.ncbi.nlm.nih.gov/). For each phage the accession numbers relative to the DNA sequences and the size in Kb are shown on the left. The level of nucleotide identity between the two phages is reported by a grayscale gradient.

Figure 8 shows the Kaplan-Meier survival curves of Galleria mellonella larvae infected with 10⁷ cells of KKBO-1 and treated with phage suspensions at a MOI of 10 or 100 (a) or with 10⁶ (b) or 10⁷ (c) cells of the 04C62 strain and treated with phage suspensions at the same MOIs.

Results obtained with larvae infected with the same amount of bacterial cells but not treated with phage, or injected with buffers only are also shown.

EXAMPLE 1 : Isolation and characterization of the bacteriophage (pBOIE

METHODS

The bacteriophage, named φΒΟ1 Ε, was obtained from hospital waste waters collected at the Policlinico Santa Maria alle Scotte University Hospital, Siena (Italy), taken upstream of the treatment plant, using the top-agar overlay method and the isolate of K. pneumoniae KKBO-1 as test strain (27). Specifically, wastewaters were centrifuged at 3850xg at 25 ° C for 10 minutes and the supernatant was then filtered at 0.45 μηι. The suspension thus obtained was filtered again at 0.22 μηι to eliminate bacterial contamination and diluted 1 :100 in sterile ddH₂0. An aliquot of 100 μΙ of this dilution was mixed with 200 μΙ of a bacterial suspension of the test strain obtained by concentrating an over-night (O/N) culture in LB medium of the test strain to 1/10 of the initial volume. The suspension buffer was SM (NaCI 5.8 g; MgS0₄ 7 H₂0 2 g; Tris-HCI (1 M, pH = 7.5) 50 ml; ddH₂0 to 1 liter). The mixture was left for 20 minutes at room temperature without stirring to favour the adsorption of phage particles on the test strain. After this period, the suspension was added to 5 ml of top agar (obtained by dissolving agar in LB broth to reach a final concentration of 0.7% w/v), mixed and immediately poured onto LBA 90 mm Petri dishes. The plates were then incubated at 37 ° C until the appearance of phage plaques (usually 6-8 hours).

Three single phage plaques, distinct by different morphologies, have been picked-up from the 90 mm Petri dish through plastic Pasteur pipette and each one was suspended for 20 minutes in 200 μΙ of SM buffer at room temperature; these suspensions were then again used for the infection of the test strain. The picking of a single phage plaque, its elution and subsequent infection, performed as described above, was repeated two more times for each of the three selected plaques in order to obtain pure phage preparations. The titration of the suspensions was carried out by the spot method, in which an aliquot of 400 μΙ of bacterial inoculum grown O/N and suspended in 1/10 of its initial volume in SM buffer was mixed with 5 ml of top agar and immediately poured onto 90 mm Petri dishes containing LBA. Once the top agar solidified, the plates were divided into several sections, onto which 5 μΙ of appropriate 10 fold dilutions of the phage suspension to be titrated were deposited. After the adsorption of the phage suspensions to the dishes, the plates were incubated at 37° C until the appearance of lysis plaques. The medium scale production of phage particles was performed by inoculating each out of 10 Petri dishes (90 mm) with a mixture of 200 μΙ of the test strain grown O/N and suspended in 1/10 of its initial volume in SM buffer and 100 μΙ of phage preparation ( 0⁸ PFU/ml). After a contact time of 20 minutes, 5 ml of top agar were added to the mix and poured on to 90 mm Petri dishes containing LBA. After incubation of the plates for 6-8 hours at 37 ⁰ C, 5 ml of SM buffer were added to each plate and, using a scraper, the layer of top-agar was detached. The plates were then left for 2 hours at room temperature with gentle bouncing and subsequently the top-agar layer and the SM buffer was recovered in 50 ml Falcon tubes and centrifuged at 3850xg, at 4 ° C for 20 minutes. The supernatant was finally filtered at 0.22 pm and subsequently titrated using the spot method previously described.

When necessary, the phage preparations were concentrated with the following procedure.

To a volume of 30 ml of phage preparation, placed in a centrifuge tube, 7.5 ml of a 20% solution of PEG-8000 and 2.5 M NaCI were added.

The tube was kept on ice for 45-60 minutes and centrifuged at 1 1.000xg for 20 minutes.

After discarding the supernatant, the centrifugation step was repeated for 2-3 times, in order to remove all the residual PEG. The pellet was suspended in 0.5-1 ml of sterile STE buffer (10 mM Tris-HCI (pH = 8.0); 0.1 mM EDTA (pH = 8.0), 100 mM NaCI), transferred to 1.5 ml tubes and centrifuged at 14,000χρ/ for 10 minutes.

At this point the supernatant was transferred to a new tube and titrated again.

The extraction and purification of the DNA of the three selected phage preparations was performed using the kit Wizard^® DNA Clean-Up System (Promega, Madison, Wl, USA), following the instructions provided by the manufacturer. After the purification step the DNA was eluted in 50 μΙ of ddH₂0 and aliquots of 5 μΙ were separated by electrophoresis on agarose gel (0.75% w/v) to check the integrity of the obtained preparation and to have a rough estimate of the size of the genome in analysis and of its concentration. The exact concentration of the phage DNA was carried out using a NanoDrop device (Nanodrop Technologies Inc., Wilmington, USA), following the instructions provided by the manufacturer. A volume equivalent to 250 ng of each of the three preparations of phage DNA was used for restriction analysis using the Hindi 11 enzyme (New England Biolabs, Boston, MA, USA), following the protocol and the conditions suggested by the supplier of the enzyme. The enzymatic restrictions thus obtained were resolved by electrophoresis on agarose gel (1 % w/v), using the marker Lambda Hind 111 DNA (Promega) as molecular weight standard.

The phage suspension brought to a concentration of 10¹² PFU/ml was used for the transmission electron microscopy (TEM) characterization. In detail, 10 μΙ of this suspension were let to adsorb on a carbon-coated matrix and then stained with 2% uranyl acetate for 15 seconds. The product thus obtained was washed 2 times with ddH₂0 and then displayed by using a transmission electron microscope FEI Tecnai 12 equipped with a CCD camera model Osis Morada 2X4K.

A volume of phage DNA preparations containing 20 ng of DNA was used for the complete high-throughput genomic sequencing with a Next- Generation Sequencing (NGS) method, by using the lllumina Nextera™ kit, a paired-ends approach (2x250 bp) and the MiSeq instrument (lllumina Inc., San Diego, CA, USA). The determination of the ends of the phage genome was performed by sequencing using the Sanger method directly from extracts of bacteriophage DNA, using primers B01 E_2F (5'- TTGACTACGTCGGGATAGGC-3' (SEQ ID NO:1 )) and B01 EJ R (5'- AGCACTAGCGATAGCCAGTG-3' (SEQ ID NO:2)).

The host range of the <pB01 E phage was determined using 55 clinical isolates of K. pneumoniae resistant to carbapenems characterized by different capsular types, using the method previously described (Table 1) (28).

Table 1. Isolates selected for the host spectrum assay. For each isolate the identifier, the capsular genotype together with the corresponding deduced phenotype, the results of MLST analysis (ST) and the possible lysis observed after the infection with φΒΟΙ Ε are reported (0=not lysed; 1 =lysed). capsular Deduced ST

Identifier genotype³ capsular serotype lysis

MOKIe 9 G wz/154 KKB04 512 1

6303 WZ/29-K41 K41 (KK207) 258 0

AN-1 wz/154 KKB04 512 1

AN-2 wz/154 KKB04 512 1

01 C03 WZ/29-K41 K41 (KK207) 258 0

01 C06 WZ/29-K41 K41 (KK207) 258 0

02C06 wz/154 KKB04 258 1

01 C22 wz/ 54 KKB04 512 1

02C01 wz/154 KKB04 512 1

06C02 WZ/29-K41 K41 (KK207) 258 0

06C04 wz/154 KKB04 512 1

06C05 wz/154 KKB04 258 1

06C07 WZ/29-K41 K41 (KK207) 258 0

06C19 wz/154 KKB04 512 1

07C06 wz/154 KKB04 258 1

07C07 WZ/29-K41 K41 (KK207) 258 0

08C02 wz/154 KKB04 512 1

08C04 wz/154 KKB04 258 1 C06 wz/154 KKB04 258

C04 wz/154 KKB04 512

C09 wz/154 KKB04 512

C07 wz/154 KKB04 512

OKIe 86 WZ 38-K38 K38 641

C29 WZ/137-K17 K17 101

C72 WZ/137-K17 K17 101

C73 WZ/137-K17 K17 101

C05 wz/154 KKB04 512

C10 wz/154 KKB04 258

C18 wz/154 KKB04 258

C05 wz/154 KKB04 512

C12 wz/154 KKB04 258

C01 wz/154 KKB04 512

C22 wz/154 KKB04 258

C24 wz/154 KKB04 258

C09 wz/154 KKB04 512

C11 wz/154 KKB04 258

C14 wz/154 KKB04 258

C09 wz/154 KKB04 258

C24 wz/154 KKB04 512

C10 wz/154 KKB04 258

C13 wz/154 KKB04 258

C02 wz/154 KKB04 258

C20 wz/154 KKB04 258

C21 wz/154 KKB04 258

C06 wz/154 KKB04 512

C08 WZ/29-K41 K41 (KK207) 258 0C12 WZ/29-K41 K41 (KK207) 258 0C35 wz/154 KKB04 512 1 04C38 wz/154 KKB04 512 1

04C49 wz/154 KKB04 258 1

05C15 wz/154 KKB04 512 1

3266 WZ/24-K24 K24 15 0

41229752 wz/154 KKB04 554 1

MOKIe 81 WZ/96-K38 K38 37 0

12C47 WZ/137-K17 K17 101 0 a capsular genotyping has been performed using the method described by Brisse and colleagues(29).

Capsular serotype was deduced, were possible, by using data obtained by capsular genotyping. Sequence type (ST) was assigned as previously described (http://bigsdb.web.pasteur.fr/klebsiella/primers_used.html). The stability of φΒ01 E at different temperatures was determined by diluting the phage particles to a final concentration of 10⁸ PFU/ml in a final volume of 1 ml of SM buffer. These aliquots were incubated at 25 °C (control), 40 °C, 50 °C and 60 °C, for 10, 20, 40 and 60 minutes. After the incubation the phage suspensions were serially diluted and titrated as described above. The assays for the determination of stability at different temperatures were performed in triplicate.

The stability of cpBOI E at different pH values was determined by diluting the phage particles to a final concentration of 10⁸ PFU/ml in aliquots of SM buffer previously brought to different pH using either 1 M NaOH or 1 M HCI, so as to create a pH range from 3 to 1 1 with intervals of 1 unit. The samples thus obtained were incubated for 60 minutes at 25 °C and then serially diluted and titrated as previously described. The assays for the determination of pH stability were carried out in triplicate.

The burst volume (burst size) of the phage in analysis, representing the average number of phages released by each bacterium at the time of lysis was determined by inoculating the test strain in a 50 ml Erlenmeyer flask containing 5 ml of medium LB. The inoculum prepared in this way was grown under agitation in aerobic conditions at 37 ° C until the reach of the exponential growth phase, equivalent to an absorbance (A₆oo) of 0.3- 0.4. An aliquot of 1 ml of the inoculum in the exponential growth phase was then centrifuged at 13000xg for 5 minutes and the obtained pellet was suspended in 1 ml of SM buffer. A portion of 0.9 ml of this suspension was placed in contact with 0.1 ml of the phage lysate at a concentration of 1x10⁷ PFU/ml, in order to obtain a multiplicity of infection (MOI) of 0.01 , and incubated for 10 minutes in a thermostatic bath at 37 ⁰ C to favour the adsorption of phage on the cells of the test strain. After this stage, the suspension was centrifuged at 13000xg for 2 minutes and the pellet thus obtained was suspended in an isovolume of SM buffer. This last step was repeated to remove any non-adsorbed phages. This preparation was diluted 1 : 10000 in LB medium and an aliquot of 10 ml was incubated in a thermostatic bath at 37 °C. Timely (10, 20, 25, 30, 40, 50, 60 and 70 minutes), aliquots of 0.1 ml were taken and added to 4.5 ml of top agar. Each suspension was then mixed, immediately poured onto LBA plates and incubated O/N at 37 °C. The latency period is defined as the period between the time of infection (excluding the 10 minutes required for the adsorption of phage and the 4 minutes of centrifugation later) and the beginning of the production of phages (28). The burst-size was finally computed as the ratio between the final number of released phage particles and the initial number of infected bacterial cells during the latency period.

To verify whether the bacteriophage was actually lytic (virulent) a lysogeny test was made according to the following protocol: after a test of spot lysis, the plates were left to incubate for 3 days to check the possible appearance of resistant colonies within lysis area. The colonies possibly grown were cultured, harvested by centrifugation, and tested for their ability to be infected by phiBOI E.

RESULTS

Screening for phage particles possibly present in the hospital waste waters and able to lyse the test strain KKBO-1 gave a positive result, with numerous lysis plaques of different sizes (Fig. 1 ). Three plaques, characterized by different diameters, have been taken, purified and amplified for further analysis, as described in methods. The restriction analysis of phage DNA obtained from the three selected preparations, showed an identical profile, suggesting that the three candidates in analysis were identical to each other (Fig. 2). These results have led to select only one phage preparation for subsequent analysis, identified as cpBOI E. Furthermore, the fragments generated after restriction with Hindi 11 allowed to estimate that the size of the phages in the analysis was approximately 40-45 Kb.

The results of analysis by TEM showed a morphology typical of the members of the virus family Podoviridae, Caudovirales order, characterized by a head with icosahedral symmetry with a diameter of about 50 nm and a short tail of 8-10 nm (Fig. 3). This result agrees both with the size estimated by the restriction analysis, and with the usual genome size of the Podoviridae family (about 38-45 kb).

Results of spot test, carried out with a collection of 55 K. pneumoniae clinical isolates, have shown that cpBOI E is characterized by lytic specificity towards isolates having CPS_KKBO-4, all of which were lysed (n = 40/55), while isolates having different CPS are not sensitive to the action of the phage particles (n = 15/55) (Table 1 ). The ability of <pB01 E to exclusively lyse isolates with capsular type CPSKKBO-4 (i.e. those belonging to the clone of KPC-Kp ST258-2), but not those belonging to the clone ST258-1 (with capsular type C SKK207), suggests that the bacterial structures recognized by the phage in analysis are components of the capsular polysaccharide, given that the main difference between the two clones is the production of a different type of capsular polysaccharide (25,30).

The results of the stability towards temperature and pH demonstrate that the phage φΒΟΙ Ε retains a good infective capacity in a wide range of conditions, given the fact that only after incubation for 60 minutes at 60 °C, or after incubation at pH <4 or pH> 9 there is a remarkable fall (≥ 2 log) of the infective ability (Fig. 4 and 5). Since no lysis plaques were observed in the lysogeny experiments, the phage cpBOI E appears to be lytic.

The results of experiments to determine the burst-size of phage cpBOI E, reported in Figure 6, show that this phage has a relatively short latency period of about 10 minutes. The period of increasing of the phage progeny is about 20 minutes before the plateau level is reached at about 40 minutes. The computed burst-size is found to be about 300 phage particles released for each infected bacterium and was calculated considering the time interval between 40 and 60 minutes. It should be noted that the increase of the phage titer observed after 60 minutes is the beginning of the second cycle of replication and should not be considered in the calculation of the burst-size.

The results of NGS sequencing and the resolution of the termini of the phage genome conducted via Sanger sequencing, show that the bacteriophage in analysis has a linear dsDNA genome of about 44 Kb with a GC content of 53.8% and has direct repeated sequences of about 200 bp at the 5 '- and 3 -ends. Bioinformatics analysis have revealed 59 open reading frames all encoded in the same strand of DNA (Genebank, accession number KM576124). The search in INSDC databases, carried out on 28.07.2015 with the BLAST software using the default parameters and the nr database, shows that cpBOI E is a new bacteriophage which has limited homology with 5 phages of Klebsiella pneumoniae previously described, as detailed in Table 2. Table 2. Overall nucleotide identity of phage <pBO1 E with homologous bacteriophages identified by the BLAST search. The reported identity has been computed with the ClustalW2 software (http://www.ebi.ac.uk/Tools/msa/clustalw2/) by using the complete DNA sequences of different phages downloaded from the database and the default parameters of the software.

Phage Identifier Version Overall nucleotide

(Reference) identity with <pB01 E

KP34 (31 ) GQ413938.2 78.8%

Gl:291195543

vB_KpnP_SU552A (32) KP708986.1 77.3%

Gl:762086021

vB_KpnP_SU503 (32) KP708985.1 77.2%

Gl:762085962

NTUH-K2044-K1-1 (33) AB716666.1 76.4% Gl:662242378

F19 (Unpublished) KF765493.2 75.6%

Gl:612405031

As it can be observed from Table 2, the closest homologues at the nucleotide level is seen with the phage KP34, with which <pB01 E has a nucleotide identity of about 79% (Fig. 7) (31 ). It should be noted that, as it is apparent from papers in which bacteriophages homologues with φΒΟΙ Ε are described, none of these has been described as lytic for isolates of Klebsiella pneumoniae having the capsular type characteristic of the isolates belonging to ST258-2.

In conclusion the bacteriophage cpBOI E, object of the present invention, is a new bacteriophage able to specifically lyse isolates of K. pneumoniae belonging to ST258-2 and characterized by a new capsular type.

EXAMPLE 2: Cloning and characterization of φΒΟΙΕ depolymerase, spanin and holin

METHODS

Genes of phiBOI E encoding putative depolymerase, spanin and holin, the proteins involved in the disruption of the bacterial cell envelope, were predicted using the PSI-BLAST network service (http://blast.ncbi. nlm.nih.gov/Blast.cgi?CMD=Web&PAGE=Proteins&PRO GRAM=blastp&RUN_PSIBLAST=on) by comparison with previously characterized homologous proteins. Results from this analysis indicated orf56, orf57 and orf59 as putative phage spanin, holin and depolymerase, respectively. Gene candidates were cloned into pGEM vector using the pGEM®-T Easy Vector System (Promega, Madison, Wisconsin, USA) and PCR product obtained with primers

59_Ndel_1_F (5 -

GAGCCATATGAATTTAGTAAAAGCAAAGTATCCG-3' (SEQ ID NO:3)) and

59_Xhol_1_R (5'- CCGCTCGAGGAAAGCTGCCTGGGTATC-3' (SEQ ID NO:4)) or Ndel_span_hol_Fw (5'- GAGCCATATGCATAAACTGGTCGCTGGG-3'(SEQ ID NO:5))

and Xhol_span_hol_Rev (5'- CCGCTCGAGTTAGTCCTTAAACTCATATTTAATAG-3'(SEQ ID NO:6)), used to amplify or†59 (primer pair 59_Ndel_1_F and 59_Xhol_1_R) or or†56-or†57 (primer pair Ndel_span_hol_Fw and Xhol_span_hol_Rev), respectively. Nucleotide sequence of the cloned genes were confirmed by double strand DNA sequencing using the amplification primers. pGEM-T Easy derivatives were then digested with Ndel and Xhol and the obtained products containing or†59 or orf56-orf57 were subcloned into pET-24a(+) expression vector to obtain pET-24-o/f59 and pET-24-o/ 56- or†57, respectively. pET-24a(+) derivatives were transformed in E. coli DH5a and BL21 (DE3).

Recombinant His-tagged proteins were expressed by induction with 1 mM IPTG at 35°C for 3 hours, followed by a sonication to obtain rough soluble fractions. E coli BL21 (DE3) carrying pET-24a(+) expression vector without insert was used as negative control. To observe enzyme activity, a spot test was performed. Briefly, LB agar plates was overlaid with top agar that had been inoculated with 100 μΙ of a O/N bacterial culture of Klebsiella pneumoniae BO1 strain and, once the top agar had solidified, 10 μΙ of each soluble fraction were spotted onto the plate. The spot test has been done also mixing the depolymerase and spanin-holin soluble fractions (5 μΙ + 5 μΙ). After O/N incubation at 35°C, plates were observed for formation of semi-clear spots.

RESULTS

Semi-clear spots were observed for all the tested soluble fractions, but negative controls. Soluble fractions containing depolymerase were able to lyse bacteria more efficiently than those containing spanin and holin. The combination of the depolymerase fractions with those containing spanin-holin resulted in increase of the lysis activity.

EXAMPLE 3: Assessment of the use of ψΒΟΙΕ as protective agent against infections sustained by strains of K. pneumoniae expressing a CPSKKBO-4 capsular polysaccharide.

METHODS

Larvae of G. mellonella were obtained from Sa.gi.p (Sa.gi.p, Ravenna, Italy) and used after one O/N incubation at 25°C. Larvae were inspected to select candidates weighing approximately 450-600 mg for the phage therapy assays. G. mellonella larvae were surface-sterilized with a cotton swab dipped in 70% ethanol (Sigma-Aldrich) and injected with 10 μί of inoculum containing either 10⁷ cells of K. pneumoniae KKBO-1 or 10⁶ or 10⁷ cells of K. pneumoniae 04C62 into the larval haemolymph behind the last proleg by using a 30-gauge syringe (Hamilton, Reno, NV). After 30 minutes posf-infection, a group of larvae were injected at the same site, but on the opposite side to the bacterial injection, with 10 ul of a φΒΟ1 Ε preparation at a concentration to obtain Multiplicity Of Infection (MOI) of 10 or 100. A total of 10 larvae were used for each condition. Positive (larvae infected with K. pneumoniae KKBO-1 or 04C62 and treated with SM buffer) and two negative control groups (one group injected with SM and PBS buffers and one group injected with PBS and phage suspension) were also included for each condition. Larvae were placed into Petri dishes and incubated at 35±2 °C in the dark, in humidified atmosphere, with food, and daily examined for pigmentation and mobility. Time of death was recorded at 24, 48 and 72 hours. For each experiment the injected inoculum was checked by plating appropriate serial dilutions of bacterial suspensions and by enumerating colonies after a 12-16 hours of incubation at 35±2 °C. Five independent experiments were performed for each different bacterial inoculum/phage titer combination. Data from independent experiments were finally combined and the protection of larvae from death by cpBOI E were assessed by log-rank (Mantel-Cox) test, p values <0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, USA).

RESULTS

To verify in vivo the efficacy of φΒΟΙ Ε in the protection against infection, the wax moth larvae model was employed. In this model, KKBO- 1 and 04C62 exhibit an LD50 at 72 h of 6.02 ± 0.09 and 6.1 ±0.05, respectively (34). Results of phage protection were recorded at 24, 48 and 72 h post infection. Protection with φΒΟΙ Ε showed a significant impact on G. mellonella larval rescue from lethal infection (Fig. 8). In fact, by using KKBO-1 at a dose of 10⁷ CFU and a MOI of 10 the protection effect didn't reach statistical significance (p=0.4698) while it was protective with the same strain and a MOI of 100 with the same inoculum size (p=0.0023; Fig. 8a). Conversely, by using the hypermucoviscous 04C62 strain at a inoculum size of either 10⁶ or 10⁷ both MOI 10 and 100 were effective in death prevention (Fig. 8b and 8c).

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Claims

1 ) Bacteriophage VIR RE RSCIC 299 able to lyse Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide_, variants of said bacteriophage and/or components of said bacteriophage or variants of said components, said variants or components being able to lyse Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide.

2) Pharmaceutical composition comprising or consisting of the bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined in claim 1 together with one or more excipient and/or adjuvant.

3) Pharmaceutical composition according to claim 2, further comprising one or more antimicrobial drugs, bacteriophages different from VIR RE RSCIC 299 or components of said bacteriophage different from VIR RE RSCIC 299 and/or probiotic microorganisms.

4) Pharmaceutical composition according to claim 2, further comprising one or more disinfecting compounds.

5) Bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined in claim 1 or composition as defined in anyone of the claims 2-3 for use as a medicament.

6) Bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined in claim 1 or composition as defined in anyone of the claims 2-3 for use in prevention or treatment of the infection or colonization of tissues by Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide, such as, but not limited to, strains of sequence type ST258-2.

7) Use of the pharmaceutical composition according to claim 4 for the sanitization of objects' surfaces.

8) Combination of the bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined in claim 1 together with one or more antimicrobial drugs, bacteriophages different from VIR RE RSCIC 299 or components of said bacteriophage different from VIR RE RSCIC 299 and/or probiotic microorganisms, said combination being for separate or sequential use in prevention or treatment of the infection or colonization of tissues by Klebsiella pneumoniae expressing a CPS«KBO-4 capsular polysaccharide_, 5 such as strains of sequence type ST258-2.

9) Use of the bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined in claim 1 , for the in vitro diagnosis of the infection or colonization of tissues by Klebsiella pneumoniae expressing a CPSKKBO-40 capsular polysaccharide, such as strains of sequence type ST258-2, or for in vitro bacteriotyping of Klebsiella pneumoniae.

1 0) Method for the in vitro diagnosis of the infection or colonization of tissues by Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide, such as sequence type ST258-2, or for in vitro5 bacteriotyping of Klebsiella pneumoniae, said method comprising or consisting of a) contacting a bacteria culture with the bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined in claim 1 and b) observing the possible lysis of the cultured bacteria, said bacteria culture being obtained0 by a biological sample of a patient in case of in vitro diagnosis or a bacteria culture of Klebsiella pneumoniae isolated from environment, wherein the infection or colonization of tissues by Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide is detected, or the bacteriotyping of the Klebsiella pneumoniae as Klebsiella pneumoniae 5 expressing a CPSKKBO-4 capsular polysaccharide occurs, when a lysis of the culture is observed.

1 1 ) Kit for the in vitro diagnosis of the infection or colonization of tissues by Klebsiella pneumoniae expressing a CPSKKBO-4 capsular polysaccharide, such as sequence type ST258-2, or for in vitro o bacteriotyping of Klebsiella pneumoniae, said kit comprising or consisting of the bacteriophage VIR RE RSCIC 299, variants thereof and/or components of said bacteriophage or variants of said components as defined in claim 1 .