Kit for concentrating and detecting a target bacterial strain in a sample, methods, products and uses thereof

Abstract

The present disclosure relates to a kit for concentrating and detecting a target bacterial strain in a sample. Furthermore, the present disclosure relates to methods, products and uses of said kit and products, in particular in food, clinical and environmental samples.

Description

D E S C R I P T I O NKit for concentrating and detecting a target bacterial strain in a sample, methods, products and uses thereof TECHNICAL FIELD

[0001] The present disclosure pertains to the field of biotechnology and microbiology, specifically to a kit designed for the concentration and precise detection of a target bacterial strain within various sample types. The invention encompasses methods, products, and uses directed towards enhancing the velocity, sensitivity, accuracy, and efficiency of detecting specific bacterial strains in different food, environmental, clinical, or industrial samples. BACKGROUND

[0002] Listeria monocytogenes (Lm) is the aetiological agent of the vexing infectious disease, listeriosis, a severe, albeit sporadic (foodborne) illness associated with a high case-fatality rate (20-30%). This ubiquitous bacterium may thrive in a plethora of hostile environments, namely agricultural and aquatic ecosystems, soil, and decaying vegetation, withstanding a multitude of biotic and abiotic stressors. Moreover, Lm is endowed with the ability to switch from an environmental, innocuous, saprophyte to an intracellular virulent pathogen. This physiological state transition is triggered by environmental signals, through the activation of an intricate regulatory network that coordinates the expression of primary virulence determinants (genes). Hence, early, on-site, bacteria detection / diagnosis is of paramount importance to curtail the pathogen’s dissemination and persistence.

[0003] The conventional microbiological-based (culture-dependent) procedures for the detection of pathogenic bacteria in food matrices or biological specimens, although reliable and accurate, are tedious and lengthy. Therefore, in recent years, considerable effort has been devoted to developing expeditious and accurate detection / diagnostic systems. In particular, biosensors have emerged as valuable detection platforms to circumvent the shortcomings of the classic techniques.

[0004] Biosensors based on hybrid organic-inorganic nanoflowers (HNFs) are highly versatile systems and have garnered significant interest as notably sensitive platforms for detecting bacteria pathogens, due to their high surface-to-volume ratio and specific functionalization capabilities. HNFs are intricate structures created at the nanoscale that often resemble theshape of flower petals. The unique properties of nanoflowers stem from their intricate hierarchical assembly and architecture, which can be tailored for specific applications. The controlled manipulation of materials at the nanoscale allows the precise engineering of nanoflowers with desired properties, including, improved catalytic activity, and unique optical or electrical characteristics. Different enzymes may be utilized as the protein component of HNFs, displaying improved catalytic activity and stability in comparison with the free form of the enzyme.

[0005] Biorecognition elements are pivotal components dictating the performance of biosensors, allowing the selective and accurate detection of target bacteria in complex sample matrices. The integration of enzymes, antibodies, oligonucleotides or bacteriophages into biosensing platforms has been extensively documented.

[0006] Bacteriocins are scarcely exploited biorecognition components for the specific detection and quantification of target bacteria. These peptides may be immobilized on a plethora of biosensing platforms, including optical, electrochemical, or mass-based biosensors.

[0007] Hitherto, only one work documented the utilization of a bacteriocin (nisin) in a distinct nanoflower-like structure (AgPW@PDA@Nisin) constructed from polyoxometalate. In particular, AgPW@PDA@Nisin demonstrated an improved antimicrobial activity against S. aureus. Those nanocomposites may be utilized in food preservation.

[0008] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure. GENERAL DESCRIPTION

[0009] The present disclosure relates to an improved method for concentrating and detecting a desired bacterium in a sample, wherein such a method has high specificity, high sensitivity, versatility and is faster than the methods described in the prior art.

[0010] The present disclosure relates to a method and to a specific detection of bacterial strains using a novel two-component system: bacteriophage-modified magnetic particles and a unique complex (multi-component) magnetic detection particle; in particular nanoflower- based particle. The bacteriophage-modified magnetic particles consist of a magnetic core with a polyethyleneimine shell functionalized with bacteriophages and surface-blocking agents like BSA. The immobilized bacteriophages bind specifically to target bacterial cells, enablingefficient isolation and concentration of bacteria from diverse sample types, including food and biological samples.

[0011] The present disclosure also relates to uniquely engineered detection particles featuring a nanoflower core (Cu₃(PO₄)₂), gold nanoparticles, β-glucosidase enzyme, and biorecognition molecules like sakacin X. These multi-functional particles amplify the detection signal through a two-step mechanism that is unprecedented in the field. First, the immobilized sakacin X, a scarcely explored bacteriocin, specifically binds to the bacteria captured by the phagomagnetic particles, forming a highly selective complex. This specific interaction facilitates the proximity required for efficient signal transduction. Second, the entrapped β-glucosidase enzyme within the copper phosphate nanoflower produces a colorimetric response in the presence of a specific reagent, such as p-nitrophenyl β-D-glucopyranoside. The groundbreaking synergistic combination of sakacin X-functionalized, enzyme entrapping nanoflowers with bacteriophage-modified magnetic particles of the present disclosure surprisingly allows for a streamlined, high-sensitivity detection process that is faster, easier to use, and more versatile than traditional methods. This disruptive two-component system represents a significant advancement over prior art by integrating novel functional elements into a cohesive and highly efficient detection system.

[0012] An aspect of the present disclosure relates to a bacteriophage-modified magnetic particle for concentrating a target bacterial strain in a sample comprising a magnetic core and a polyethyleneimine shell; wherein the polyethyleneimine shell is functionalized with a plurality of bacteriophages and a plurality of a surface-blocking agent; wherein the plurality of bacteriophages is bound to the polyethyleneimine shell; wherein the plurality of surface-blocking agents is adsorbed to the polyethyleneimine shell; wherein the plurality of bacteriophages is able to bound to the target bacterial strain.

[0013] The bacteriophage-modified magnetic particle for directly isolating and concentrating a target bacterial strain of the present disclosure surprisingly offers several advantages, including high specificity and high sensitivity, even in samples with low bacterial concentrations, and broad applicability across clinical, food safety, and environmental monitoring sectors. Notably, the phagomagnetic particle eliminates the need for lengthy sample pre-processing or enrichment procedures, while simultaneously reducing matrix interference, significantly reducing detection time while maintaining high accuracy. Its ease ofuse, coupled with the portability of the system, makes it an ideal solution for rapid on-site bacterial detection, offering a significant improvement over prior art in terms of speed, sensitivity, and operational simplicity.

[0014] In a preferred embodiment for better results, the plurality of bacteriophages is immobilized in the magnetic particle such that a majority of the phages are oriented in a head- in configuration relative to the magnetic particle such that a head of the bacteriophage is coupled to the magnetic particle and the tail of the bacteriophage is free to attach to bacterial cells of the target strain in a sample containing the target bacteria.

[0015] In an embodiment for better results, the bacteriophage has a genome that has at least 80% sequence identity with the genome of bacteriophage P100 as set forward in GenBank under Accession No. DQ004855 (version DQ004855.1; Definition: Listeria phage P100, complete genome). In a preferred embodiment, the bacteriophage has a genome that has at least 85% sequence identity with the genome of bacteriophage P100 as set forward in GenBank under Accession No. DQ004855; preferably at least 90% sequence identity; more preferably at least 95% sequence identity; even more preferably 100% sequence identity.

[0016] In an embodiment, the bacteriophage has a genome that has at least 80% sequence identity with the genome of bacteriophage Los 1 (DSM 103938) as set forward in GenBank under Assembly Accession No. GCA_002614145.1 (NCBI RefSeq assembly: GCF_002614145.1). In a preferred embodiment, the bacteriophage has a genome that has at least 85% sequence identity with the genome of bacteriophage Los 1 (Campylobacter phage vB_CjeM_Los1) as set forward in GenBank under GenBank assembly accession No. GCA_002614145.1; preferably at least 90% sequence identity; more preferably at least 95% sequence identity; even more preferably 100% sequence identity.

[0017] In an embodiment for better results, the bacteriophage has a genome that has at least 80% sequence identity with the genome of bacteriophage Campylobacter phage vB_CjeM_Los1, as set forward in GenBank under Accession No. as set forward in GenBank under Accession No. KX879627 (Version KX879627.1; Definition: Campylobacter phage vB_CjeM_Los1, complete genome), preferably at least 85% sequence identity, preferably at least 90% sequence identity; more preferably at least 95% sequence identity; even more preferably 100% sequence identity.

[0018] In an embodiment, the bacteriophage has a genome that has at least 80% sequence identity with the genome of bacteriophage Listeria phage P100 (as set forward in GenBank under Assembly Accession No. GCA_002629965.1), preferably at least 85 % sequence identity;more preferably at least 90% sequence identity; more preferably at least 95% sequence identity; even more preferably 100% sequence identity.

[0019] In an embodiment for better results, the surface-blocking agent is selected from BSA (Bovine serum albumin) or casein, or mixtures thereof. In a preferred embodiment, the surface-blocking agent is BSA. The surface-blocking agent minimizes nonspecific adsorption to the polyethyleneimine shell, thereby enhancing specificity for the target bacterial strain.

[0020] In an embodiment for better results, the plurality of bacteriophages is immobilized in the magnetic particle such that a majority of the phage are oriented in a head-in configuration relative to the magnetic particle such that a head of the bacteriophage is coupled to the magnetic particle and the tail of the bacteriophage is free to attach to bacterial cells of the target strain in a sample containing the target bacteria.

[0021] In an embodiment for better results, the diameter of the magnetic particle ranges from 1-3 μm; preferably 2 μm.

[0022] In an embodiment, the magnetic core of the magnetic particle comprises a material selected from the list consisting of: Fe3O4, γ-Fe₂O₃, CoFe₂O₄, NiFe₂O₄, MnFe₂O₄, FePt, FeCo; preferably Fe3O4.

[0023] Another aspect of the present disclosure relates to a copper detection particle , preferably a copper nanoflower-based detection particle ,for detecting a target bacterial strain in a sample, said copper detection particle , preferably nanoflower-based detection particle, comprises a Cu3(PO4)2 nanoflower, a gold nanoparticle, a β-glucosidase enzyme and a biorecognition molecule, wherein: the plurality of biorecognition molecules is electrostatically immobilized on the Cu3(PO4)2nanoflower through the gold nanoparticles; the β-glucosidase enzyme is entrapped in the Cu3(PO4)2 nanoflower; the biorecognition molecule has an amino acid sequence that has at least 80% sequence identity with the amino acid sakacin X wherein the sequence of the amino acid sakacin X is selected from SEQ ID 1, SEQ ID 2; the copper detection particle is able to bind to the target bacterial strain and change the colour of the sample by adding a suitable colorimetric reagent.

[0024] Nanoflower is a newly developed class of a nanoparticle showing structural similarity to plant flowers. Organic-inorganic hybrid nanoflowers present simple methods of synthesis to improve the stability and efficiency of the surface reaction. Nanoflowers are composed of several layers of petals to encompass a larger surface area in a small structure for multipleapplications in catalysis, biosensors and delivery of drugs (Jianxiong Chen, Zitao Guo, Yu Xin, Zhenghua Gu, Liang Zhang, Xuan Guo, Organic–inorganic hybrid nanoflowers: A comprehensive review of current trends, advances, and future perspectives, Coordination Chemistry Reviews, Volume 489, 2023, 215191, ISSN 0010-8545, https: / / doi.org / 10.1016 / j.ccr.2023.215191.

[0025] In an embodiment for better results, the biorecognition molecule has an amino acid sequence that has at least 85% sequence identity with the amino acid sakacin X wherein the sequence is selected from SEQ ID 1, SEQ ID 2; preferably at least 90% sequence identity; more preferably at least 95% sequence identity; even more preferably 100% sequence identity.

[0026] In an embodiment for better results, the suitable colorimetric reagent is p-nitrophenyl ^-D-glucopyranoside.

[0027] Another aspect of the present disclosure relates to a kit for detecting and concentrating a target bacterial strain in a sample comprising the bacteriophage-modified magnetic particle herein described and the copper detection particle herein described.

[0028] Another aspect of the present disclosure relates to a method for obtaining the bacteriophage-modified magnetic particle herein described comprising the following steps: providing magnetic particles comprising a magnetic core and a polyethyleneimine shell; adding a plurality of bacteriophages specific for a target bacterial strain to obtain a first product of magnetic particles comprising a magnetic core and a polyethyleneimine shell functionalized with a plurality of bacteriophages; re-suspending the first product in an aqueous solution of a blocking agent to obtain bacteriophage-modified magnetic particles comprising a magnetic core and a polyethyleneimine shell, wherein the polyethyleneimine shell is functionalized with a plurality of bacteriophages and a plurality of a surface-blocking agent; wherein the concentration of the blocking agent in the aqueous solution ranges from 3-7 % (w / v).

[0029] In a preferred embodiment, the concentration of the blocking agent in the aqueous solution is 5 % (w / v).

[0030] In a preferred embodiment, the aqueous solution of the blocking agent is a BSA (bovine serum albumin) solution in PBS (phosphate-buffered saline).

[0031] In a preferred embodiment, the step of re-suspending the first product in an aqueous solution of a blocking agent to obtain the bacteriophage-modified magnetic particles is performed under stirring and during a period ranging from 2-6h; more preferably 4h.

[0032] Method for synthesizing a copper nanoflower-based detection particle as described in the present disclosure comprising the following steps: providing an aqueous solution by mixing copper sulphate with phosphate-buffered saline to initiate the formation of copper phosphate naoflowers; wherein the β-glucosidase enzyme is introduced at a concentration ranging from 0.1 to 1.0 mg mL-1, allowing the enzyme to become entrapped within the forming nanoflowers. adding a plurality of gold nanoparticles to coat the surface of the nanoflowers; functionalizing the gold nanoparticle-coated nanoflowers by immobilizing biorecognition molecules, wherein the biorecognition molecules are electrostatically bound to the nanoflowers via the gold nanoparticles and are selected from sakacin X or sequences having at least 80% sequence identity to sakacin X.

[0033] In an embodiment for better results, the concentration of the CuSO4 is 120 mM and the concentration of β-glucosidase enzyme is 0.5 mg mL-1.

[0034] In an embodiment for better results, during the functionalization step, the concentration of the sakacin X used to immobilize on the nanoflowers is 30 ng mL-1.

[0035] Another aspect of the present disclosure relates to a method for concentrating and detecting a target bacterial strain in a sample, the method comprising the steps of: mixing the bacteriophage-modified magnetic herein described with the sample to form a first reaction mixture under conditions suitable to allow the bacteriophages to bind to the target bacterial strain present in the sample; applying a magnetic field to the first reaction mixture to collect, and separate, bacteriophage- modified magnetic particles bounded to the target bacterial strain; mixing the bacteriophage-modified magnetic particles bound to the target bacterial strain with the copper detection particle, preferably copper nanoflower-based detection particle, herein described to form a second reaction mixture under conditions suitable to allow the biorecognition molecule of the copper detection particle to bind to the bacteria bonded to the bacteriophage-modified magnetic particle;applying a magnetic field to the second reaction mixture to collect, and separate, copper detection particles bonded to the bacteria bonded to the bacteriophage-modified magnetic particles; mixing the copper detection particles bonded to the bacteria bonded to the bacteriophage- modified magnetic particles with a suitable colorimetric reagent.

[0036] In an embodiment for better results, the sample is a food sample, an environmental sample or a biological sample; preferably a food sample.

[0037] In a preferred embodiment for better results, the suitable colorimetric reagent is p- nitrophenyl ^-D-glucopyranoside.

[0038] The method for concentrating and detecting a target bacterial strain in a sample herein disclosed present several advantages: High specificity: The method specifically targets the desired bacteria, which allows the method to selectively isolate and pre-concentrate the desired bacteria from a sample; High efficiency: The method can efficiently isolate and pre-concentrate the desired bacteria from a sample, even when the bacteria are present in low concentrations; Versatility: The method can be used in a variety of applications, such as clinical diagnostics, food safety testing, and environmental monitoring; Ease of use: The method is easy to use, due to the colorimetric detection; Quickness: the method concentrates and detects the desired bacteria in a faster method than the methods described in the prior art.

[0039] In an embodiment for better results, the ^-GLU-Cu3(PO4)2-AuNPs-sX-NFs&P100-PEI-MP herein disclosed have proven to be an improved capture and signal regulator system in the Lm detection, owing to the integration of a signal amplification (^-Glucosidase), and biorecognition (bacteriocin sakacin X and bacteriophage P100) elements. This novel system is a versatile, simple, expeditious, affordable, portable platform for the accurate detection of pathogenic bacteria.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of the invention.

[0041] Figure 1: Effect of BSA and casein concentrations on the capture efficiency (columns; 15 and 30 min-first and second column, respectively) of blank PEI-MPs and on the specific capture efficiency (solid line; 15 and 30 min, first and second column, respectively) of P100- MPs for L. monocytogenes (103CFU mL-1) separation, for a blocking time of 1 h (1% and 2% BSA: 1 h and 8 h; and 5% BSA: 4h). Error bars were estimated as the standard deviation of three independent experiments. Different lowercase letters indicate statistical significance (p < 0.05).

[0042] Figure 2: Evaluation of the influence of the MPs amount (40, 80, 120, 160 μg) and 3 -1 incubation time in the Lm ScottA (10 CFU mL in PBS (pH 7.4), MOI 1,000, 37 °C) capture efficiency.

[0043] Figure 3: SEM micrograph concerning the examination of the capture performance of 3 -1 the phagomagnetic platform (Lm ScottA (10 CFU mL in PBS (pH 7.4), MOI 1,000, 37 °C).

[0044] Figure 4: Evaluation of the influence of the temperature (4, 11, 25, 30, 37 °C) in the Lm 3 -1 ScottA (10 CFU mL in PBS (pH 7.4), MOI 1,000) capture efficiency. 3

[0045] Figure 5: Evaluation of the influence of the pH value (5 to 8) on the Lm ScottA (10 CFU -1 mL , MOI 1,000) capture efficiency.

[0046] Figure 6: Evaluation of the biofunctionalized P100-MP sensitivity for distinct bacterial 6 -1 loads (10-10 CFU mL , in 10 mM phosphate buffer pH 7.4, 37 °C).

[0047] Figure 7: Evaluation of the biofunctionalized P100-PEI-MP specificity for distinct Lm 3 -1 serotypes (4 strains per serotype; 10 CFU mL , in 10 mM phosphate buffer pH 7.4, 37 °C).

[0048] Figure 8: Assessment of the biofunctionalized P100-MP selectivity using distinct Gram- 3 -1 positive and -negative bacteria (10 CFU mL , MOI 1,000).

[0049] Figure 9: Validation of the P100-MP applicability towards Lm isolation (37 °C) from 3 -1 distinct food matrices spiked with Lm ScottA (10 CFU mL ). For the individual assessment of each of the 4 parameters (pH, salt, fat, protein content), the matrices were grouped into 2 clusters, according to their intrinsic composition. Each data point represents one food matrix. The lines indicate the mean for each cluster.

[0050] Figure 10A-C: Purification of sakacin x. Ion exchange (A) and hydrophobic interaction (B) chromatograms. (blue) protein detection (abs 280 nm); (green) high salt buffer concentration; (red) conductivity. (C) MS / MS spectrum f or the purified peptide.

[0051] Figure 11A-B: Minimum inhibitory concentration of sakacin X against Lm serotype 4b (A) and serotype 1 / 2a (B).

[0052] Figure 12: SEM analysis of the morphology of sakacinX-^-GLU-Cu3(PO4)2nanoflowers (A-D) and the combined detection system comprising the phagomagnetic platform and sX-^- GLU-Cu3(PO4)2 (E and F).

[0053] Figure 13: Evaluation of the analytical sensitivity of the hybrid nanoflower ^-GLU- Cu3(PO4)2-AuNPs-sX in solutions with different Lm loads (10 and 102CFU mL-1).

[0054] Figure 14: Assembly of the phagomagnetic particles.

[0055] Figure 15A-B: Phagomagnetic Lm isolation.

[0056] Figure 16: Nanoflowers assembly.

[0057] Figure 17A-B: Enzymatic platform for Lm detection. DETAILED DESCRIPTION

[0058] The present disclosure relates to a kit for concentrating and detecting a target bacterial strain in a sample. Furthermore, the present disclosure relates to methods, products and uses of said kit and products, in particular in food, clinical and environmental samples.

[0059] The present disclosure also relates to a method for the rapid, specific, and sensitive detection of bacterial strains using a dual-component system comprising bacteriophage- modified magnetic particles and copper nanoflower-based detection particles. The bacteriophage-modified magnetic particles consist of a magnetic core with a polyethyleneimine (PEI) shell functionalized with bacteriophages and surface-blocking agents, enabling efficient isolation and concentration of target bacteria. Copper nanoflower-based detection particles, featuring a Cu₃(PO₄)₂ nanoflower core, gold nanoparticles, β-glucosidase enzyme, and biorecognition molecules, amplify detection through a colorimetric response.

[0060] This system offers high specificity, versatility, and ease of use, with applications in clinical diagnostics, food safety, and environmental monitoring. The method improves on prior art by providing faster detection, even at low bacterial concentrations, and enables portable, cost-effective bacterial detection through a simple, colorimetric readout. EXAMPLE 1

[0061] A novel phagomagnetic platform (P100-PEI-MP) was coupled to hybrid copper phosphate nanoflowers (entrapping the β-glucosidase enzyme), functionalized with the scarcely exploited bacteriocin sakacin X (^-GLU-Cu3(PO4)2nanoflowers&AuNPs&sakacinX) to detect Listeria monocytogenes in food matrices and biological specimens. Surprisingly, the solution developed and herein described is able to detect Listeria monocytogenes in food matrices and biological specimens in a rapid and accurate way, with a high analytical sensitivity and specificity.

[0062] The conjugated platform developed (^-GLU-Cu3(PO4)2-AuNPs-sX&P100-PEI-MP) has proven improved potential as a novel capture and signal regulator system in the detection of bacteria, in particular Lm, owing to the integration of signal amplification (^-glucosidase), and biorecognition (sakacin X and phage P100) elements. Materials and Methods Reagents and solutions

[0063] MgSO4.7H2O, TRIS, and gelatin from porcine skin, were acquired / purchased from Sigma-Aldrich (Germany), Na2HPO4.2H2O and NaH2PO4from Riedel-de Haën, NaOH from WVR chemicals and NaCl from Panreac. All the chemicals were analytical grade and used as received without further purification. PhageGuard L (formerly LISTEX™ P100) bacteriophage was purchased from Micreos Food Safety. Micromer®-M magnetic particles with 2 µm of size, polyethyleneimine coating (PEI-MPs) was purchased from Micromod®. A MagJET separation rack from Thermo Scientific™ was used to perform magnetic separation of the MPs between incubation and washing steps. A PCMT Thermo-shaker was used for all incubation steps with temperature control.

[0064] Brain heart infusion (BHI) broth and BHI agar were acquired from Biokar Diagnostics (France). LC soft agar culture medium (10 g L-1tryptone, 5 g L-1yeast extract, 10 g L-1glucose, 7.5 g L-1NaCl, 10mM MgSO4, 10mM CaCl2, 0.4% (w / v) agar).

[0065] Bovine Serum Albumin (BSA) solutions were prepared with BSA ≥98%, heat shock fraction, protease-free, essentially globulin-free, pH 7. A 0.5 mg L-1β-Glucosidase (from almonds, lyophilized powder, ≥2 units / mg solid) (Sigma-Aldrich) solution was prepared in a PBS 100 mM solution. A 20 mM solution of nitrophenyl-β-D-Glucopyranoside was prepared using a nitrophenyl-β-D-Glucopyranoside ≥98% (TLC), from Sigma-Aldrich. Several 4-Nitrophenol standard solutions were prepared with 4-Nitrophenol, PESTANAL®, analytical standard from Sigma-Aldrich.Microorganisms and inoculum preparation Bacterial strains and culture conditions

[0066] Listeria monocytogenes ScottA (phage P100-susceptible) was used as the reference strain to develop, optimize the phagomagnetic separation procedure and perform downstream applications. Additionally, a panel of L. monocytogenes (representatives of the most relevant serotypes) were comprehensively selected to validate the platform performance.

[0067] Stock cultures were preserved in BHI broth supplemented with 20% (v v-1) glycerol. Prior to each experiment, the bacterial strains were streaked onto BHI agar and incubated overnight at 37 °C. Afterwards, a single colony was inoculated into BHI broth and grown at 37 °C until the late exponential phase, and sub-cultured (1%, v v-1) onto fresh medium, under the indicated culture conditions. Bacteriophage characterization

[0068] The bacteriophage used in the present example (Listeria phage P100; or just identified as P100) is better characterized in GenBank, under Accession No. DQ004855 (version DQ004855.1; Definition: Listeria phage P100, complete genome), or in NCBI RefSeq assembly GCF_002629965.1 (submitted GenBank assembly: GCA_002629965.1). Bacteriophage titration by the double-layer method

[0069] Phage Listex™ P100 stock solution presented an initial titre of 1011plaque forming units (PFU) mL-1and was stored in the original saline buffer at 4 °C.

[0070] The phage titration was performed according to the double-layer method (plaque assay), as formerly described by Kropinski et al. (2009). Briefly, phage samples (MP- immobilized or in their free form) were serially 10-fold diluted in SM buffer. The host culture (100 μL of overnight grown L. monocytogenes) and aliquots of 100 μL of the decimal phage dilution were mixed with 3 mL of molten LC soft agar. The suspension was poured onto BHI agar plates and incubated at 30 °C. Plaque forming unit enumeration was performed 24h post- infection. Preparation of bacteriophage-functionalized magnetic particles (P100-PEI-MPs )

[0071] Commercial magnetic particles (MPs), with 2 μm diameter (^), composed by a magnetite core (comprising Fe3O4) and a polyethyleneimine shell, and the validated phageP100 were employed in the preparation of bacteriophage-biofunctionalized magnetic particles (P100-MPs). For this purpose, a three-step protocol encompassing the sterilization of MPs, the bacteriophage physical / electrostatic immobilization and blocking of P100-MPs active binding sites was applied. To perform the P100 physical immobilization, phage particles (1x109PFU mL-1, 0.01M citrate buffer pH 5) or buffer (blanks) were directly added into sterilized MPs and allowed to react overnight under continuous mixing (350 rpm, 4 °C). Then, P100-MPs were magnetically separated, the supernatant was collected into a sterile eppendorf for enumeration of the amount of P100 phage that had not been adsorbed. Afterwards, the separated P100-MPs were resuspended in a BSA solution (5% w v-1in 0.01M PBS) during 4h (350 rpm, 4 °C) to block the remaining active binding surface spots / sites on MPs surface. Finally, BSA / P100-MPs were re-suspended in 500μL of SM buffer (100 mM Sodium chloride, 10 mM Magnesium sulphate, 50 mM Tris-HCl, pH 7.5 and 0.01% (w / v) gelatin). One washing step was performed after each magnetic separation. All samples were stored at 4 °C until microbiologic experiments. Influence of Non-Specific Adsorption: Surface Blocking Step Optimization

[0072] The P100-MPs surface blocking step was optimized according to critical variables such as concentration and incubation time of the blocking solution. This study evaluated and compared two different standard blocking agents (BSA and casein) with distinct sizes and interaction strengths on hydrophilic surfaces, to maximize the blocking surface. Phagomagnetic Separation Protocol: Capture Efficiency Effect of pH and temperature on Lm capture efficiency

[0073] The effect of P100-MPs amount (40, 80, 120, 160 µg), pH (5-8) and temperature (4, 11, 25, 30 and 37 °C) on capture efficiency and specific capture efficiency were also investigated. For this purpose, a stationary-phase culture of L. monocytogenes was 100-fold diluted in BHI and grown at 37 °C to exponential phase (optical density at 600^nm (OD600 nm) equal to 0.6). The cells were harvested by centrifugation (4,000 x g, for 10 min, at room temperature) and washed thrice with PBS. To ascertain the colony-forming units (CFU) of the initial inoculum the obtained bacterial cell suspension was 10-fold serially diluted in PBS and plated onto BHI agar. For magnetic separation and pre-enrichment, 500 μL of the L. monocytogenes suspension (103CFU mL-1in PBS) were added into 120 µg P100-modified magnetic particles (P100-MPs) previously washed thrice with 10 mM PBS pH 7.4 and incubated under orbital shaking at 4, 11, 25, 30 and 37 °C.

[0074] Afterwards, we sought to investigate the effect of pH on the performance of the phagomagnetic detection platform. For this purpose, the bacterial pellet (prepared as above detailed) was suspended in 10 mM of sodium acetate (pH 5.0 and 6.0) and potassium phosphate (pH 7.0 and 8.0) (Sigma-Aldrich, Germany) solutions. The protocol was performed at 37 °C, for 20 min. Specificity and sensitivity of the magnetic separation protocol

[0075] The specificity of the newly proposed phagomagnetic platform was evaluated by resorting to a panel of 16 L. monocytogenes comprising representatives of the most relevant serotypes (1 / 2a, 1 / 2b, 1 / 2c, and 4b). Listeria monocytogenes strains isolated from a wide spectrum of food specimens were also included according to the proposed food-focused application of the assay. Moreover, a panel of Gram-positive and Gram-negative bacteria was included in order to evaluate the specificity of the detection platform to distinguish / discriminate L. monocytogenes from closely related Listeria species (L. aquatica), competitive microbiota (Lactobacillus plantarum, Pseudomonas aeruginosa) and other bacterial cultures (Escherichia coli, Enterococcus faecalis, Salmonella enterica serovar Typhimurium and Enteritidis, Staphylococcus aureus, Campylobacter coli and jejuni) were evaluated.

[0076] In order to evaluate the sensitivity (limit of detection) of the magnetic separation protocol proposed herein, a bacterial cell suspension was prepared as previously detailed and decimal serial dilutions were performed in phosphate buffer (10 mM, pH 7.4) to obtain different bacterial loads (1-106CFU mL-1). Twelve hundred micrograms of the P100-MPs were incubated with five hundred microliters of the bacterial suspension. Validation of the phagomagnetic separation protocol applicability in complex food matrices and biological samples

[0077] The proof-of-concept of the optimized phagomagnetic protocol for the rapid and accurate detection of L. monocytogenes was performed in distinct complex food matrices (some of which commonly associated with listeriosis outbreaks) and biological samples (horse blood, artificial urine). Twenty-five grams of solid matrices or 25 millilitres of liquid matrices were aseptically transferred to sterile Falcon tubes, spiked with decimally diluted L. monocytogenes cell suspensions to obtain a final bacterial load of 103CFU mL-1, and thoroughly homogenized. The Listeria inoculum volume corresponded to 1% of the total sample volume.Three independent replicates were prepared for each sample and spiked food matrices were incubated at 25 °C, for 1h in order to provide the temperature adaptation (pre-equilibration).

[0078] The previously optimized magnetic separation protocol was performed to capture and pre-concentrate bacterial cells from those artificially contaminated samples. In brief, 10 mL of the spiked and un-spiked samples were divided into 1 mL aliquots, and 240 µg of P100-MPs were added to each aliquot, and the mixture was incubated at 25 °C under orbital shaking for 30 min. The same protocol was also conducted with magnetic particles devoid of P100 (negative control). Colorimetric phagomagnetic assay: ^-GLU-Cu3(PO4)2nanoflowers&AuNPs&sakacinX as enzymatic labels

[0079] According to the prior art, the phagomagnetic platform analytical performance can only be assessed through classic microbiological-based methods (culturing the captured bacterial cells for quantification). Hence, to further improve the method’s sensitivity and obtain a simple and visual detection method, the present disclosure discloses the combination of the novel phagomagnetic platform (P100–PEI–MP) to hybrid copper nanoflowers (entrapping the β-glucosidase enzyme), functionalized with the bacteriocin sakacin X (^-GLU- Cu3(PO4)2NF&AuNPs&sX). Functionalization of ^-GLU-Cu3(PO4)2nanoflowers&AuNPs with sakacin X Chemical synthesis of Sakacin

[0080] Sakacin X can be obtained by several methods described in the prior art. Sakacin X may be obtained by solid-phase peptide synthesis (SPPS) using the Fmoc / tBu (Fmoc-SPSS) strategy, as described in a) Bédard, F., Hammami, R., Zirah, S. et al. Synthesis, antimicrobial activity and conformational analysis of the class IIa bacteriocin pediocin PA-1 and analogs thereof. Sci Rep 8, 9029 (2018). https: / / doi.org / 10.1038 / s41598-018-27225-3 ; or b) Collins, J. M., Porter, K. A., Singh,peptide synthesis (HE-SPPS). Org. Lett.16, 940–943. doi: 10.1021 / ol4036825).Sequence of Sakacin X Seq. Sequence Organism Molecul Qualifier Residues ID name name e type Molecule Type 1 Sakacin X Lactobacill Amino protein MEAIKKLDLQAMKGIVGG us sakei 5 acid KYYGNGLSCNKSGCSVDWSKAISIIGNN AVANLTTGGAAGWKS 2 Sakacin X Lactobacill Amino protein KYYGNGLSCNKSGCSVDWSKAISIIGNN – mature us sakei 5 acid AVANLTTGGAAGWKS peptide Sakacin X purification and identification

[0081] Sakacin X, the biorecognition element to be utilized on the functionalization of the hybrid copper nanoflowers, was purified and identified by nano liquid chromatography mass spectrometry.

[0082] For this purpose, ammonium sulfate was added to the bacterial crude extract to bring the final salt concentration to 40% (w / v) of saturation and the precipitation was performed at 4 °C for 4 hours. The precipitate was resuspended in 10% of the starting volume in 50 mM sodium acetate pH 4.4. Sample conductivity was measured and the value recorded was 14.7% (corresponding to nearly 150 mM), while the conductivity of the sodium acetate buffer pH 4.4 was 2.6%. Hence, the sample was 4-fold diluted in the referred buffer. Afterwards, ion exchange chromatography (IEC) was performed and the peptide was eluted with a linear salt gradient over a range of 20 to 50% 1M NaCl in 50 mM acetate buffer pH 4.4, at a flow rate of 1 mL min-1. The IEC fractions with the highest antilisterial activity were pooled and submitted to a final polishing through hydrophobic interaction chromatography (HIC).

[0083] Protein identification was performed by nano LC-MS / MS. The raw data was processed using Proteome Discoverer 2.3 software (Thermo Scientific). The Sequest HT search engine was used to identify tryptic peptides. The ion mass tolerance was 10 ppm for precursor ions and 0.02 Da for fragment ions. Cysteine carbamidomethylation was defined as constant modification. Methionine oxidation was defined as variable modification. Peptide confidence was set to high. The processing node target decoy PSM validator was enabled with maximum delta Cn 0.05 and decoy database search target FDR 1%. Protein label free quantitation wasperformed with the Minora feature detector node at the processing step. The precursor abundance was based on intensity. Determination of the minimum inhibitory concentration of the purified sakacin X

[0084] In order to assess the threshold of bacteriocin concentration that could be utilized on the functionalization of the NFLs, without compromising the Lm viability, the minimum inhibitory concentration of sakacin X was determined using the broth microdilution susceptibility method. In brief, 96-well microplates were pre-treated with BSA (1% (w / v) BSA in PBS, incubation at 37 °C, for 30 min) to prevent the peptide adsorption. Overnight cultures of Listeria monocytogenes strains were sub-cultured into fresh medium and allowed to grow at 37 °C to exponential phase (optical density at 600^nm (OD600 nm) equal to 0.6) and diluted to an initial inoculum load of 105CFU mL-1. Sakacin X was 2-fold serially diluted and added to the target strain. The microplate was incubated at 37 °C for 18h. Synthesis and characterisation of ^-GLU-Cu3(PO4)2nanoflowers-AuNPs-sakacinX

[0085] For the preparation of ^-GLU-Cu3(PO4)2-AuNPs-sakacinX, 40 μL of 120 mM CuSO4 were added to 1460 μL 100 mM PBS pH 7.4, containing β -glucosidase (β -GLU, 0.5 mg mL-1) in order to form the Cu3(PO4)2nanoflowers. This solution was left, for 4 hours, at 25°C, without stirring and protected from light. The formed β-GLU-Cu3(PO4)2nanoflowers were centrifuged (5000 rpm, 10 minutes) and washed thrice with distilled water to remove the excess of enzyme.

[0086] The collected nanoflowers (NFLs) were resuspended in half of the initial volume with a AuNPs (gold nanoparticles) solution (2 mL of AuNPs Dropsens solution and 1 mL of distilled water). The AuNPs are used for the electrostatic immobilization of the bacteriocin (sakacin X). After a gentle manual stirring, the tube was incubated during 4 hours at room temperature, protected from light. AuNPs-β-GLU-Cu3(PO4)2nanoflowers were collected through centrifugation (6000 rpm, 15 minutes), washed once with distilled water and with 10 mM PBS pH 5.4.

[0087] Afterwards, a bacteriocin solution (sub-inhibitory concentration) was added to the AuNPs-β-GLU-Cu3(PO4)2 nanoflowers and left to react in a thermo-shaker overnight (4^C, 350 rpm). ^-GLU-Cu3(PO4)2nanoflowers-AuNPs-sakacinX were collected through centrifugation (6000 rpm, 10 minutes) and washed twice with 10 mM PBS. Redispersion of the collected nanoflowers was performed in 100 mM PBS and maintained at 4^C until further use. Themorphology and size of pre-formed ^-GLU-Cu3(PO4)2-AuNPs-sX nanoflowers was assessed by scanning electron microscopy (SEM). Analytical performance of the ^-GLU-Cu3(PO4)2nanoflowers-AuNPs-sakacinX for Lm detection– colorimetric phagomagnetic assay

[0088] Upon Lm cells adsorption on the surface of the phagomagnetic platform (Lm-P100-PEI- MPs), an equivalent volume of the hybrid nanoflowers (formerly synthesized, dilution 1:2 in PBS pH 5.4) was added and the mixture was incubated for 10 minutes, 350 rpm, at 37 °C. Afterwards, the sandwich complex integrating PEI-P100-Lm-bacteriocin-NFLS was magnetically separated and washed twice with 100 mM Tris. Subsequently, 190 μL of p-nitrophenyl β-D- glucopyranoside in phosphate-citrate buffer pH 5.5 (20 mM) was added and incubated for 20 min, at 37 °C. Afterwards, 190 μL of 1M NaOH solution (final concentration 0.5 M) was added to stop the enzymatic reaction. The enzymatic activity of the ^-glucosidase-based hybrid nanoflowers was spectrophotometrically (colorimetry) measured based on the conversion of the chromogenic substrate p-nitrophenyl ^-D-glucopyranoside into p-nitrophenol. Results In-depth investigation of the main parameters governing the bacterial capture efficiency

[0089] Phagomagnetic separation performance of P100-PEI-MPs was evaluated and optimized concerning the following parameters / variables, namely MPs surface blocking, P100- MPs quantity, pH of capture solution, as well as the time and temperature of incubation with Listeria cells. For this purpose, different capture assays were performed, in which the capture efficiency of the magnetic probes (over time) was evaluated. As a comparison, control experiments, comprising particles devoid of the listeriophage P100 were concomitantly carried out. Influence of non-specific adsorptions: surface blocking step optimization

[0090] In an attempt to hamper / hinder the non-specific L. monocytogenes attachment to the PEI polymeric surface, without compromising the phagomagnetic platform sensitivity, different blocking agents were adsorbed onto the unmodified surface of PEI-MPs. Hence, the efficiency of two proteins (BSA and casein) with different molecular weights and adsorption strengths on PEI hydrophilic surface was evaluated. For this purpose, a surface blocking procedure optimization was performed concerning pivotal parameters, namely the proteins concentration and incubation time, in order to maximize the particles surface coverage while guaranteeing a scarce steric hindrance to the immobilized phages.

[0091] Figure 1 illustrates the effect of the different concentrations of BSA and casein (% w / v) on the specific capture and non-specific adsorption of the phagomagnetic platform.

[0092] As a whole, the results disclosed that the two proteins elicited dissimilar non-specific adsorption and the superior performance / efficiency of BSA as a blocking agent in comparison to casein (p < 0.05) was highlighted. In fact, BSA electrostatic adsorption onto the P100-PEI-MP surface effectively impeded non-specific Listeria anchorage to the positively charged polymeric surface. The attachment of BSA, a large globular protein (66 kDa), onto PEI surface is expected to be triggered by electrostatic interactions, since under the neutral condition (pH 7) herein employed, the protein presents a negative charge (isoelectric point (IEP) of 4.5-5.0). In fact, BSA adsorption was documented as a pH dependent phenomenon and at the neutral condition herein employed maximum protein adsorption should be observed. Analogously, casein (IEP of 4.6) biomolecules electrostatically adsorb onto PEI surface, owing to the presence of negatively charged phosphoseryl residues. Nonetheless, this blocking agent proved scarce efficacy in hampering Listeria non-specific attachment / binding.

[0093] According to the capture efficiency and specific capture values obtained for the PEI particles devoid of phage P100, 5% (w / v) BSA (4h) evidenced a higher efficiency (significant differences (p > 0.05) were observed either at 15 or 30 min). This superior performance indicates a higher coverage of adsorbed BSA on the hydrophilic polycationic surface of PEI. Moreover, a longer incubation time improved the blocking efficiency of BSA for a concentration of 10 mg mL-1, but not for 20 mg mL-1, nor 50 mg mL-1. The results disclosed herein pointed out that the BSA-PEI-MP surface complex was formed preferentially at a higher concentration of BSA (> 10 mg mL-1) or with longer incubation times (8h), correlating to the most effective blocking conditions (BSA 5% (w / v), for 4 h). Following exposure of the non- biofunctionalized control (blank) PEI particles (devoid of P100 listeriophage) to the bacterial suspension, a significant difference (p < 0.05) on the capture efficiency was attained in comparison with the obtained with BSA. This finding corroborated the blocking capacity of this protein, guaranteeing the specific biorecognition of the P100 magnetic platform.

[0094] With respect to the capture efficiency values obtained for the PEI particles devoid of phage P100 and blocked with 0.5 and 1% (w / v) casein (1h), a prominent Listeria non-specific adsorption was observed. The protein size has been documented as a critical factor, and proteins with a lower molecular weight are expected to present a higher adsorption. Considering that BSA possesses a molecular weight 2.7-fold higher than casein, the latter was predictable to be a more effective blocking agent. Nonetheless, the aforementioned differences may rely on the physical and bioelectric characteristics of those proteins. Caseinproteins are prone to cluster and hence may form large micellar structures, which may hinder Listeria attachment to P100 (lower specific capture). One may surmise that the higher attachment of Listeria to the casein blocked PEI particles devoid of P100 may be attributed to the conformational flexibility of the protein in comparison to the rigid, globular BSA structure. This may contribute to a structural rearrangement of casein exposing a more hydrophobic surface eliciting Listeria attachment.

[0095] Since PEI presents a positive charge, when in contact with protein solutions, the proteins will be adsorbed onto the polymer surface, with the negative moiety of the protein oriented towards the particle (the PEI coating) and the remaining positively charged moiety outwards. In this sense, and considering that the bacterial phospholipidic membrane is negatively charged, there will be attachment / adhesion of the bacterium to the proteins. Nonetheless, BSA comprises three globular domains, of which (at neutral pH) two are negatively charged, and one is neutral. In this sense, when BSA adsorbs to a positively charged surface by one of its negatively charged domains, there is still one negative domain which will act as a repulsive force against Listeria adhesion. Moreover, in comparison with globular proteins (namely BSA), casein is more flexible and with time conformational alterations may occur, which can further increase the hydrophobicity of the surface and therefore the potential for Listeria adhesion. Al-Makhlafi (1994) reported that, when in contact with a hydrophilic surface, both BSA and casein were adsorbed with the hydrophilic (negative) moiety of the proteins oriented facing the surface, while the hydrophobic portions were oriented to the surrounding solution, which increased the hydrophobicity of the surface. Conversely, the increase in hydrophobicity increased Listeria adhesion. Influence of the phagomagnetic platform quantity, incubation time, pH and temperature on the capture efficiency performance

[0096] The efficiency of phage adsorption to the bacterial host membrane relies on a plethora of physicochemical parameters, namely environmental pH, temperature, ionic strength (particularly divalent cations calcium and magnesium), along with incubation time and P100- MPs quantity. P100-PEI-MPs quantity

[0097] The influence of different quantities of P100-PEI-MPs (40, 80 and 120 µg) on the Lm capture efficiency were evaluated following a 60-minute contact. The P100-PEI-MPs amount unveiled to be the most important parameter on the analytical performance of the magnetic platform, dictating its sensitivity.

[0098] The results indicated a concomitant improvement on the bacterial capture efficiency with the increment on the quantity of the particles (and hence the total number of the available ligand (adsorbed P100) and consequently of the tail binding sites of the immobilized phages) up to 160 μg (p < 0.05). The results evinced the occurrence of a saturation of the magnetic particles for the highest quantities of MPs (120 and 160 μg, 100% of CE), and hence no significant differences (p > 0.05) were observed.

[0099] Concerning bacteria capture kinetics, 10 minutes of contact sufficed to achieve 90% of CE and the optimum incubation time was established to be 20 min. The Lm attachment to the magnetic particle was corroborated through SEM analysis. Temperature

[0100] The influence of the incubation temperature on the analytical performance of the phagomagnetic platform was also investigated. The highest efficiency occurred at 37 °C (98.65 %).

[0101] The intrinsic capability of phages to adsorb and infect a narrow host bacterial spectrum mostly depends on the specific recognition by phage-encoded affinity proteins. Most phages harbor a complex baseplate at the distal end of their tail that coordinates host adsorption. The phages baseplate holds dedicated receptor binding proteins (RBPs), commonly identified as tail fibers or globular tail spikes, mediating the phage adsorption to the host- specific receptor. Temperature may alter the formation capacity of the complex comprising listerial peptidoglycan-anchored cell wall teichoic acid (WTA) and tailspike proteins (TSP) of phage P100. Peptidoglycan-anchored cell wall teichoic acid (WTA) perform as a phage receptor in L. monocytogenes. In the carbohydrate (peptidoglycan) binding sites of TSP, the complex formation depends on the energetic contribution of hydrogen bond formation and desolvation of the partners and, to a more considerable (larger) extent, on the van der Waals interactions. Beyond the optimum temperature of 37 °C, lower temperatures may trigger higher protein solvation which may interfere with the interaction between receptor-anti-receptor. Instead, and even when the interaction energies are low, a temperature decrease may also be adverse to the formation and stability of those bonds. In this perspective, the minor Brownian movement of phages and host bacteria may difficult the phage adsorption owing to this energetic barrier. A slightly reduced adsorption efficiency of phage P100 to L. monocytogenes at 30 °C may indicate that the expression of rhamnose in the WTA may be down-regulated. Nonetheless, since the magnetic platform is still capable to adsorb to L. monocytogenes with asignificant efficiency, one may surmise that accessible rhamnose I is still sufficient to enable P100 interaction with N-acetylglucosamine (GlcNAc) residue. Environmental pH

[0102] The effect of the environmental pH (a factor which may hamper phage stability and may dictate the P100-Lm interaction) was evaluated, with the purpose of mimicking the alkalinity / acidity of distinct food systems or biological fluids. The bacteriophage P100 has formerly demonstrated capability to endure hostile environments.

[0103] Poly(ethylenimine) is an amino-rich highly hydrophilic cationic polyelectrolyte, and as aforementioned, a low pH value will ensure the correct immobilization of P100 on the PEI-MPs surface, through the negatively charged head. The results highlighted the versatility of the phagomagnetic platform to be utilized in a broad range of pH values (5 to 9), with a superior performance occurring at pH values of 7 (CE of 98.7 %) and 8 (CE of 98.4 %). Fister et al. (2016) documented the remarkable stability of phage P100 over a pH range of 4 to 10. The authors reported a significant reduction in P100 titre only when the pH values were lower than 2 or higher than 12. Hence, an alkaline environment appears to favor the electrostatic adsorption of the positively charged P100 tail to the wall teichoic acids (WTA) of L. monocytogenes, structurally comprised by anionic glycopolymers, which present a high negative charge irrespective of the pH value. Phagomagnetic separation protocol sensitivity and selectivity

[0104] The phagomagnetic platform proposed herein disclosed was surprisingly found to be highly sensitive, presenting a separation limit of 1.4 CFU mL-1, with a comparable capture efficiency performance (and respective specific capture efficiency) up to 106CFU mL-1(99- 100%). Zhou et al. also exploited the phage P100 as a biorecognition element to propose a phagomagnetic protocol towards L. monocytogenes isolation in PBS. The authors documented a significantly lower capture efficiency (40 to 50 %) in comparison to the value obtained herein.

[0105] The selectivity of the phagomagnetic platform towards L. monocytogenes capture was also investigated. The method achieved the highest CE value for Lm serotypes 4b (97.6-100%) and 1 / 2a (91.2-94.2%) (Fig.7). The most abundant Listeria cell-wall glycopolymers are teichoic acids. Two distinct structural classes of Listeria teichoic acids have been described, namely the polyribitolphosphate-based wall teichoic acid (WTA) (covalently attached to the peptidoglycan) and the polyglycerolphosphate-based lipoteichoic acid (LTA) (anchored to the cytoplasmic membrane). The vast majority of listeriophages adsorb specifically to WTA structures. Thedistinct glycosylation patterns of WTA result in a high degree of surface variation across the Listeria serotypes, which in turn confers distinct effects on bacteriophage susceptibility. Listeria monocytogenes strains belonging to the serogroups 1 / 2 and 4 possess distinct WTA substitution pattern, namely N-acetylglucosamine and L-rhamnose. Wall teichoic acids are negatively charged and contribute to a prominent cell hydrophilicity owing to the significant content of phosphate moieties. The availability of these negatively charged phosphate groups on the wall teichoic acids influence / govern / dictate the interaction degree of phage-bacterium.

[0106] The specificity of the P100-PEI-MPs was assessed against a panel of Gram-positive and Gram-negative bacteria (Fig. 8). The CE values obtained for non-target bacteria were residual (0.1-1.05 %). Validation of P100-MP applicability in distinct food and biological samples

[0107] The inherent heterogeneity and complexity of food matrices, which comprise a plethora of constituents, namely organic compounds and inorganic elements (minerals), competitive microflora, along with particulate matter, poses a challenge in the development of an effective foodborne pathogen detection protocol. In fact, some of those components may constitute critical interferents in magnetic separation procedures (food particulate and lipid content). Hence, in order to circumvent those constraints, a pre-analytical processing of the food sample is of paramount importance for bacterial capture, concentration and isolation from the complex matrix, ergo enhancing the sensitivity of the detection method.

[0108] The phagomagnetic platform applicability was validated in a panoply of food matrices commonly associated with listeriosis outbreaks and possessing distinct physicochemical characteristics, namely pH, salt, fat and protein content. In fact, Listeria-P100 interaction may be influenced by those variables and the presence of interferents may hamper the virion diffusion and listerial WTA recognition and attachment.

[0109] Biofunctionalized P100-MPs performance in acidic matrices corroborated the aforementioned results concerning the pH influence on the interaction of L. monocytogenes and P100-PEI-MPs. A high fat content also negatively impacted the CE of P100-MP, hence constituting critical interferents in the bacterial adsorption to the phagomagnetic particles. Amongst the four evaluated parameters. the variation in the salt and protein contents presented the lowest impact in the P100-MP performance.

[0110] The lower performance obtained in high fat content food matrices is in accordance with what has been previously documented by other authors. In milk, such performance may be resultant from the difficulty in the contact between the (immobilized) phages and thebacteria, which can be hindered by the presence of fat globules. Zhou et al. (2019) also studied the isolation of L. monocytogenes from food matrices (namely, ground beef and whole milk) using magnetic particles with bacteriophage P100, and found that the capture efficiency was much lower in ground beef samples. Based on those findings, as well as on similar results obtained by Shan et al. with the same pathogen and matrices (ground beef and whole milk, CE of 25.1 and 91.2%, respectively), using immunomagnetic nanobeads, it was theorized that the higher amounts of fat in the ground beef could result in fouling and, consequently, lower availability of the phages to adsorb to the target bacterium. As a whole, the food composition / physicochemical features proved to be of utmost importance towards an efficient isolation of L. monocytogenes from distinct matrices. Colorimetric phagomagnetic assay: ^-GLU-Cu3(PO4)2-AuNPs-sakacinX nanoflowers as enzymatic labels Functionalization of ^-GLU-Cu3(PO4)2nanoflowers&AuNPs with sakacin X Sakacin X purification and identification

[0111] Sakacin X, the biorecognition element to be conjugated with the hybrid copper nanoflowers, was purified and identified by nano liquid chromatography mass spectrometry.

[0112] A two-step purification procedure composed by ion-exchange chromatography (IEX) and hydrophobic ionic chromatography (HIC) was performed and the peptide was eluted as a single symmetrical peak, presenting a remarkable antilisterial activity and a high degree of purity. The rationale for the establishment of this purification scheme relied on the peptide properties, namely net positive charge (cation) of the peptide and hydrophobicity. The MS / MS analysis indicated that the molecular weight of the peptide was 4364.87 Da.

[0113] In order to assess the threshold of bacteriocin concentration that could be utilized on the functionalization of the NFLs, without compromising the Lm viability, the minimum inhibitory concentration (MIC) of the peptide was determined. The efficacy of the purified sakacin X against L. monocytogenes serotype 1 / 2a and serotype 4b was evaluated in an in vitro study based on the determination of the MIC and it was found that sakacin X was more active against sv. 4b (MIC 39.1 ng mL-1) than against sv. 1 / 2a (MIC) (156.3 ng mL-1). On the light of these findings, the peptide was immobilized on the NFLs surface at a sub-inhibitory concentration (30 ng mL-1) in order to prevent Lm death. Morphology characterization of the hybrid nanoflowers

[0114] Figure 12 (A-D) displays SEM micrographs of sX-^-GLU-Cu3(PO4)2 hybrid nanoflowers prepared by one-pot biomineralization synthesis. Most of the nanoflowers exhibited a uniform spherical-like shape, with an average diameter of 3 μm. Moreover, a hierarchical branched architecture, in which each branch provided a large and porous surface area (displaying a peony-like morphology), increasing the active sites available for bacteriocin immobilization (electrostatic adsorption). Consequently, a heightened colorimetric signal is expected, which will improve the method’s sensitivity.

[0115] The micrographs of ^-GLU-Cu3(PO4)2-AuNPs-sx&P100-PEI-MPs (E-F) evinced the higher diameter of the hybrid nanoflowers (3 μm) in comparison to the phagomagnetic platform (2 μm). Analytical performance of the hybrid nanoflowers for Lm detection

[0116] In the current work, the synthesized ^-GLU-Cu3(PO4)2-AuNPs-sX nanoflowers were utilized with the dual-purpose of bacteria (Lm) attachment (owing to the functionalization with the bioreceptor sakacin X) and enzymatic activity (^-glucosidase) to attain the signal output amplification.

[0117] Listeria monocytogenes electrostatically adsorbed on the surface of the phagomagnetic platform (Lm-P100-PEI-MP) was quantitatively determined by the enzymatic- based nanoflower system. When ^-GLU-Cu3(PO4)2-AuNPs-sX nanoflowers are added, a sandwich complex was formed with Lm-P100-PEI-MP, in which the bacteria were entrapped between the two biorecognition elements (bacteriophage and bacteriocin). The results indicated the remarkable analytical performance of the proposed scheme to detect as low as 10 CFU mL-1, being observed a direct correlation between the Lm concentration and the optical density value.

[0118] The integration of ^-GLU-Cu3(PO4)2-AuNPs-sX hybrid nanoflowers as enzymatic labels for the selective quantification of viable L. monocytogenes cells was accomplished.

[0119] The ^-GLU-Cu3(PO4)2-AuNPs-sX-NFs&P100-PEI-MP herein disclosed have proven to be an improved capture and signal regulator system in the Lm detection, owing to the integration of a signal amplification (^-Glucosidase), and biorecognition (bacteriocin sakacin X and bacteriophage P100) elements. This system is a versatile, simple, expeditious, affordable, portable platform for the accurate detection of pathogenic bacteria.EXAMPLE 2

[0120] In example 2, the platform (PEI-MP-BSA(5%)) was also validated concerning the isolation and pre-concentration of Campylobacter jejuni (Gram-negative), achieving CE values of 90%. In example 2, all the substances, materials and methods of examples 1 were used, except the bacteriophage: in example 2 the bacteriophage used was Los 1 (Campylobacter phage vB_CjeM_Los1) (GenBank under GenBank assembly No. GCA_002614145.1, specific for C. jejuni) to capture viable cells of C. jejuni. It was the first time that a phagomagnetic platform displaying a whole phage (Los 1, specific for C. jejuni) was used to capture viable cells of C. jejuni.

[0121] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0122] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable.

[0123] The following dependent claims further set out particular embodiments of the disclosure.

Claims

C L A I M S1. Bacteriophage-modified magnetic particle for concentrating a target strain bacterium in a sample comprising, wherein said bacteriophage-modified magnetic particle comprises a magnetic core and a polyethyleneimine shell; wherein the polyethyleneimine shell is functionalized with a plurality of bacteriophages and a plurality of a surface-blocking agent; wherein the plurality of bacteriophages is bound to the polyethyleneimine shell; wherein the plurality of surface-blocking agents is adsorbed to the polyethyleneimine shell; wherein the plurality of bacteriophages is able to bind to the target bacterial strain.

2. Bacteriophage-modified magnetic particle according to the previous claim wherein the bacteriophage has a genome that has at least 80% sequence identity with the genome of a bacteriophage selected from the list consisting of: bacteriophage P100 (as set forward in GenBank under Accession No. DQ004855, version DQ004855.1), bacteriophage Los 1 (as set forward in GenBank under Assembly Accession No. GCA_002614145.1), bacteriophage Campylobacter phage vB_CjeM_Los1 (as set forward in GenBank under Accession No. KX879627, version KX879627.1); bacteriophage Listeria phage P100 (as set forward in GenBank under Assembly Accession No. GCA_002629965.1).

3. Bacteriophage-modified magnetic particle according to any of the previous claims wherein the surface-blocking agent is selected from BSA (Bovine serum albumin) or casein, or mixtures thereof.

4. Bacteriophage-modified magnetic particle according to the previous claim wherein the surface-blocking agent is BSA.

5. Bacteriophage-modified magnetic particle according to the previous claim wherein the plurality of bacteriophages is immobilized in the magnetic particle such that a majority of the phage are oriented in a head-in configuration relative to the magnetic particle such that a head of the bacteriophage is coupled to the magnetic particle and the tail of the bacteriophage is free to attach to bacterial cells of the target strain in a sample containing the target bacteria.

6. Bacteriophage-modified magnetic particle according to any of the previous claims wherein the bacteriophage has a genome that has at least 80% sequence identity with the genome of bacteriophage P100 as set forward in GenBank under Accession No. DQ004855.

7. Bacteriophage-modified magnetic particle according to the previous claim wherein the bacteriophage has a genome that has at least 85% sequence identity with the genome of bacteriophage P100 as set forward in GenBank under Accession No. DQ004855; preferably at least 90% sequence identity; more preferably at least 95% sequence identity; even more preferably 100% sequence identity.

8. Bacteriophage-modified magnetic particle according to any of the previous claims 1-2 wherein the bacteriophage has a genome that has at least 80% sequence identity with the genome of bacteriophage Los 1 as set forward in GenBank under GenBank assembly No. GCA_002614145.

1.

9. Bacteriophage-modified magnetic particle according to the previous claim wherein the bacteriophage has a genome that has at least 85% sequence identity with the genome of bacteriophage Los 1 as set forward in GenBank under GenBank assembly accession number No. GCA_002614145.1; preferably at least 90% sequence identity; more preferably at least 95% sequence identity; even more preferably 100% sequence identity.

10. Bacteriophage-modified magnetic particle according to any of the previous claims wherein the diameter of the magnetic particle ranges from 1-3 μm; preferably 2 μm.

11. Bacteriophage-modified magnetic particle according to any of the previous claims wherein the magnetic core of the magnetic particle comprises a material selected from the list consisting of: Fe3O4,γ-Fe₂O₃, CoFe₂O₄, NiFe₂O₄, MnFe₂O₄, FePt, FeCo; preferably Fe3O4.

12. Copper detection particle for detecting a target bacterial strain in a sample, wherein said copper detection particle comprises a Cu3(PO4)2 nanoflower, a plurality of gold nanoparticles, a plurality of β-glucosidase enzymes and a plurality of biorecognition molecules, wherein:the plurality of biorecognition molecules is electrostatically immobilized on the Cu3(PO4)2nanoflower through the gold nanoparticles; the β-glucosidase enzyme is entrapped in the Cu3(PO4)2 nanoflower; the biorecognition molecule has an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of sakacin X wherein the sequence is selected from SEQ ID 1, SEQ ID 2; the copper detection particle is able to bind to the target strain bacterium and change the colour of the sample by the addition of a suitable colorimetric reagent.

13. Copper detection particle according to the previous claim wherein the biorecognition molecule has an amino acid sequence that has at least 85% sequence identity with the amino acid sequence of sakacin X wherein the sequence of sakacin X is selected from SEQ ID 1, SEQ ID 2; preferably at least 90% sequence identity; more preferably at least 95% sequence identity; even more preferably 100% sequence identity.

14. Copper detection particle according to any of the previous claims 12-13 wherein the suitable colorimetric reagent is p-nitrophenyl ^-D-glucopyranoside.

15. Kit for detecting and concentrating a target bacterial strain in a sample comprising the bacteriophage-modified magnetic particle according to any of the previous claims 1—11 and the copper nanoflower-based detection particle according to any of the previous claims 12-14.

16. Method for obtaining a bacteriophage-modified magnetic particle as described in any of the previous claims 1-11 comprising the following steps: providing magnetic particles comprising a magnetic core and a polyethyleneimine shell; adding a plurality of bacteriophages specific for a target strain bacterium to obtain a first product of magnetic particles comprising a magnetic core and a polyethyleneimine shell functionalized with a plurality of bacteriophages; re-suspending the first product in an aqueous solution of a blocking agent to obtain bacteriophage-modified magnetic particles comprising a magnetic core and a polyethyleneimine shell, wherein the polyethyleneimine shell is functionalized with a plurality of bacteriophages and a plurality of a surface-blocking agent; wherein the concentration of the blocking agent in the aqueous solution ranges from 3-7 % (w / v).

17. Method according to the previous claim wherein the concentration of the blocking agent in the aqueous solution is 5 % (w / v).

18. Method according to any of the previous claims 16-17 wherein the aqueous solution of the block agent is a BSA solution in PBS.

19. Method according to any of the previous claims 16-18 wherein the step of re-suspending the first product in an aqueous solution of a blocking agent to obtain the bacteriophage- modified magnetic particles is performed under agitation and during a period ranging from 2-6h; more preferably 4h.

20. Method for synthesizing a copper detection particle, preferably copper nanoflower-based detection particle, as described in any of the previous claims 12-14 comprising the following steps: providing an aqueous solution by mixing copper sulphate with phosphate-buffered saline to initiate the formation of copper phosphate naoflowers; wherein the β-glucosidase enzyme is introduced at a concentration ranging from 0.1 to 1.0 mg mL-1, allowing the enzyme to become entrapped within the forming nanoflowers. adding a plurality of gold nanoparticles to coat the surface of the nanoflowers; functionalizing the gold nanoparticle-coated nanoflowers by immobilizing biorecognition molecules, wherein the biorecognition molecules are electrostatically bound to the nanoflowers via the gold nanoparticles and are selected from sakacin X or sequences having at least 80% sequence identity to sakacin X.

21. Method according to the previous claim wherein the concentration of the CuSO4is 120 mM and the concentration of β-glucosidase enzyme is 0.5 mg mL-1.

22. Method according to any of the previous claims 20-21 wherein during the functionalization step, the concentration of the sakacin X used to immobilize on the nanoflowers is 30 ng mL-1.

23. Method for concentrating and detecting a target strain bacterium in a sample, wherein the method comprises the steps of: mixing the bacteriophage-modified magnetic particle according to any of the previous claims 1-11 with the sample to form a first reaction mixture under conditions suitable to allow the bacteriophages to bind to the target bacterial strain present in the sample;applying a magnetic field to the first reaction mixture to collect, and separate, bacteriophage-modified magnetic particles bound to the target bacterial strain; mixing the bacteriophage-modified magnetic particles bound to the target bacterial strain with the copper detection particle, preferably copper nanoflower-based detection particle, according to any of the previous claims 12-14 to form a second reaction mixture under conditions suitable to allow the biorecognition molecule of the copper detection particle to bind to the bacteria bonded to the bacteriophage-modified magnetic particle; applying a magnetic field to the second reaction mixture to collect, and separate, copper nanoflower-based detection particles bonded to the bacteria bonded to the bacteriophage-modified magnetic particles; mixing the copper nanoflower-based detection particles bonded to the bacteria bonded to the bacteriophage-modified magnetic particles with a suitable colorimetric reagent.

24. Method according to the previous claim wherein the sample is a food sample, an environmental sample or a biological sample; preferably a food sample.

25. Method according to any of the previous claims 23-24 wherein the suitable colorimetric reagent is p-nitrophenyl ^-D-glucopyranoside.

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