Phage luminescent detection platform for rapid typing of klebsiella pneumoniae capsular types

Abstract

This invention provides a reporter phage, its construction method, and its applications. The reporter phage comprises a nucleic acid sequence encoding a reporter protein and at least one receptor-binding protein, wherein the receptor-binding protein is partially or entirely engineered to enable the reporter phage to specifically infect specific capsular types of Klebsiella pneumoniae. The reporter phage provided by this invention enables sensitive and specific detection of Klebsiella pneumoniae in complex genetic and multi-microbial environments. In particular, the scalable and iteratively evolving reporter phage platform provided by this invention offers a universal diagnostic tool for addressing capsular diversity, guiding phage-based therapies, and providing information for epidemiological surveillance.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the construction and application of a phage luminescence detection platform for rapid typing of Klebsiella pneumoniae capsular type. Background Technology

[0002] Klebsiella pneumoniae ( Klebsiella pneumoniae , K. pneumoniae Klebsiella pneumoniae is a key opportunistic pathogen that can cause serious, life-threatening illnesses, including lung infections, purulent liver abscesses, and endophthalmitis. It is also the second most common cause of nosocomial urinary tract infections (UTIs) worldwide. A key virulence determinant contributing to these diverse and serious infections is the capsular polysaccharide (CPS) of Klebsiella pneumoniae, a thick, surface-exposed layer of high-molecular-weight polysaccharide that envelops the bacteria and protects them from host immune clearance and environmental stress.

[0003] Serological typing is a traditional capsular typing method based on antigen-antiserum reactions to distinguish capsular (K) antigens, and has long been used for classification. K. pneumoniae Classical serological typing identifies nearly 80 chemically distinct capsular serotypes (K antigens). In contrast, modern genotyping methods based on K locus genetic characterization have identified 186 different K locus types. Capsular typing results are crucial for effective clinical diagnosis and treatment, further influencing global epidemiological surveillance, source tracing, rational vaccine design, and antibody- or phage-based therapies. K. pneumoniae The distribution of capsular forms exhibits significant heterogeneity, varying considerably depending on geographical region and anatomical site of infection (especially bloodstream and urinary tract infections). In China, K47 and K64 are carbapenem-resistant. K. pneumoniae The most common capsule type in CRKP. Together, they account for 76% of all clinical isolates and 76.3% of urinary tract infection isolates (Hu F, et al. Carbapenem-resistant). Klebsiella pneumoniae capsular types, antibioticresistance and virulence factors in China: a longitudinal, multi-centrerest. Nature Microbiology (2024;9:814-29.), this high prevalence highlights the clinical threat posed by this multidrug-resistant bacterial group. Furthermore, K1, K2, and K57 are often associated with highly virulent bacteria. K. pneumoniae (hvKP)-related capsular types, among which K1 and K2 are the main highly virulent capsular types found in Chinese patients with urinary tract infections and have the potential to cause invasive urinary tract infections.

[0004] However, the application of serological typing is limited by its reliance on expensive antisera, technically demanding procedures, and susceptibility to cross-reactivity due to high antigenic similarity between strains. Furthermore, nucleic acid-based molecular techniques for capsular typing typically require sequence information covering approximately 10-30 kbp of the genome, necessitating whole-genome sequencing or multiple rounds of Sanger sequencing, which is time-consuming. Another key drawback of molecular techniques is their inability to distinguish between live and dead cells. These limitations underscore the need to develop novel detection methods or strategies that specifically target the capsular region.

[0005] Bacteriophages are viruses that specifically target bacteria, with a range of specificity for attaching to and infecting host cells from species-specific to single strains. Therefore, phage therapy offers a highly specific alternative to traditional antibiotics with minimal interference to the host microbiota. Furthermore, the precise specificity of phage-bacteria interactions makes phages a valuable tool for bacterial detection and identification. Integrating reporter genes (such as NanoLuc or NLuc) into the phage genome can significantly improve the detection sensitivity of phages. NLuc is a small, highly stable, and representative luciferase that produces a strong, ATP-independent glow-type luminescence signal upon substrate addition; its excellent brightness and stability are particularly advantageous for detecting low bacterial loads. Utilizing these properties, patent application CN119120396A discloses a recombinant mycobacterial fluorescent phage for rapid detection of mycobacteria. Patent application CN119464443A discloses a reporter phage T4::Nluc for the detection of clinical urinary tract infections caused by Escherichia coli. However, due to the host specificity of bacteriophages, these reporter phages only cover a small subset of each bacterial species. Current research involving reporter phages has not yet seen applications for rapid typing of multiple capsular serotypes of Klebsiella pneumoniae. Therefore, there is an urgent need to develop a detection phage capable of efficiently identifying and distinguishing different capsular serotypes of Klebsiella pneumoniae for rapid capsular typing of the bacterium. Summary of the Invention

[0006] bacteriophages, especially those that infect K. pneumoniae The host specificity of bacteriophages is mainly determined by their receptor-binding protein (RBP), resulting in strict host specificity. K. pneumoniae The RBP of a bacteriophage typically recognizes CPS and has depolymerase activity. It defines the host range of the bacteriophage by cleaving specific repeating units within the host CPS. Therefore, the RBP is the main factor determining the capsule specificity of Klebsiella pneumoniae bacteriophages.

[0007] In order to solve one of the aforementioned technical problems existing in the prior art, the present invention firstly... nlucA reporter gene was inserted into a K2-targeting phage, resulting in a K2-specific reporter phage, ΦRCIP0109:: nluc The reporter phage exhibits high specificity and sensitivity. Using this phage as a framework, this invention, based on RBP structural unit modules capable of recognizing multiple capsule types, generates a modular set of reporter phages and compositions by freely switching and combining RBPs and replacing them with variants targeting different K types. These reporter phages demonstrate high specificity and robust performance in the detection of various sample types, including standard culture conditions and urinary tract infections. Furthermore, this invention demonstrates that directed evolution can enhance the infectivity of the phage, thereby improving the sensitivity of the reporter phage. In summary, this invention establishes a scalable and iteratively evolving reporter phage platform, successfully achieving rapid typing of multiple capsular types of Klebsiella pneumoniae in samples from different sources. This platform provides a universal diagnostic tool for addressing capsule diversity, guiding phage-based therapies, and providing information for epidemiological surveillance.

[0008] In a first aspect, the present invention provides a reporter phage comprising a nucleic acid sequence encoding a reporter protein and at least one receptor-binding protein. The receptor-binding protein is partially or entirely engineered so that the reporter phage can specifically infect specific types of Klebsiella pneumoniae.

[0009] In some embodiments, the capsule type is selected from one or more of K1 to K79.

[0010] In some preferred embodiments, the capsule type is selected from one or more of K1, K2, K5, K10, K20, K25, K27, K47, K54, K57 and K64.

[0011] In some preferred embodiments, the capsule type is selected from one or more of K1, K2, K47, K57 and K64.

[0012] In some embodiments, the reporter protein is selected from luciferase, fluorescent protein, alkaline phosphatase, β-lactamase, or β-galactosidase.

[0013] In some preferred embodiments, the luciferase is selected from firefly luciferase, kidney luciferase, or nanoluciferase.

[0014] In some preferred embodiments, the luciferase is selected from nanoluciferase.

[0015] In some embodiments, the reporting phage is selected from the Myotail Phage Family, Longtail Phage Family, or Shorttail Phage Family.

[0016] In some preferred embodiments, the reporter phage is a bacteriophage belonging to the family Brachyphageidae.

[0017] In some embodiments, the reporter phage has a genome comprising the nucleotide sequence shown in SEQ ID NO.1 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO.1.

[0018] In some implementations, the reporting phage includes variants.

[0019] In some implementations, the variant is obtained by directing the evolution of the parental reporter phage.

[0020] In some embodiments, the variant comprises a mutant of the receptor-binding protein.

[0021] In some preferred embodiments, the mutant of the receptor-binding protein comprises a mutation in which the Y at position 376 from the N-terminus to the C-terminus of the amino acid sequence shown in SEQ ID NO.2, relative to its parent receptor-binding protein, is changed to H.

[0022] In some preferred embodiments, the amino acid sequence of the mutant of the receptor-binding protein is shown in SEQ ID NO. 3.

[0023] In a second aspect, the present invention provides a composition comprising at least one of the reporter phages described in the first aspect.

[0024] Thirdly, the present invention provides a method for preparing reporter phages.

[0025] In some embodiments, the method includes: (a) An expression cassette is inserted into the non-coding region downstream of the major capsid protein gene in the parental bacteriophage genome to obtain a recombinant vector, and (b) the recombinant vector is introduced into host bacteria for culture to obtain the reporter bacteriophage. The parental bacteriophage can specifically infect a specific capsular type of Klebsiella pneumoniae, and the expression cassette contains a nucleic acid sequence encoding a reporter protein and a ribosome binding site (RBS) sequence, and the host bacterium is the corresponding specific capsular type of Klebsiella pneumoniae.

[0026] In some alternative implementations, the method further includes purifying the reporter phage.

[0027] In some embodiments, the purification includes centrifugation, chromatography, gel filtration, and / or precipitation steps.

[0028] In some embodiments, the centrifugation step includes density gradient centrifugation, preferably cesium chloride density gradient centrifugation.

[0029] In some implementations, step (a) includes: (a1) The parental phage genome is provided in the form of multiple fragments, wherein an expression cassette is inserted in the fragment containing the downstream non-coding region of the major capsid protein gene. (a2) Provides linear carriers, and (a3) The recombinant vector is prepared by seamless cloning the linear vector and the plurality of fragments.

[0030] In some embodiments, the parent phage is Klebsiella pneumoniae phage RCIP0109.

[0031] In some embodiments, the reporter protein is a nanoluciferase.

[0032] In some embodiments, the expression cassette contains the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO.4.

[0033] In some optional embodiments, the method further includes obtaining a reporter phage that specifically infects another or more specific types of Klebsiella pneumoniae by replacing part or all of the receptor-binding protein of the reporter phage with a phage receptor-binding protein that specifically infects another or more specific types of Klebsiella pneumoniae.

[0034] Fourthly, the present invention provides a method for detecting and / or quantifying a specific capsular type of Klebsiella pneumoniae present in a sample, wherein the method comprises: The sample or bacteria derived therefrom are contacted with the reporter phage described in the first aspect, the composition described in the second aspect, or the reporter phage prepared by the method described in the third aspect, wherein the reporter phage specifically infects the specific capsular Klebsiella pneumoniae and produces a reporter protein; and The amount of the reporter protein produced is measured, and the measured amount is correlated with the presence and / or quantity of the specific capsular type of Klebsiella pneumoniae.

[0035] In some implementations, the time from contacting the sample with the reporter phage to detecting the presence and / or quantity of a specific capsular Klebsiella pneumoniae is between 5 minutes and 3 hours.

[0036] In some implementations, the time from contacting the sample with the reporter phage to detecting the presence and / or quantity of a specific capsular Klebsiella pneumoniae is between 10 minutes and 3 hours.

[0037] In some embodiments, the sample is selected from any one of environmental samples, food samples, clinical samples, or hospital waste resource samples.

[0038] In some preferred embodiments, the sample is selected from clinical samples.

[0039] In some embodiments, the clinical sample is obtained from tissue, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, lavage fluid, bronchial lavage fluid, swabs from the skin or mucous membrane surface, cultures thereof, or any combination thereof.

[0040] In some preferred embodiments, the clinical sample is obtained from urine, irrigation fluid, sputum, saliva, mucus, cultures thereof, or any combination thereof.

[0041] Fifthly, the present invention provides a reagent kit or system.

[0042] In some embodiments, the kit or system comprises the reporter phage described in the first aspect or the composition described in the second aspect.

[0043] In some embodiments, the kit or system comprises a reporter phage prepared by the method described in the third aspect.

[0044] In some embodiments, the kit or system further includes a detection reagent, wherein the detection reagent contains a substrate for reacting with the reporter protein to detect the reporter protein.

[0045] In some embodiments, the kit or system further includes a solid matrix capable of supporting attachment of at least one target microorganism.

[0046] In some implementations, the system is selected from microfluidic chips.

[0047] In some implementations, the system may be fully automated, semi-automated, or guided by a user via a computer.

[0048] Klebsiella pneumoniae ( Klebsiella pneumoniaeK2 is an important opportunistic pathogen that can cause serious, life-threatening infections. Its capsular polysaccharide is a key virulence factor, and capsular typing is crucial for surveillance, source tracing, and the development of vaccines or phage therapies. Providing information for accurate and rapid clinical diagnosis and treatment highlights the need for novel targeted capsular detection methods. This invention utilizes the high specificity of phage receptor-binding proteins to construct a method that specifically detects K2 phages. K. pneumoniae The report phage of the strain ΦRCIP0109:: nluc ΦRCIP0109:: nluc Using this as a base, by exchanging receptor-binding proteins, its detection range is extended to four other clinically important K types (K1, K47, K57, and K64). These five reporter phages achieve 100% specificity against clinical isolates, can detect bacterial concentrations as low as 10 CFU / mL, have a detection time of 3.5–5 hours, and can successfully distinguish host strains in urine in multimicrobial environments.

[0049] Furthermore, this invention, through directed evolution, introduces mutations into the receptor-binding protein, resulting in an evolved reporter phage with a luminescence intensity 10 to 100 times higher than its ancestral phage. This invention establishes a scalable platform, thereby constructing a platform that can be extended to the detection of other capsule types and other pathogens, possessing clear potential for integration into point-of-care diagnostics to precisely guide targeted antimicrobial therapy or phage therapy.

[0050] Compared with the prior art, the beneficial effects of the present invention are at least as follows: 1. A modular bioluminescent reporter phage platform was constructed for rapid typing of Klebsiella pneumoniae capsules.

[0051] 2. By exchanging receptor-binding proteins, the detection capability of reporter phages is extended to a variety of clinically important K-types. The modular assembly platform for RBPs provided by this invention successfully achieves precise customization of the phage host range by freely switching and combining RBPs among phages.

[0052] 3. The reporter phage of the present invention can achieve sensitive and specific detection of Klebsiella pneumoniae in complex genetic and multi-microbial environments.

[0053] 4. The receptor-binding protein mutation guided by directed evolution in this invention significantly enhances the adsorption capacity of bacteriophages, thereby increasing detection sensitivity. Attached Figure Description

[0054] Figure 1 The report phage Φ109:: is shown. nlucThe construction process; (A) Genomic homologous comparison of phage T7 and T7-like phage Φ109; (B) Φ109:: nluc A schematic flowchart of the construction process; abbreviation in the diagram: YAC, yeast artificial chromosome; nluc NanoLuc luciferase.

[0055] Figure 2 The report phage Φ109:: is shown. nluc Characterization; (A) Wild-type phage Φ109 and reporter phage Φ109:: nluc (A) Genomic homologous comparison; (B) Spotting experiment evaluation of Φ109:: nluc (c) Lytic activity and bioluminescence of wild-type bacteriophage on host bacteria (Kp8-41); (d) Monitoring OD over time 600 Compare Φ109:: nluc Bacterial lysis kinetics of wild-type bacteriophage Φ109 infection; (D) Comparison of Φ109:: nluc One-step growth curves of wild-type phages propagated on Kp8-41; data are mean ± SD of three biological replicates. Mann-Whitney U The test compares the two groups. Significant differences are expressed as follows: ns Not significant ( p ≥0.05); Abbreviation: OD 600 Optical density at 600 nm; SD, standard deviation.

[0056] Figure 3 The use of reporter phage Φ109:: was shown. nluc Results of sensitive and specific detection of Klebsiella pneumoniae type K2; (A) Φ109:: nluc Infecting host strain Kp8-41 within 6 hours, different bacterial concentrations (10) 1 -10 7 The detection sensitivity kinetics of (CFU / mL) were determined, with the gray dashed line representing the detection baseline (mean background + 3SD). RLU values ​​below this threshold were considered negative signals; (B) Φ109:: was determined by simple linear regression. nluc Linear dynamic range after 1 h of incubation; (C) Φ109:: nluc Specificity assessment of the target K2 strain and non-K2 strain groups after 3 h of incubation; data points with RLU values ​​below the detection baseline are shown in light yellow and defined as negative, while positive signals are shown in blue; data are expressed as mean ± SD. n =3); Abbreviations: SD, standard deviation; RLU, relative light unit.

[0057] Figure 4This demonstrates the implementation of Φ109:: via RBP switching. nluc Modular expansion results of detection range; (A) Engineered report of phages within 6 h at different bacterial concentrations (10 1 -10 7 Sensitivity kinetics (CFU / mL). The gray dashed line represents the detection baseline; (B) Evaluation of engineered reporter phage specificity against non-target strains (representing 17 other K types) and their specific target strains; data are expressed as mean ± SD (CFU / mL). n =3); Abbreviations: SD, standard deviation; RLU, relative light unit.

[0058] Figure 5 The performance results of the reporter phage toolkit in simulated urine and multimicrobial environments are shown; (A) the sensitivity kinetics of the reporter phage against five target K-type (K1, K2, K47, K57, and K64) strains in simulated urine, i.e., the RLU values ​​monitored over time for each corresponding host strain; (B) the specificity assessment of the reporter phage toolkit at 1 h and 3 h time points in a multimicrobial co-culture model; data are expressed as mean ± SD. n =3); Abbreviations: SD, standard deviation; RLU, relative light unit.

[0059] Figure 6 This demonstrates that directed evolution enhances Φ109:: nluc Detection performance; (A) Ancestor bacteriophage (Anc) and evolved bacteriophage (Evo) at different bacterial concentrations (10 4 -10 7 (A) Comparison of detection kinetics at CFU / mL; (B) Evaluation of the detection enhancement effect of a group of different clinical isolates with K2-type capsules; (C) Determination of the adsorption efficiency of ancestral phages and evolved phages on host Kp8-41 by measuring unadsorbed phage particles within 5 min. The results were analyzed using two-way ANOVA and post-hoc tests (Sidak correction), and significance was expressed as: p ≤ 0.05, p ≤ 0.001, or ns Not significant; (D) Analysis of SNP-induced mutations in the RBP2 gene. The upper figure shows a schematic diagram of the RBP2 locus at the Y376H mutation site, and the lower figure shows the RBP2 structure predicted by AlphaFold3. Data are expressed as mean ± SD. n = 3); (E) using Φ109_R57:: nluc and Φ109_R57:: nluc-E7 linear range assessment at 0.5 h; abbreviations: RBP, receptor-binding protein; SNP, single nucleotide polymorphism; SD, standard deviation; RLU, relative optical unit.

[0060] Figure 7 Φ109:: is shown nluc Further characterization of infection kinetics and detection performance; (A) Unpurified lysate and Φ109:: purified by sucrose gradient. nluc Comparison of background luminescence, results using the Mann-Whitney method. U The test compares two groups, and the significance is expressed as: p ≤0.05; (B) Density gradient purification for Φ109:: nluc The effect of detection sensitivity is shown in the figure, which displays the curves for purified phage (blue squares) and unpurified control (gray squares); (C) Target K2 strain at 10 7 Bioluminescence detection at CFU / mL, with the black dashed line representing the detection baseline; (D) Four other K2 strains at 10 3 -10 5 (E) Detection kinetics within 2 h at CFU / mL concentration range; (F) Determination of the limit of detection (LOD) for four other K2 strains, with the gray dashed line representing the baseline, and data expressed as mean ± SD from three biological replicates; (G) Serial dilutions of Φ109:: nluc Lytic activity against five target K2 strains; data are expressed as mean ± SD from three biological replicates; abbreviations: LOD, limit of detection; SD, standard deviation; results are presented in Mann-Whitney U The test compares two groups; significance is expressed as... p ≤ 0.05.

[0061] Figure 8a - 8c Further characterization of engineered RBP-exchange reporter phages is shown; (A) A schematic flowchart of RBP exchange via homologous recombination, from the chassis (reporter phage Φ109:: nluc (A) Generate four engineered reporter phages, which are then restarted and proliferated in the host strain; (B) Evaluate the engineered reporter phages (Φ109_R1::) using growth curves. nluc Φ109_R47:: nluc Φ109_R57:: nluc and Φ109_R64:: nluc ) and chassis bacteriophage Φ109:: nluc(C) Comparison of lytic activity on their respective target hosts; (D) Lytic activity and bioluminescence spectra of engineered reporter phages; (E) Determination of the linear dynamic range of engineered reporter phages after 1 h of incubation using simple linear regression; (F) Demonstration of the lytic activity of serially diluted RBP exchange reporter phages against five strains of each target K type using spotting method; (G) Comparison of Φ109_R57:: using one-step growth curve determination. nluc With Φ109:: nluc and Φ109_R1:: nluc Growth characteristics; (G) Using RBP exchange reporter phage at 10 7 Bioluminescent detection of target strains at CFU / mL, with the black dashed line representing the baseline; (HK) K1, K47, K57 and K64 four capsular strains at 10 3 -10 5 Detection kinetics within 2 h in the CFU / mL concentration range; (L) Engineered report phage detection limits for four other strains of each K type, with gray dashed lines representing the baseline, and data expressed as mean ± SD from three biological replicates;

[0062] Figure 9 Further evaluation results of the phage reporting toolkit in synthetic urine and multimicrobial conditions are shown; (A) phage reporting performance against target bacteria (10) in synthetic urine. 3 -10 4 (CFU / mL) luminescence; (B) without K. pneumoniae (c) A multimicrobial mixture was used as a negative control for the reporter phage toolkit; (d) Targeted detection of five target bacteria by five reporter phages, with four non-target urinary tract pathogens at three concentrations (10... 3 -10 4 CFU / mL)-induced luminescence signal inhibition rate; (D) Φ109:: nluc The adsorption efficiency in synthetic urine cultures containing a single microorganism (Kp8-41 only) and multiple microorganisms (Kp8-41 and four other urinary tract pathogens) was determined. Results were analyzed using two-way ANOVA and post-hoc tests (Sidak corrected). Significance was expressed as mean ± standard deviation. p ≤ 0.001, or ns Not significant.

[0063] Figure 10a - 10b The evolved reporter bacteriophage Φ109:: is shown. nluc -E7 screening and characterization; (A) After 7 rounds of directed evolution, based on the luminescent Φ109:: nluc(A) Variant system screening; (B) Area under the luminescence curve of phages from 7 evolutionary rounds (correlation with A); (C) Φ109:: nluc -E7 at lower bacterial loads (10 1 -10 3 (CFU / mL) detection sensitivity; (D) Confirmation of Φ109:: nluc - Host specificity assessment of E7, data expressed as mean ± SD from three biological replicates; (E) Ancestor reporter phage (Anc, Φ109:: nluc ) and its evolved variant (Evo, Φ109:: nluc Comparison of EOPs between -E7) n =13); the results were obtained using the Mann-Whitney... U The test included comparisons between the two groups; (F) assessing the lytic activity of the ancestral and evolved phages against the host strain Kp8-41 using growth curves; and (G) determining the lytic activity of the ancestral and evolved phages using one-step growth curves over 80 min. nluc Replication kinetics; (H) by measuring unadsorbed phage particles within 5 min, determining the Φ109:: of each generation. nluc Adsorption efficiency on host strain Kp8-41; (I) Electrostatic potential diagrams show the charge changes caused by Y376H substitution; negatively charged (ancestor) and positively charged (mutant) sites are colored red and blue, respectively; (J) After 7 rounds of directed evolution, based on the luminescent Φ109_R57:: nluc Variant system screening; (K) Area under the luminescence data curves of phages from 7 evolutionary rounds; (L) Determination of Φ10⁹:: of each generation by measuring unadsorbed phage particles at 1 min. nluc Adsorption efficiency on host strain Kp142; results were analyzed using two-way ANOVA and post-hoc tests (Sidak correction); significance is expressed as... p ≤0.05, p ≤0.01, or ns Not significant; abbreviations: AUC, area under the curve; SD, standard deviation; EOP, plaque formation efficiency; RBP, receptor-binding protein. Detailed Implementation

[0064] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0065] Definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. The terminology used to describe this invention is intended only to describe a particular implementation and not to limit the scope of the teachings.

[0066] Unless the context clearly indicates otherwise, references to a specific quantity herein include their plural forms. For example, the term "cell" includes one or more such cells and equivalents known to those skilled in the art, etc.

[0067] The term “and / or” as used herein should be understood to mean any one of the options or any combination of two or more of the options.

[0068] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0069] The term "phage" as used in this article includes bacteriophages, Klebsiella pneumoniae bacteriophages (e.g., those targeting classic Klebsiella pneumoniae), and more. Klebsiella pneumoniae , cKp), highly virulent Klebsiella pneumoniae (hypervirulent Klebsiella pneumoniae Bacteriophages can be one or more of the following: carbapenem-resistant highly virulent Klebsiella pneumoniae CR-hvKp phage, and any other virus capable of invading living bacteria and other microorganisms. Bacteriophages have a simple structure, primarily composed of a protein coat and nucleic acid, the genetic material. Most bacteriophages have a tail structure, connected to a apex of the head, typically composed of a tail spike, tail fibers, and tail tube. Although the tail structure and composition vary among different types of bacteriophages, they all contain a RBP composed of specific protein subunits, responsible for binding to receptors on the host surface.

[0070] The term "receptor-binding protein (RBP)" as used in this article refers to the collective term for the structures required for bacteriophages to bind to the bacterial surface. A single bacteriophage can possess multiple RBPs, whose main function is to recognize and bind to specific bacterial receptors. RBPs with hydrolytic functions can hydrolyze bacterial surface structures, assisting in the injection of nucleic acids into the host bacteria. According to statistics on RBPs in bacteriophage populations, most RBPs are tail spines, tail filaments, and the substrate in the tail structure; in a few bacteriophages without a tail structure, the capsid protein can serve as an adsorption structure. The structure of RBPs is highly template-like, consisting of a conserved N-terminal (Amino-terminal), a flexible linker in the middle, and a C-terminal (Carboxy-terminal) that can adsorb and hydrolyze bacterial receptors. High specificity is both an advantage and a disadvantage of bacteriophages as biological agents (acting only on the target and possessing safety). RBPs are crucial for researching bacteriophage biotechnology and optimizing bacteriophage biological agents: the host specificity of RBPs allows for bacterial identification and capsule serotyping; through editing and acclimation of RBPs, the target host range of bacteriophages can be artificially selected. In some embodiments, the reporter phage described in this invention can have a wider or broader target host range compared to the parent phage.

[0071] Phage engineering methods mainly include traditional homologous recombination technology, Red system-based recombination, CRISPR-Cas-based phage engineering, and phage reactivation using assembled phage genomic DNA. Applications of genetically engineered phages in bacterial infection include expanding or redirecting the host range of genetically engineered phages. RBP bioengineering is an important potential tool for controlling the host range of phages in biomedical applications. Existing descriptions of engineering methods, as known to those skilled in the art, can be used for the construction of the engineered phages described in this invention, such as those described in Chinese patent application CN109952373A, and are incorporated herein by reference.

[0072] As used herein, the term "parental phage" can refer to a wild-type phage found in any environment or an engineered phage. In some embodiments, the parental phage may be specific to at least some desired target host bacteria and may be further modified according to the methods of this application to alter or customize its target host range. In some embodiments, the receptor-binding protein of the parental phage may be replaced to recognize a target host that is different from some or all of the host species of the parental phage, thereby generating a reporter phage with a customized host range. In some embodiments, the parental phage is phage RCIP0109.

[0073] In some embodiments, the bacteriophage is selected from the families Myoviridae, Siphoviridae, Podoviridae, Ackermannviridae, and Herelleviridae. Myoviridae refers to any bacteriophage with an icosahedral head and a long, contractile tail, encompassing bacteriophages within both Myoviridae and Herelleviridae families. The Podoviridae family is divided into two subfamilies, Autographivirinae and Picovirinae, comprising 11 genera. The head of Podoviridae is an equidistant or elongated icosahedron, assembled from scaffold proteins and capsid proteins; a few bacteriophages contain head fibers, which facilitate attachment to bacteria. The bacteriophage tail connects to one apex of the head and typically consists of structures such as a tail spike, tail fibers, and a tail tube. Although different types of short-tailed bacteriophages differ in their tail structure and composition, they all contain RBPs composed of specific protein subunits, which are responsible for binding to host surface receptors.

[0074] The term "Klebsiella pneumoniae" is used in this article. Klebsiella pneumoniae "Klebsiella pneumoniae" is a Gram-negative bacterium belonging to the family Enterobacteriaceae and the genus Klebsiella. Klebsiella Klebsiella pneumoniae (spp.) commonly infests the skin, respiratory tract, and intestines of humans and animals. Infections with Klebsiella pneumoniae can involve multiple organs and sites, potentially causing various types of infectious diseases such as pneumonia, meningitis, urinary tract infections, sepsis, and purulent liver abscesses. It is one of the most common and important pathogens causing hospital-acquired infections and community-acquired infections. Based on virulence characteristics, Klebsiella pneumoniae can be classified into Classic Klebsiella pneumoniae (Classic...) K. pneumoniae (cKP), Hypervirulent Klebsiella pneumoniae K. pneumoniaeKlebsiella pneumoniae (cKP) or carbapenem-resistant highly virulent CR-hvKp group. hvKP is generally prevalent in young, healthy individuals in the community, while cKP is mainly seen in secondary infections in immunocompromised individuals in hospitals. hvKP is a highly invasive and pathogenic pathogen that can lead to more severe and disseminated infections. With bacterial evolution and the overuse of some antibiotics, drug-resistant bacterial infections have become one of the major public health challenges of the 21st century. The drug resistance characteristics of Klebsiella pneumoniae are closely related to the antibiotic resistance genes (ARGs) encoded by plasmids. Due to plasmid and genetic factors, Klebsiella pneumoniae continues to accumulate ARGs under inappropriate antibiotic use, leading to the emergence of multidrug-resistant (MDR) bacteria, especially extensively drug-resistant (XDR) bacteria and "superbugs," which pose a serious threat to the treatment of clinical infections. The emergence and spread of extensively drug-resistant strains is an urgent problem to be solved in the field of Klebsiella pneumoniae control. Klebsiella pneumoniae possesses various virulence factors, including siderophores, fimbriae, capsular polysaccharides (CPS), and lipopolysaccharides (LPS). CPS is considered the most important virulence factor in Kp, and there are nearly 80 capsular serotypes of Klebsiella pneumoniae. Compared to other capsular serotypes, K1, K2, K5, K20, K54, and K57 are highly virulent strains, commonly found in pneumonia cases. In some embodiments, the capsular serotype of Klebsiella pneumoniae described in this invention is selected from K1 to K79, as described by Dai P et al. (Dai P, Hu D. The making of hypervirulent Klebsiella pneumoniae. J Clin Lab Anal. 2022; 36: e24743. doi: 10.1002 / jcla.24743), which is incorporated herein by reference.

[0075] As used herein, "phage genome" includes naturally occurring phage genomes and their derivatives. Typically, the derivatives have the ability to reproduce in the same host as the naturally occurring phage. In some embodiments, the difference between a naturally occurring phage genome and a derived phage genome lies at least in the deletion or addition of at least one nucleotide from at least one end (if the genome is linear) or at least one point (if the genome is circular) of the phage genome.

[0076] As used herein, the terms “polynucleotide” or “nucleic acid” refer to any RNA or DNA, which may be unmodified or modified. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA as a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions.

[0077] As used herein, the term "reporter protein" may include bioluminescent proteins, fluorescent proteins, or chemiluminescent proteins. Examples of bioluminescent proteins include, but are not limited to: jellyfish luciferin, firefly luciferase, kidney luciferase, red luciferase, luxAB, or nanoluciferase. Examples of chemiluminescent proteins include, but are not limited to, β-galactosidase, β-lactamase, horseradish peroxidase, or alkaline phosphatase. Examples of fluorescent proteins include, but are not limited to: sfGFP, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, mMidoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, and Superfolder. GFP, mAzamiGreen, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, mKusabira-Orange, mKO, mKO2, mOrange, mOrange2, mRaspberry, mCherry, Ds Red, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS-mKate2, PA-GFP, PAmCherry, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange or Dronpa.

[0078] As used herein, the term "luciferase" commonly includes, but is not limited to, luminescent proteins that emit light as a byproduct of substrate catalysis, the light of which can be measured experimentally. Luciferases are a class of luminescent proteins derived from many sources, including firefly luciferase (from the species *Fireflya chinensis*), *Gnaphalium luciferase* from the marine pansy, click beetle luciferase, marine copepod luciferase, and deep-sea shrimp nanoluciferase. Firefly luciferase catalyzes the oxidation of luciferin to oxidized luciferin, resulting in the emission of light photons, while other luciferases (e.g., *Gnaphalium chinensis*) emit light by catalyzing coelenterate. Different filtering systems can be used to read the wavelengths of light emitted by different forms and variants of luciferase. The amount of luminescence is proportional to the amount of luciferase expressed in the cell, and luciferases have been used as sensitive reporter proteins to assess the effect of stimulus-induced biological responses. In some embodiments, the reporter protein is a luciferase. The luciferase can be naturally occurring, such as *Synostemma pentaphyllum* luciferase, firefly luciferase, *Russian luciferase* luciferase, or *Reniformis luciferase* luciferase. In some embodiments, the reporter protein can be a modified luciferase, such as nanoluciferase (NanoLuc, NLuc). When the reporter protein is a luciferase, in some embodiments, analyzing a sample to detect the presence and / or amount of luciferase to determine the presence and / or quantity of a specific *Klebsiella pneumoniae* species includes binding a reporter protein produced by a specific *Klebsiella pneumoniae* species to a luciferase substrate. In some embodiments, the luciferase substrate is selected from luciferin or derivatives thereof, and coelenterate or derivatives thereof. In some embodiments, the luciferase substrate is selected from furazolidone. Small phages package smaller genomes and therefore have lower tolerance to additional transgenes. Smaller reporter proteins are a more suitable choice for modifying phages, especially those with smaller genomes. OpLuc and NLuc proteins are only about 20 kDa, while FLuc is about 62 kDa. The T7 genome is approximately 40 kbp, while the T4 genome is approximately 170 kbp. In some implementations, using a smaller reporter protein allows multiple copies of the reporter gene to be inserted into the phage genome, thereby further amplifying the signal.

[0079] As used herein, the term "host bacterium" and related expressions refer to the microorganisms that a bacteriophage can infect. Bacteriophages can lyse host cells and release new virions upon lysis, transfer genes between hosts, and form lysogenic bacteria, which can alter host function. As used herein, the expression "host range" and related terms and expressions refer to the range or number of hosts that can be infected by a bacteriophage. In other words, "host range" describes the range of organisms (genus, species, strain, or other taxa) that a bacteriophage can infect. Some bacteriophages have a narrow host range, capable of infecting only a few strains within the same species. Other bacteriophages can infect many hosts, sometimes spanning different genera. The breadth of a particular bacteriophage's host range can be attributed in part to the specificity of the bacteriophage's receptor-binding protein (RBP), biochemical interactions during infection, the presence of a relevant prophage or specific plasmid, and host bacteriophage resistance mechanisms. The terms "altered host range" and related expressions are used herein to refer to the artificially altered or engineered host range of a bacteriophage. Such host variability can be achieved through at least some of the methods described in this application, as well as others. In some embodiments, the host range of a bacteriophage is determined by its highly specific RBP, which typically recognizes only one type of host. In Kp, there are many capsule types, and a bacteriophage typically recognizes only one of them.

[0080] As used herein, "specifically" is a description well-known in the art, meaning that when the reporter phage of the present invention is administered to an organism, it preferentially binds to the target host bacterium; or, in other words, the reporter phage of the present invention is distributed only in the target host bacterium, i.e., a specific capsular type, such as K2 capsular Klebsiella pneumoniae or K20 capsular Klebsiella pneumoniae. In some embodiments, the reporter phage of the present invention exhibits multispecificity, meaning that when administered in vivo, the reporter phage specifically infects multiple capsular types of Klebsiella pneumoniae.

[0081] As used in this article, the terms "amino acid" and "amino acid residue" refer to all naturally occurring L-α-amino acids. These amino acids are identified by single-letter or three-letter names: Asp, D, aspartic acid; Ile, I, isoleucine; Thr, T, threonine; Leu, L, leucine; Ser, S, serine; Tyr, Y, tyrosine; Glu, E, glutamic acid; Phe, F, phenylalanine; Pro, P, proline; His, H, histidine; Gly, Glycine; Lys, K, lysine; Ala, A, alanine; Arg, R, arginine; Cys, C, cysteine; Trp, W, tryptophan; Val, V, valine; Gln, Q, glutamine; Met, M, methionine; Asn, N, asparagine.

[0082] As used herein, the term "identity" refers to the degree of matching between two or more nucleotide or amino acid sequences. Two or more sequences used for comparison are identical at a site when they share the same nucleotide or amino acid monomeric subunit (e.g., each of two or more amino acid sequences has a lysine residue at a site). The percentage of identity between two or more sequences is a function of the number of identical sites shared by the two sequences relative to the total number of sites used for comparison × 100. For example, if six out of ten sites in two or more sequences match, the two or more sequences have 60% identity. Typically, comparisons of two or more sequences are performed in a manner that yields the maximum identity. For example, the percentage identity between two or more nucleotide or amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller, incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Furthermore, the percentage of identity between two or more nucleotide sequences or amino acid sequences can be determined using the Needleman and Wunsch algorithm in the GAP program, which has been incorporated into the GCG software package, with a Blossum 62 matrix or a PAM250 matrix, and vacancy weights of 16, 14, 12, 10, 8, 6 or 4, and length weights of 1, 2, 3, 4, 5 or 6.

[0083] As used herein, the term "purification" of a bacteriophage refers to the measurable increase in concentration of a bacteriophage through any purification process (including, but not limited to, isolation from a culture, e.g., isolation from a culture after propagation and / or amplification, centrifugation, etc.) thereby partially, substantially, almost completely, or completely removing impurities, such as host cells and host cell components. Those skilled in the art will understand the amount of purification required for a given application. For example, purified bacteriophages used for detection typically must meet compliant process standards and possess high purity. In some embodiments, the reporter bacteriophage is a purified reporter bacteriophage. Absolute purity is not required for purification; a purified molecule is one in which the molecule is more enriched than it is in its natural environment, for example, in which the molecule constitutes at least 50%, at least 60%, at least 80%, at least 90%, at least 99%, or higher of the total amount of similar molecules in the sample. For example, a purified sample of a reporter bacteriophage is a sample in which the reporter bacteriophage constitutes at least 50% of all bacteriophages in the sample.

[0084] As used herein, the terms "solid support" or "solid matrix" refer to a substrate and / or surface to which biomolecules can bind. For example, a solid matrix may be a assay plate (e.g., a microtiter plate or a porous plate).

[0085] As used herein, “RLU” refers to a relative unit of light measured by a photometer (e.g., BioTek) or a similar instrument that detects light. For example, the detection of a reaction between luciferase and a suitable substrate (e.g., NLuc and furazolidone) is typically reported in the detected RLU. In some implementations, other machines or devices may also be used. For example, a spectrophotometer, a CCD camera, or a CMOS camera can detect color changes and other light emissions. An absolute RLU is important for detection, but a high signal-to-noise ratio (e.g., >2, >2.5, or >3) is also required to reliably detect small numbers or even single cells.

[0086] As used herein, the term "sample" refers to a clinical sample obtained from a subject or isolated microorganisms. In some embodiments, the sample is a clinical sample obtained from a biological source, such as tissue, body fluid, or microorganisms collected from a subject. Sample sources include, but are not limited to, mucus, sputum, bronchoalveolar lavage fluid, bronchial lavage fluid, whole blood, body fluids, cerebrospinal fluid, urine, plasma, serum, or tissue.

[0087] In some embodiments, the kits or systems described herein (e.g., automated systems or kits) include components for performing the detection methods described herein. In some embodiments, a reporter phage is included in the system or kit according to the present invention. In some embodiments, the detection methods are particularly suitable for automated systems and / or kits. In some embodiments, the system or kit may be fully automated, semi-automated, or user-guided via computer. In some embodiments, the systems or kits described herein include systems or kits for rapid detection of a specific capsular type of Klebsiella pneumoniae in a sample, comprising: components for incubating the sample with a reporter phage specific to the target microorganism; components for capturing the microorganism from the sample on a solid support; and components for detecting the reporter protein. In some embodiments, the same components may be used for the capture and / or incubation and / or washing steps.

[0088] The present invention firstly nluc A reporter gene was inserted into a K2-targeting phage, resulting in a K2-specific reporter phage, ΦRCIP0109:: nlucThis phage exhibits high specificity and sensitivity. Subsequently, using this phage as a base, its RBP was replaced with variants targeting different K types, thereby generating a modular reporter phage toolkit capable of detecting multiple capsule types. These reporter phages demonstrated high specificity and robust performance in both standard culture conditions and urinary tract infection simulation samples. Furthermore, this invention demonstrates that directed evolution can enhance the infectivity of phages, thereby improving the sensitivity of reporter phages. In summary, this invention establishes a scalable, iteratively evolving reporter phage platform for rapid capsule typing of Klebsiella pneumoniae, providing a universal diagnostic tool for addressing capsule diversity, guiding phage-based therapies, and providing information for epidemiological surveillance.

[0089] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0090] Example Unless otherwise stated, the present invention will be carried out using conventional techniques of biology, cell culture, molecular biology, etc., which are described in the literature or performed in accordance with product instructions. For example, see J. Sambrook's *Molecular Cloning: A Laboratory Manual* (4th edition, Science Press). Unless otherwise specified, the materials, reagents, or instruments used in the examples are all commercially available conventional products.

[0091] Materials and Methods Strains and culture conditions Unless otherwise stated, all microbial strains used in this invention are listed in Table 1 below. Unless otherwise stated, *Klebsiella pneumoniae* (…) K. pneumoniae Acinetobacter baumannii ( Acinetobacter baumannii ), Escherichia coli ( Escherichia coli ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa Clinical isolates of Staphylococcus aureus were cultured in Luria-Bertani (LB) broth or on LB agar plates at 37°C. Liquid cultures were incubated with shaking at 200 rpm. Staphylococcus aureus ) was cultured in brain and heart infusion medium (BD, USA), and Saccharomyces cerevisiae ( Saccharomyces cerevisiae Strain BY4741 was cultured in yeast extract peptone glucose broth (YPD) (Solepro, Beijing) or agar solid medium at 30°C. All strains were stored at -80°C in stock form of glycerol (25% v / v).

[0092] Table 1 bacteriophage proliferation Prepared using the agar covering method K. pneumoniae Phage lysis buffer. Log-phase host bacteria are mixed with the corresponding phage (approximately 10...). 7 Mix PFU, add 5 mL of melted 0.6% soft agar (cooled to approximately 50°C), and pour onto a 1.5% agar plate. Incubate the plate overnight at 37°C until confluent plaques form. To recover phage particles, add 5 mL of SM buffer (supermicro buffer) to the plate surface and incubate overnight with gentle shaking at 4°C. Then collect the lysis mixture, add 1% chloroform, and vortex. Remove bacterial debris and agar residue by centrifugation. Filter the supernatant through a 0.22 µm membrane, and store the clear phage lysis buffer at 4°C.

[0093] Plaque formation efficiency determination The phage suspension was serially diluted 10-fold, and 5 μL of each dilution was spotted onto bacterial colonies. After incubation at 37°C overnight, plaque formation on the plates was examined. Each... K. pneumoniae The plaque formation efficiency (EOP) of the strains was calculated by comparing the phage titer (PFU / mL) on the test strain with the titer on the propagating host. After substrate addition, bioluminescent signals of the plaques were captured using a Tanon-5200 Multicolor fluorescent gel imaging system (Tanon, Shanghai). All experiments were performed in triplicate.

[0094] Growth curve Bacterial growth dynamics were continuously monitored using an automated growth curve analyzer (Cerillo, USA). The culture was grown to OD... 600 To achieve a multiplicity of infection (MOI) of approximately 0.3, 100 μL of the sample was transferred to each well of a 96-well microplate. Phage suspension was added to the experimental wells to achieve an MOI of 0.01–0.1, while an equal volume of SM buffer was added to the control wells. The microplates were incubated with continuous shaking at 37°C in an analyzer. The optical density (OD) was measured every 3 minutes. 600 The experiment lasted for 6 hours. All experiments were repeated three times.

[0095] One-step growth curve The one-step growth curve was performed following a modified version of a previously published method (Fulgione A, et al. Biomimetichydroxyapatite nanocrystals are an active carrier for Salmonellabacteriophages. Int J Nanomedicine 2019;14:2219-32.). The procedure was as follows: 4 mL of fresh bacterial culture (10... 8 Phages were infected with cFU / mL at an MOI of 0.1 and incubated statically at 37°C for 5 min to allow adsorption. The mixture was then centrifuged at 13,000 × g for 3 min to remove unbound phages. The precipitate was immediately resuspended in 5 mL of preheated LB medium and incubated at 37°C with shaking at 200 rpm for 60–80 min. 500 μL aliquots were collected every 5 min and filtered through a 0.22 μm membrane to obtain cell-free lysates for subsequent phage titer determination. All experiments were performed in triplicate.

[0096] Adsorption experiment The phage adsorption assay was performed according to the previously described method (Ngiam L, et al. The presence of plasmids in bacterial hosts alters phage isolation and infectivity. ISME Communications 2022; 2), with slight modifications. The procedure was as follows: purified phage lysate was added at an MOI of 0.1 to 20 mL of medium in mid-logarithmic growth (OD2). 600 =0.3, ~1×10 8 The bacterial host culture was inoculated with CFU / mL. Immediately take 1 mL of the mixture (t=0) to determine the initial phage titer. P 0 Subsequently, equal volumes of samples were collected from the mixture at time points of 1, 2, 5, 10, 15, and 20 min. Each sample was processed immediately after collection by centrifugation at 13,000 × g for 2 min to precipitate bacterial cells and adsorbed phages. The titer of unadsorbed phages in the supernatant was determined using a spotting method. P f The adsorption percentage at time T is calculated using the following formula: Adsorption rate (%) = [1 - ( P f / P 0[ ] × 100%. For clarity, this embodiment shows data at three representative time points (1, 2, and 5 min) that best illustrate the key stages of the adsorption experiment in the main figure. All experiments were performed in triplicate.

[0097] Phage genomic DNA extraction and bioinformatics analysis Genomic DNA was extracted from newly proliferating phage particles using the λ phage rapid genomic DNA extraction kit (Zhuangmeng Biotechnology, ZP317-1) according to the manufacturer's instructions. Whole-genome sequencing was performed on an Illumina NovaSeq 6000-PE150 platform (Novogene). Quality-filtered reads were de novo assembled using SPAdes (v3.15.5), and contigs were reoriented based on homology and coverage depth, starting from the terminal enzyme large subunit (TerL). The genome was annotated using Prokka (v1.13.1). Single nucleotide polymorphisms (SNPs) were identified by aligning post-evolutionary isolate reads to the ancestral genome at a minimum depth of 10× using Snippy (v4.0.0) (https: / / github.com / tseemann / snippy). All reported variants were confirmed by Sanger sequencing using the primer pairs listed in Table 2.

[0098] Table 2 Primer Information Chassis report phage construction Reporter phages were constructed using a yeast-based assembly strategy. Using the genome of phage RCIP0109 as a template, it was divided into five linear DNA fragments by PCR, with primers designed to generate overlapping ends. To obtain the linearized yeast artificial chromosome vector (YAC) and... nluc The fragment amplified the CEN / ARS replication element and LEU2 selection marker (along with its promoter) from plasmid pRS415, and amplified the fragment from plasmid pNL2.1. nluc The expression cassette and specific primer information are shown in Table 2 above.

[0099] These seven linearized fragments were converted into equimolar amounts. Saccharomyces cerevisiae Competent cells. After recovery at 30°C for 1 h, the transformation mixture was plated on SD / -Leu agar plates (Coolaber) and incubated at 30°C for 72 h to obtain recombinant clones. Single clones were picked and inoculated into 5 mL of SD / -Leu liquid medium and cultured overnight at 30°C. Yeast genomic DNA was then extracted using the YeaStar Genomic DNA Kit (Zymo Research) according to the manufacturer's instructions. The recovered DNA was introduced via electroporation. E. coliDH10B electroporated competent cells (Zhuangmeng Biotechnology, ZC1021D). Phage particles were collected by vortexing and centrifugation after incubation at 37°C for 1 h and lysis with 1% chloroform. 0.5 mL of the supernatant was then mixed with 200 μL of freshly cultured host bacteria in 5 mL of 0.6% soft agar and poured onto LB agar (1.5%) plates. The plates were incubated at 37°C for 18 h to examine plaque formation. Successful construction and validation of reporter phages were performed using the primer pairs listed in Table 2.

[0100] Engineering of reporter phages via RBP exchange The engineering of bacteriophage RBPs was carried out according to the previously described method (Wang C, et al. Data-Driven Engineering of Phages with Tunable Capsule Tropism for Klebsiella pneumoniae). Adv Sci (Weinh) 2024;11:e2309972.). A screening and validation process integrating bioinformatics mining and cluster analysis was used to identify different candidate RBPs and to enable phages with different RBPs to specifically target corresponding bacterial capsule types. The specific steps are as follows: A donor DNA fragment containing the target RBP gene and its flanking homologous arms was cloned into the recombinant vector pCOLADRed using Gibson Assembly to construct the plasmid pCOLADRed-donor. pCOLADRed-donor was electroporated into host bacteria, induced with L-arabinose, and then co-cultured to obtain recombinant chassis reporter phages. To screen for RBP exchange variants, lysis buffer was plated onto the target host strain using a double-layer agar method. Recombinant phages were isolated and purified using three rounds of plaque purification to ensure genetic homogeneity. Successful integration of the new RBP gene was verified by PCR using the primers listed in Table 2.

[0101] Phages were purified by density gradient centrifugation. Phage particles were purified using density gradient centrifugation. The specific steps were as follows: A discontinuous gradient consisting of 60%, 40%, 20%, and 10% (w / v) sucrose layers was prepared. Phage lysate was placed on the top layer of the gradient and ultracentrifuged at 45,218 × g (Optima L-100XP, Beckman Coulter) for 2 h at 4°C. The milky white band containing phage particles was collected. This band was then ultracentrifuged at 8,000 × g for 1 h at 4°C, and replaced with SM buffer to remove sucrose. Finally, the preparation was aseptically filtered (0.22 µm).

[0102] Sensitivity and specificity of bioluminescence reporter assays Bioluminescence measurements were performed using white 96-well microplates (Beyotime Biotechnology) on a Synergy H4 microplate reader (BioTek). Instrument parameters were set as follows: automatic sensitivity and 3-s integration time. Continuous measurements for 6 h were performed under shaking incubation at 37°C and 180 rpm. For most sensitivity assessments, 100 μL of host bacteria (10... 1 -10 7 CFU / mL) of bacteriophage suspension was mixed with 100 μL of phage suspension in microplates. The plates were incubated continuously in a microplate reader for 2–6 h, with luminescence values ​​recorded every 30 min. For experiments assessing enhanced sensitivity, the bacterial-phage co-culture was incubated in a microplate reader for 30–40 min, with luminescence values ​​monitored every 5 min. For specificity assessment, the mixture was incubated at 37 °C and 180 rpm for 3 h before luminescence measurement. Before measurement, the substrate was added as follows: 1 µL of 10 µM furazine (Promega) for single assays, and 1 µL of 1 mM furazine for continuous assays. The concentration of all bacterial inoculum was determined by OD0.05. 600 The viable counts were determined by correlating them with those obtained from plate cultures of serially diluted solutions. The background threshold was set to a signal-to-noise ratio (SNR) of 3, based on the standard definition of mean background plus three times the standard deviation (3σ). All experiments were performed in triplicate.

[0103] Detection in pure cultures and multimicrobial artificial urine matrices For sensitivity assessment, K. pneumoniae The overnight culture was mixed with artificial urine to achieve 5 × 10⁻⁶. 3 Up to 5 × 10 5 Urine samples were prepared to a final concentration of CFU / mL. The mixture was incubated at 37°C with shaking (200 rpm) for 1 h to allow bacterial acclimatization. Subsequently, 100 μL of urine sample was transferred to a microplate and mixed with 100 μL of reporter phage. Bioluminescence kinetics were continuously monitored for 6 h after substrate addition. For specificity assessment, a solution containing... E. coli , A. baumannii , P. aeruginosa and S. aureus (Each adjusted to 2 × 10) 5 A multimicrobial mixture (CFU / mL) was used as a negative control. After the same pre-incubation, 100 μL of the bacterial mixture was mixed with 100 μL of reporter phage in microplates, and the luminescence value was measured after 1 h and 3 h of incubation. The luminescence signal inhibition rate was calculated using the following formula: Signal inhibition rate (%) = (MFU / mL) / (CFU / mL) * ... 对照 -MFI 干扰 ) / MFI 对照 ×100%. Among them, MFI 对照The mean luminescence intensity represents the control group, which consists of host strains cultured in pure synthetic urine; MFI 干扰 The value represents the average luminescence intensity of the interference group, which consists of host strains co-cultured with other urinary tract pathogens in pure synthetic urine. The CFU / mL values ​​of all bacterial inoculum are expressed by converting OD... 600 The viable counts were determined by correlating the results with those obtained from plate cultures of serially diluted solutions. The background threshold was defined according to the standard of the average background signal plus 3σ, corresponding to an SNR of 3. All experiments were performed in triplicate.

[0104] Directed Evolution To enhance the reporting phage Φ109:: nluc To assess the sensitivity of the target strain, directed evolution was performed. The specific steps are as follows: Φ109:: nluc Add 4 mL of the host strain Kp8-41 (OD1000) in the logarithmic phase. 600 The mixture was incubated in LB broth (0.3 g / L) with an MOI of 0.1. The mixture was incubated at 37°C and 200 rpm for 24 h with shaking. After incubation, 1% chloroform was added and the mixture was vigorously vortexed to lyse the bacterial cells. The lysate was then centrifuged at 4°C and 8,000 × g for 10 min to remove cell debris. 400 μL of the supernatant was used to initiate the next passage. This process was repeated a total of seven passages, and the final evolved population was named Φ109:: nluc -E7. Luminescence was monitored in independent lineages over seven generations, with each generation evaluated for 60 minutes. Luminescence data for each generation was quantified by calculating the area under the curve (AUC). To account for random evolutionary events, three independent lineages were passaged in parallel.

[0105] Prediction and Structural Analysis of RBP Variants AlphaFold3 was used to predict wild-type and mutant RBPs (from phage ΦRCIP0109::). nluc A three-dimensional structural model of the complex was constructed. The confidence level of the predicted complex was evaluated based on the interface prediction template modeling (ipTM) score. PyMOL (v3.1) was used for structural visualization, calculation of the global root mean square deviation (RMSD) between wild-type and mutant structures, and analysis of surface electrostatic potential.

[0106] Statistical Analysis and Data Visualization Statistical analysis was performed using GraphPad Prism (v10.1.2). Genome comparison plots were generated using EasyFig (v2.2.2). Data in the plots are expressed as mean ± standard deviation (SD). The limit of detection (LOD) was determined as the lowest value that could be detected in ≥95% of replicates. K. pneumoniae Concentration. Data via Mann-WhitneyU The analysis was performed using the test and one-way ANOVA. If the ANOVA was significant, a post-hoc test (Sidak corrected) was used to compare with the control group. Significance was expressed as: ns Not significant. p ≤0.05, p ≤0.01, or p ≤0.001.

[0107] Example 1: Genetically engineered reporter phage RCIP0109:: nluc Construction As Przondovirus Representative of the genus Brachyta, K. pneumoniae Bacteriophage RCIP0109 (hereinafter referred to as "Φ109") is characterized by its specific targeting of K2 strains, compact genome (41,152 bp), and genome homology with that of bacteriophage T7. Figure 1 A) It was selected as the chassis for further engineering modifications, which is beneficial for genetic manipulation. Through whole-genome sequencing and gene annotation prediction, it was identified as... Autographiviridae division Przondovirus A short-tailed bacteriophage of the genus. Autographiviridae Bacteriophages, represented by the T7 model, possess early genes transcribed by the host RNA polymerase, as well as mid-to-late-stage genes specifically transcribed by the phage's own RNA polymerase. They have linear double-stranded DNA genomes with short direct repeat sequences at the ends. Comparison of the linear genomes of Φ109 and T7 phages reveals that they both follow a highly ordered and conserved gene module arrangement, linearly arranged according to transcription time. From left to right, these regions are: early regions encoding proteins that interact with the host and shut down host functions; mid-stage regions encoding DNA metabolism-related enzymes (such as DNA polymerase, ligase, and endonucleases); and late-stage regions encoding structural proteins (capsid, tail) and cell lysis proteins. Figure 1 A). Based on the highly homologous T7 RNA polymerase gene and the lytic life cycle, Φ109 can be classified as a T7-like phage.

[0108] This embodiment employs a yeast-based phage assembly platform suitable for T7-like phage engineering. To ensure correct and high-level expression of the engineered phage targeting K2 serotype Klebsiella pneumoniae for rapid detection, the entire 56 bp untranslated region downstream of the major capsid protein on the Φ109 genome was replaced with the selected insertion point. nluc Reporter genes. This allows the ribosome binding site (RBS) to be located. nlucThe expression cassette is directly inserted into the non-coding region downstream of the major capsid protein gene in the Φ109 genome. Figure 1 B). Due to limitations in the amplification system, the linear genome of Φ109 phage was divided into five segments: A, E, and F. The C segment ended with an exogenously added RBS sequence. nluc The gene (516 bp) was fused together, and the start portion of fragment A was connected to the end portion of the pRS415 plasmid fragment. Figure 1 B). Six fragments were amplified in vitro using PCR for circularization in yeast cells, with each fragment retaining sticky ends for efficient ligation. The resulting fragments were assembled in yeast cells to obtain YAC-Φ109:: nluc carrier ( Figure 1 B). Correct recombinants were screened using auxotrophic plates, and Φ109:: was achieved through growth competition within *E. coli* and phage genome packaging cleavage. nluc The bacteria were released via chloroform-induced lysis. Reactivation in the K2 host strain Kp8-41 yielded reporter phage 109:: nluc This bacteriophage can produce NLuc, which generates a strong signal upon addition of a substrate, as shown in the figure (). Figure 1 B). Figure 1 The construction process of the reporting phage Φ109::nluc is shown.

[0109] YAC-Φ109:: nluc The vector is assembled in yeast cells and then introduced into the host strain for rebooting. Generate -Φ109: :nluc Then, NLuc is generated during the replication process.

[0110] Example 2: 109:: nluc Genomic and biological characteristics characterization Whole-genome sequencing results showed that the integrity of the Φ109 phage backbone was preserved, and nluc Correct gene integration ( Figure 2 A). Crucially, Φ109:: nluc Its lytic activity is similar to that of its ancestral bacteriophages, and it can also produce a strong bioluminescent signal on spotted bacterial motility after substrate addition. Figure 2 B). Through growth curve analysis, Φ109:: nluc It exhibited lytic activity comparable to wild-type Φ109 when infecting host Kp8-41. Figure 2 C). Furthermore, compare Φ109:: nluc A one-step growth experiment comparing the proliferation efficiency of the two plants with that of their parent Φ109 showed no significant difference. Figure 2 D). From Figure 2 It can be seen that, nlucThe gene insertion did not adversely affect the infectivity or replication of the phage. Therefore, this example demonstrates the development of a functional reporter phage Φ109:: with high lytic activity and strong luminescent activity. nluc .

[0111] Example 3: Using Φ109:: ​ Sensitive and specific detection of Klebsiella pneumoniae type K2 To minimize background luminescence caused by residual free NLuc enzyme in the lysis buffer, Φ109:: was purified by sucrose density gradient ultracentrifugation. ​ This purification step reduced background noise by 10 times. ​ A). Simultaneously, to evaluate the purification effect, purified and unpurified Φ109:: ​ Detection performance of the lysis buffer. Results showed that, compared to the unpurified counterpart, the purified Φ109:: ​ The signal strength was increased by 10 times, and the detection limit (LOD) was increased by 100 times. ​ B).

[0112] To characterize the detection kinetics and sensitivity range, this embodiment uses Φ109:: ​ and concentration range from 10 1 Up to 10 7 Bioluminescence kinetics were determined using Kp8-41 series dilutions at CFU / mL. Specifically, high bacterial concentrations (10⁻⁶ CFU / mL) were used. 7 The limit of detection (10 CFU / mL) was detected within 0.5 h and reached the limit of detection (10 CFU / mL) at 3.5 h. ​ A). Compared with the previous 10 2 Compared to the 1.5 h detection time for CFU / mL samples, this represents a 2 h delay ( ​ A). By plotting the relationship between bacterial concentration and fluorescence signal after 1 h incubation and performing linear fitting, a standard curve for quantitative detection was constructed, and its correlation coefficient (R²) was [value missing]. 2 The bacterial concentration was 0.9571 within the detection time. 2 Up to 10 6 A strong linear correlation was observed between CFU / mL and fluorescence signal. ​ B). Within this linear range, detection time (0.5–1.5 h) can serve as a direct indicator for distinguishing samples with different bacterial concentrations. To validate the application of this method in clinical isolates, four additional K2 strains were evaluated. Φ109:: ​ All K2 strains were successfully detected; 10 3 Up to 10 5 Bacterial load of CFU / mL was identified within 1 h. ​C-7D), the detection limit is reached within 3-3.5 hours. ​ E). In addition, Φ109:: nluc The detection performance on different K2 Klebsiella pneumoniae strains correlated well with their respective EOP values. Figure 7 F). Next, using a strain cohort containing 8 target K2 strains and 51 non-target strains representing 17 different capsule types, the Φ109:: nluc The specificity was such that strong luminescence signals were observed only in samples containing the K2 strain, while all signals produced by non-K2 strains were at or below the background threshold. Figure 3 C). From Figure 3 and Figure 7 The results show that this embodiment demonstrates that the reporter phage Φ109:: nluc It can serve as a highly specific and sensitive biosensor for the K2 strain.

[0113] Example 4: Engineered Φ109:: nluc As a tool for detecting important clinical capsule types To expand the detection capabilities of the detection platform to cover different capsule types, this embodiment utilizes bacteriophage Φ109:: nluc As a chassis, it reorients its host tropism by exchanging its RBPs through homologous recombination. Specific RBP genes for these different capsule types have been identified in previous studies. This embodiment successfully obtained four engineered reporter phages, specifically targeting strains K1, K47, K57, and K64, and named them Φ109_R1:: nluc Φ109_R47:: nluc Φ109_R57:: nluc and Φ109_R57:: nluc (Figure 8A). Importantly, unlike the parent Φ109, these four engineered phages exhibited efficient lysis against their respective target hosts, enabling specific detection of these four additional capsule types (Figure 8B). All engineered reporter phages exhibited strong lysis activity comparable to the parent phages and produced progeny capable of emitting detectable signals in bioluminescent imaging (Figure 8C).

[0114] Φ109_R1:: nluc Φ109_R47:: nluc and Φ109_R64:: nluc 10 were detected within 1 hour. 3 The respective target strains (K1, K47, and K64) at CFU / mL, while Φ109_R57:: nluc It takes 2 hours to detect the same bacterial load. Figure 4A). Φ109_R1:: nluc and Φ109_R47:: nluc The detection limit was reached within 2.5 h and 4.5 h, respectively, while Φ109_R57:: nluc and Φ109_R64:: nluc It will take 5 hours. Figure 4 A).

[0115] Kinetic analysis shows that Φ109_R1:: nluc Φ109_R47:: nluc and Φ109_R64:: nluc In 10 2 -10 7 It exhibited a strong linear correlation within the CFU / mL range, while Φ109_R57:: nluc The linear range is relatively narrow (10 4 -10 7 CFU / mL), at 10 2 -10 3 The signal deviated from linearity at CFU / mL (Figure 8D). The variation in the linear range of the detection method is consistent with the inter-group differences in EOP observed among the four engineered reporter phages, particularly Φ109_R57:: nluc It exhibits a relatively low EOP (Fig. 8E). Although this phage has a high replication rate (characterized by a large burst size), the stability and persistence of its progeny tend to be low, a phenomenon likely attributed to an evolutionary trade-off observed in microorganisms. Therefore, at low bacterial concentrations (≤10⁻⁶), 3 At CFU / mL, low EOP remains the main limiting factor, thus delaying the luminescence enhancement required for timely detection. Conversely, Φ109_R1:: nluc Despite the low replication rate, it exhibits an optimized linear range and a lower detection limit (Figure 8F).

[0116] This embodiment further evaluated the inclusiveness of the detection method using multiple clinical isolates. For the five strains of the target K type that could be infected by all four engineered reporter phages, all tested strains were tested at bacterial concentrations ≥10-1. 3At CFU / mL, all strains produced positive signals above baseline within 1.5 h (Fig. 8G-K). Except for the K1 type strain (whose detection time was extended to 3.5 h), the detection time for the other four strains with the other three capsule types (K47, K57, and K64) to reach the detection limit was similar to that of their respective host strains (Fig. 8L). Next, the specificity of the engineered phages was evaluated using a group of 54 clinical isolates representing 18 different capsule types. Strong luminescent signals were observed only in strains carrying K1, K47, K57, and K64 capsules, while all other strains produced signals at or below the background threshold. Figure 4 B). From Figure 4 The results show that this embodiment successfully developed a modular detection toolkit by using a reporter phage targeting K2 as a chassis, which can quickly identify K1, K47, K57 and K64 capsule types.

[0117] Based on other identified specific RBP genes, this embodiment further extended the reporter phage to specifically target strains K5, K10, K20, K25, K27, and K54, respectively, and conducted preliminary validation. All engineered reporter phages exhibited strong lytic activity comparable to the parental phages and produced progeny capable of emitting detectable signals in bioluminescent imaging (results not shown).

[0118] Example 5: Application of the phage reporting toolkit in pure culture and simulated multimicrobial urine models To evaluate the functionality of the phage reporting toolkit of this invention in simulated clinical samples, this embodiment tested its ability to detect specific capsule types in synthetic urine. The initial bacterial concentration was set at 10-1. 5 CFU / mL, consistent with the diagnostic threshold for routine urinary tract infections (UTIs). Under these conditions, the phage reporting kit successfully detected 10 of them. 5 CFU / mL of K1, K2, K47, K57 and K64 types K. pneumoniae For this strain, the luminescent signal rose above baseline within 0.5 h. Figure 5 A). Its kinetic characteristics show that RLU reaches its peak rapidly (within 1-2 h for most strains), and the inflection point dynamics in urine are very similar to those observed in LB medium. Figure 5 A, Figure 7 (D and Figures 8G-H). Next, it was investigated whether the reporter phage of this invention remained effective in urine at low bacterial concentrations. 10 [samples / samples] were tested. 3 Up to 10 4 A bacterial load of CFU / mL corresponds to a lower quantitative threshold for diagnosing catheter-related urinary tract infections. Among all tested strains, those at 10... 3 Up to 104 Consistent RLU curves were observed at all concentrations. Figure 9 A, Figure 7 D. Figure 8G-H).

[0119] Furthermore, to investigate whether multiple microbial infection niches would interfere with detection, this embodiment established a niche containing four urinary tract pathogens in synthetic urine ( E. coli EP02, S. aureus SA6453 A. baumannii Ab11 and P. aeruginosa The co-culture system of SY29 (a pathogen) may contain pathogens in urine that are present in urine. K. pneumoniae Coexistence. All reporter phages specifically recognized their host bacteria within 3 hours (average load approximately 2 × 10⁻⁶). 5 CFU / mL, no cross-reactivity was detected at bacterial loads used for urinary tract diagnosis (CFU / mL). Figure 5 B Figure 9 B). This embodiment calculated the signal inhibition rate to show a slight delay that was positively correlated with bacterial concentration ( Figure 9 C). Further analysis revealed that phage adsorption efficiency was significantly reduced in multimicrobial urine samples, which may be attributed to direct physical barriers (C). Figure 9 (D) These results indicate that non-host bacteria impede phage dissemination through direct physical barriers, acting as a size-selective barrier. Although there was a slight detection delay compared to pure cultures, multimicrobial mixtures were still able to accurately distinguish the target K-type strain.

[0120] from Figure 5 and Figure 9 The results show that this embodiment demonstrates that the phage reporting toolkit of the present invention can robustly detect all strains with K1, K2, K47, K57 and K64 capsule types in both pure cultures and simulated multimicrobial synthetic urine.

[0121] Example 6: Developing performance-optimized reporter phages using directed evolution to enhance detection capabilities To improve the detection sensitivity and speed of reporter phages, this embodiment employs directed evolution to select variants exhibiting enhanced infectivity or faster growth rates. Chassis reporter phage Φ109:: is used. nluc Phage lysates were continuously passaged for 7 days on the ancestral host strain Kp8-41. Phage lysates were collected after every 24 h of cycles. To assess the improvement in detection efficiency, three independent directed evolutionary lineages were isolated from the seventh evolutionary passage, and their performance on 10-1 phages was compared. 7 The detection sensitivity of Kp8-41 is CFU / mL. Among them, the phage variant Φ109:: nluc-E7 exhibited accelerated detection kinetics, exceeding the baseline signal threshold within 30 minutes (Figure 10A). Furthermore, quantitative analysis showed that, compared to variants obtained in earlier evolutionary rounds, Φ109:: nluc The -E7 luminescence signal produced a significantly larger area under the curve (Figure 10B). Therefore, this embodiment used luminescence kinetics to evaluate the detection performance of the evolved phage. Notably, Φ109:: nluc -E7 exhibited superior sensitivity, producing a luminescent signal 10 to 100 times higher than its ancestral strain. Meanwhile, for 10... 5 The detection time for CFU / mL was reduced from 25 min to 20 min, for 10 4 The detection time for CFU / mL has been reduced from 30 min to 20 min. Figure 6 A). Furthermore, the required detection time was also reduced at the other three bacterial concentrations (Figure 10C). Consistent with the enhanced binding affinity, this improvement extended to the original host and four other K2 strains ( Figure 6 B). The increased sensitivity resulted in shorter detection times, with reductions of 5 min and 15 min for the two high-affinity strains, respectively, and 20 min and 30 min for the two low-affinity strains, respectively. Figure 6 B). Evolved Φ109:: nluc -E7 against K2 strain ( n =8) showed complete specificity and no cross-reactivity with the group of 51 strains containing 17 other K types (Fig. 10D).

[0122] Consistently, Φ109:: nluc -E7 is standardized to 10 7 At PFU / mL, it also showed a higher EOP than the ancestral strain (Figure 10E), indicating that its infectivity may have been enhanced. Furthermore, Φ109:: nluc The lytic activity of -E7 remained unchanged, and its growth curve at the same MOI showed only minor differences compared to the ancestral phage (Fig. 10F). A one-step growth curve determination was performed to analyze its reproductive characteristics. Although there was no significant difference in burst size, Φ109:: nluc The latency period of -E7 is almost half that of its ancestral phage (Fig. 10G). Furthermore, Φ109:: nluc -E7 performs better than Φ109:: nluc The higher adsorption efficiency is consistent with its enhanced replication tendency on Kp8-41. Figure 6 C. Figure 10H).

[0123] Sequencing analysis showed that, compared with the parent strain, the mutant Φ109:: nluc A nonsynonymous point mutation exists in the RBP 2 region of -E7 (the RBP closer to the genome end), resulting in the substitution of histidine at position 376 with tyrosine (Y376H). Figure 6 D). Notably, structural models predict that this mutation will induce a significant conformational change in RBP2 (global root mean square deviation [RMSD] = 3.073 Å) and alter the local electrostatic potential by introducing a positive charge ( Figure 6 (D, Figure 10I). It is speculated that these modifications may enhance the interaction between RBP2 and the host polysaccharide receptor.

[0124] To alleviate Φ109_R57:: nluc To address the limitation of linear dynamic range, this embodiment conducted independent directed evolution experiments on the engineered reporter phage. The resulting optimized variant Φ109_R57:: nluc -E7, confirmed by a 100-fold increase in the area under the luminescence curve and a 4-fold increase in adsorption efficiency (Figure 10J-L). This optimized variant exhibited superior performance in capsule typing, at 10 3 -10 5 The luminescence intensity increased approximately 4-fold and the linear range expanded 1.5-fold within the CFU / mL range, thus enabling more precise quantification over a wider range of bacterial concentrations. Figure 6 E). These two evolved reporter phages together enabled a faster and more sensitive differentiation between K2 and K57 Klebsiella pneumoniae.

[0125] Example 7: Clinical Sample Testing Currently, there are no clinical kits for detecting or identifying Klebsiella pneumoniae capsular serotypes. Based on the recommendations of the "Expert Consensus on Laboratory Detection of Highly Virulent Klebsiella pneumoniae," this embodiment uses PCR+sequencing as a control method for capsular serotype. Clinical sputum and bronchoalveolar lavage fluid (BALF) samples were collected from 50 patients diagnosed with Klebsiella pneumoniae (Kp) infection. After pretreatment according to the method in Example 5, each sample was tested using PCR+sequencing and the system / method of this invention, respectively. The results showed that, compared to PCR+sequencing, the system / method of this invention achieved 98% accuracy in detecting Klebsiella pneumoniae capsular serotypes, and the detection time was significantly shortened. The main reason for the inconsistent results is likely false positives in PCR caused by dead or colonizing bacteria. A review of medical records revealed that all patients had received antibiotic treatment, suggesting that the PCR detection indicated residual nucleic acid from dead bacteria, while the phage negative result more accurately reflected the clinical state of no live bacteria infection. This demonstrates that positive PCR test results in clinical practice may originate from dead bacteria, colonizing bacteria, or environmental nucleic acid contamination, while a positive phage result directly proves the presence of live bacteria with replication capabilities, providing a solid basis for infection status monitoring and treatment decisions.

[0126] Rapid microbial identification can support clinical decision-making and facilitate antimicrobial drug management. Therefore, various immunological and molecular diagnostic techniques have been developed to enable point-of-care testing (POCT) of pathogens. Reporter phages carrying bioluminescent labels have also emerged as promising POCT tools with the 4S characteristics (simple, rapid, sensitive, and specific), and have been reported for the detection of a variety of bacteria. However, due to the high host specificity of phages, a major limitation of this type of research is its inability to cover different strain typing within the same bacterial species.

[0127] As our understanding of bacterial-phage interactions deepens, this apparent limitation can actually be used to detect specific subgroups of bacterial strains. For example, K. pneumoniae bacteriophage infection and K. pneumoniae Capsule type is closely related. Further mechanistic studies of phage-host interactions have shown that RBPs with depolymerase activity are a key determinant for phage recognition of specific capsule types.

[0128] This invention uses NLuc-labeled phages as a chassis to generate a set of reporter phages via RBP exchange, enabling them to collectively target capsule types K2, K1, K57, K47, and K64. The first three capsule types are associated with highly virulent Klebsiella pneumoniae, while K47 and K64 are the most common serotypes among carbapenem-resistant strains. All five reporter phages were robust in artificial urine, with ≥ 10⁻⁶ detectable within 0.5 h. 5 CFU / mL of bacteria, ≥10 were detected within 1.5 h.3 CFU / mL of bacteria. They also exhibit high specificity, with each phage selectively targeting a single capsule type. In summary, this invention establishes a modular, scalable reporter phage-based platform for rapid and sensitive typing of Klebsiella pneumoniae capsules. In this platform, characterized RBPs are used as interchangeable building blocks, enabling direct expansion to other capsule types and potentially establishing a reporter phage library covering the diversity of this pathogen's capsules in the future. Compared to other Klebsiella pneumoniae capsule typing methods, the platform of this invention offers advantages such as short detection cycles, high sensitivity and specificity, and relatively low cost.

[0129] Sensitivity is a key parameter for evaluating reporter phages, and the platform of this invention provides a strategy to enhance its performance. Directed evolution of phages can increase their infectivity, thereby improving the sensitivity of reporter phages. Notably, Φ109:: nluc -E7 has a luminescence intensity 10-100 times higher than its ancestral strains, with 10 5 The detection time for CFU / mL was reduced from 25 min to 20 min, and the detection time for 10 CFU / mL was reduced from 25 min to 20 min. 4 The detection time for CFU / mL was reduced from 30 min to 20 min. This phage evolution training on specific phages on bacterial hosts has been used to optimize therapeutic phages. For ΦER16e and ΦER46e, mutations in the RBP improved their adsorption to host cells and increased phage infectivity and population size. Consistent with these findings, the results of this invention suggest that evolutionary optimization of reporter phages can be used to improve detection sensitivity. Genomic analysis revealed that confinement regions like the RBP are evolutionary hotspots where mutations can enhance phage-CPS binding and promote adsorption, thereby overcoming diffusion limitations. These results highlight phage evolution training as a powerful tool for systematically tuning reporter phage performance in future work. AlphaFold 3 results predicted that the Y376H mutation significantly altered the electrostatic potential of the K2 binding site; these structural hypotheses provide important guidance for research on key sites related to evolution training. Further in vitro studies will further characterize the effect of this mutation on binding energy. By purifying recombinant wild-type and mutant RBP2, and then using biolayer interferometry or surface plasmon resonance to biophysically quantify the binding kinetics, these simulated interactions were verified, yielding data consistent with the predictions of AlphaFold 3.

[0130] This invention establishes a method for containing artificial urine. E. coli , A. baumannii , P. aeruginosa and S. aureus The mixture. All reporter phages were specifically detected as their corresponding host bacteria (~10⁻⁶) within 3 h.5 (CFU / mL), demonstrating the strong specificity and applicability of the reporter phage set of this invention in multi-microbial environments. Simultaneously, a prolonged detection time was observed in multi-microbial mixtures compared to pure cultures. This delay was confirmed to be driven by reduced adsorption efficiency, due to the formation of size-selective barriers by non-target bacteria, hindering phage diffusion. Therefore, evolved reporter phages with accelerated adsorption kinetics are better adapted to these mass transfer limitations and partially alleviate the analytical challenges inherent in complex biological matrices.

[0131] The platform design of this invention has broad clinical applicability, utilizing a standard microplate reader while providing visual inspection as a practical screening tool for point-of-care testing (POCT) applications. This operational flexibility is supported by the system's significant robustness under near-clinical conditions. Although host-derived components in urine (such as cell debris, soluble proteins, and endogenous proteases) may interfere through nonspecific adsorption or steric hindrance, the system's high specificity minimizes their impact. Furthermore, results from this invention demonstrate that incorporating a pre-culture procedure effectively reduces matrix effects by up to 94.4%.

[0132] In addition to its hardware versatility and analytical integrity, the phage's stability at 4°C further simplifies logistical requirements, allowing for transport via standard refrigerated packaging. To ensure long-term diagnostic reliability, production and quality control will be conducted according to standardized procedures outlined in in vitro diagnostics (IVD) regulatory guidelines. Furthermore, this platform addresses diverse clinical challenges by focusing on high-risk, highly virulent, and carbapenem-resistant lineages. K. pneumoniae The detection problem.

[0133] This platform can be further expanded to cover the remaining capsular types through modular design. Recent machine learning models predicting phage-host interactions (particularly the specific interaction between RBP and capsular proteins) provide a robust framework for accelerating the rational design of capsular-specific phages. With advancements in computational modeling of phage-host interactions, this platform provides an ideal experimental framework for validating these predictions. Combining these predictive algorithms with AI-assisted protein design and targeted mutant libraries will ultimately enable this platform to systematically target the remaining K types.

[0134] In summary, the findings of this invention demonstrate that modular engineering and evolutionary optimization of reporter phages can achieve clinically relevant results in multi-microbial environments. K. pneumoniae Rapid, sensitive, and highly specific detection of capsule types. This scalable platform provides a framework for expanding the reportable phage library to more capsule types and key pathogens, and shows clear potential for integration into point-of-care diagnostics to better guide targeted antimicrobial therapy and precision phage therapy.

[0135] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A reporter phage, characterized in that, The reporter phage contains a nucleic acid sequence encoding a reporter protein and at least one receptor-binding protein. The receptor-binding protein is partially or entirely engineered so that the reporter phage can specifically infect specific capsular types of Klebsiella pneumoniae.

2. The reporter phage according to claim 1, wherein the capsule type is selected from one or more of K1 to K79, preferably the capsule type is selected from one or more of K1, K2, K5, K10, K20, K25, K27, K47, K54, K57 and K64, and more preferably the capsule type is selected from one or more of K1, K2, K47, K57 and K64.

3. The reporter phage according to claim 1, characterized in that, The reporter protein is selected from luciferase, fluorescent protein, alkaline phosphatase, β-lactamase, β-galactosidase or any combination thereof. Preferably, the luciferase is selected from firefly luciferase, kidney luciferase or nano-luciferase. More preferably, the luciferase is selected from nano-luciferase.

4. The reporter phage according to claim 1, characterized in that, The reporter phage is selected from the Myotail Phage Family, Longtail Phage Family, or Shorttail Phage Family, and preferably the reporter phage is from the Shorttail Phage Family.

5. The reporter phage according to claim 1, characterized in that, The reporter phage has a genome containing the nucleotide sequence shown in SEQ ID NO.1 or a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence shown in SEQ ID NO.

1.

6. The reporter phage according to claim 1, characterized in that, The reporter phage contains a mutant of the receptor-binding protein, preferably the mutant of the receptor-binding protein contains a mutation in the amino acid sequence shown in SEQ ID NO. 2, where the Y at position 376 from the N-terminus to the C-terminus is changed to H relative to the parent receptor-binding protein; more preferably, the amino acid sequence of the mutant of the receptor-binding protein is shown in SEQ ID NO.

3.

7. A composition, characterized in that, The composition comprises at least one reporter phage according to any one of claims 1-6.

8. A method for preparing reporter phages, characterized in that, The method includes: (a) An expression cassette is inserted into the non-coding region downstream of the major capsid protein gene in the parental bacteriophage genome to obtain a recombinant vector; (b) the recombinant vector is introduced into host bacteria for culture to obtain the reporter bacteriophage. Optionally, (c) the reporter phage is purified. The parental bacteriophage can specifically infect a specific capsular type of Klebsiella pneumoniae, and the expression cassette contains a nucleic acid sequence encoding a reporter protein and a ribosome binding site sequence, and the host bacterium is the corresponding specific capsular type of Klebsiella pneumoniae.

9. The method according to claim 8, characterized in that, Step (a) includes: (a1) providing the parental phage genome in the form of multiple fragments, wherein an expression cassette is inserted in the fragment containing the downstream non-coding region of the major capsid protein gene. (a2) Provides linear carriers, and (a3) The recombinant vector is prepared by seamless cloning the linear vector and the plurality of fragments.

10. The method according to claim 8 or 9, characterized in that, The parental bacteriophage is Klebsiella pneumoniae bacteriophage RCIP0109, and / or The reporter protein is a nano-luciferase. Preferably, the expression cassette contains the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having at least 75% sequence identity with the nucleotide sequence shown in SEQ ID NO.

4.

11. The method according to any one of claims 8-10, characterized in that, The method optionally further includes obtaining a reporter phage that specifically infects another or one specific type of Klebsiella pneumoniae by replacing part or all of the receptor-binding protein of the reporter phage with a phage receptor-binding protein that specifically infects another or one specific type of Klebsiella pneumoniae.

12. A method for detecting and / or quantifying a specific type of Klebsiella pneumoniae present in a sample, characterized in that, The method includes: The sample or bacteria derived therefrom are contacted with the reporter phage of any one of claims 1-6, the composition of claim 7, or the reporter phage prepared by the method of any one of claims 8-11, wherein the reporter phage specifically infects the specific capsular Klebsiella pneumoniae and produces a reporter protein; and The amount of the reporter protein produced is measured, and the measured amount is correlated with the presence and / or quantity of the specific capsular type of Klebsiella pneumoniae.

13. The method according to claim 12, characterized in that, The time from contacting the sample with the reporter phage to detecting the presence and / or quantity of a specific capsular type of Klebsiella pneumoniae is between 5 minutes and 3 hours.

14. The method according to claim 12 or 13, characterized in that, The sample is selected from any one of environmental samples, food samples, clinical samples, or hospital waste resource samples. Preferably, the sample is selected from clinical samples, which are obtained from: tissue, saliva, blood, plasma, serum, feces, urine, sputum, mucus, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, lavage fluid, bronchial lavage fluid, swabs from the skin or mucous membrane surface, their cultures, or any combination thereof. More preferably, the clinical sample is obtained from urine, bronchoalveolar lavage fluid, sputum, saliva, mucus, their cultures, or any combination thereof.

15. A reagent kit or system, characterized in that, The kit or system comprises the reporter phage of any one of claims 1-6, the composition of claim 7, or the reporter phage prepared by the method of any one of claims 8-11.

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