Biosensors for staphylococcus aureus detection

Abstract

A biosensor for detecting Methicillin resistant Staphylococcus aureus (MRSA) or Methicillin sensitive Staphylococcus aureus (MSSA) are provided. The biosensor for detecting MRSA can contain a core component and an anti-penicillin-binding protein 2a (PBP2a) antibody immobilized thereon. The biosensor for detecting MSSA can contain a core component and an anti-penicillin-binding protein 2 (PBP2) antibody immobilized thereon. Also provided are methods for detecting or quantitating Staphylococcus aureus in a sample using the biosensor provided herein. The biosensor can be a bio-layer interferometry (BLI) biosensor, and the signals generated by binding of Staphylococcus aureus to the biosensor can be indicative of spectral shift, which can be measured using BLI.

Description

BIOSENSORS FOR STAPHYLOCOCCUS AU / US YTECTAOBackground

[0001] The present disclosure relates to apparatus and methods for detecting and / or quantitating a microbe in a sample.

[0002] Staphylococcus aureus is a gram-positive and catalase-positive bacterium. It is a common cause of hospital infections (such as nosocomial pneumonia, surgical site infections, postoperative wound infections, bacteremia) and food poisoning and can be life threatening if left untreated or when treated with incorrect antibiotics. For example, Staphylococcus aureus is the most common cause of nosocomial pneumonia; causes about 37% of surgical site infections in community hospitals; and causes about 6% of hospital-acquired infections. Staphylococcus bacteremia (SaB) has a mortality rate of around 25%, with higher mortality rates in patients with underlying comorbidities and methicillin-resistant Staphylococcus aureus (MRSA). According to the US Centers for Disease Control report, nearly 120,000 Staphylococcus aureus bloodstream infections and 20,000 associated deaths occurred in the United States in 2017. In addition, Staphylococcus aureus has been considered as one of the most common foodborne pathogens and is the top ordinary pathogen causing food poisoning. Rapid and accurate diagnosis of Staphylococcus aureus infection can lead to effective treatment.

[0003] MRSA is a type of Staphylococcus aureus that does not respond to the antibiotics (0- lactams) that are usually used to treat Staphylococcus infections. MRSA cannot be effectively treated with 0-lactam antibiotics such as methicillin, nafcillin, cephalosporin, or penicillin that are usually used to treat Staphylococcus aureus, and thus detecting MRSA is a determinant fortreatment decisions. A limited group of drugs (such as vancomycin, daptomycin, linezolid, sulfamethoxazole and trimethoprim, quinupristin-dalfopristin, clindamycin, and tigecycline) can be used to treat MRSA infections. MRSA can cause a life-threatening infection in the bloodstream or other organs, leading to sepsis or death. The mortality rate for people with colonized MRSA is 36%, whereas the mortality rate of people with methicillin sensitive Staphylococcus aureus (MSSA) is about 18%. Rapid and accurate diagnosis of MRSA infection not only facilitate treatment decisions but also enables early implementation of measures to prevent further MRSA transmission to other patients and staff in the hospital settings.

[0004] Thus, fast and accurate detection methods for Staphylococcus aureus, such as MRSA and MSSA, is in an urgent need for reducing Staphylococcus aureus-re a A morbidity and mortality, and further spread of MRSA. Conventional methods for detecting Staphylococcus aureus, such as culturing bacteria from specimen, chromogenic media tests, coagulase tests, matrix-assisted laser desorption / ionization coupled to time-of-flight mass spectrometry (MALDI- TOF MS), enzyme-linked immunosorbent assay (ELISA), and quantitative polymerase chain reaction (qPCR), can take some time to obtain results and / or can be inaccurate, often leading to a poor prognosis. For example, culturing bacteria from specimen (such as a wound site sample or a blood sample) can take a few days, and a drug sensitivity test to identify MRSA among Staphylococcus aureus cultures can take additional few days. Chromogenic media tests take at least 24 hours to obtain results. Coagulase tests can be faster but less specific and less reliable. MALDI-TOF MS can be time consuming. Immunological tests (such as ELISA, immunoblotting, immunoprecipitation) take at least a few hours to obtain results. Genomic tests (such as qPCR test and SureTect™) can provide specific results but can take about 6 hours to obtain results.Summary

[0005] In view of the foregoing, there is a need for methods that efficiently, accurately, and rapidly detect the presence or amount of Staphylococcus aureus, in particular methods that efficiently, accurately, and rapidly detect the presence or amount of MRSA and MSSA differentially. This disclosure is directed generally to systems and methods to address these shortcomings of the art and provide other additional or alternative advantages. The disclosure herein provides embodiments of biosensors, and methods using the biosensors, for detecting Staphylococcus aureus, in particular differentially detecting MRSA and MSSA in a sample. The biosensor for detecting Staphylococcus aureus contains a core component and an antibody immobilized thereon that specifically binds to Staphylococcus aureus, and to MRSA or MSSA in particular. The biosensor can be a bio-layer interferometry (BLI) biosensor, and the signals generated by binding of Staphylococcus aureus to the biosensor can be indicative of spectral shift, which can be measured using BLI.

[0006] In certain aspects of the present disclosure, a method for detecting Staphylococcus aureus in a sample is provided. The method includes contacting the sample with a biosensor, and detecting signals generated by binding of Staphylococcus aureus to the biosensor, thereby detecting Staphylococcus aureus in the sample. The biosensor contains a core component and an anti-penicillin-binding protein (PBP) antibody immobilized thereon, such that Staphylococcus aureus in the sample binds the anti-PBP antibody of the biosensor.

[0007] In embodiments, the anti-PBP antibody is an anti-penicillin-binding protein 2a (anti- PBP2a) antibody, such that MRSA in the sample binds to the anti-PBP2a antibody of the biosensor. In these embodiments, the method includes detecting signals generated by binding of MRSA tothe biosensor, thereby detecting MRSA in the sample. In some embodiments, the anti-PBP2a antibody is a monoclonal anti-PBP2a antibody.

[0008] In certain embodiments, the anti-PBP antibody is an anti-penicillin-binding protein 2 (anti-PBP2) antibody, such that MS SA in the sample binds to the anti-PBP2 antibody of the biosensor. In these embodiments, the method includes detecting signals generated by binding of the MSSA to the biosensor, thereby detecting MSSA in the sample. In some embodiments, the anti-PBP2 antibody is a monoclonal anti-PBP2 antibody.

[0009] In some embodiments, the method further includes, after contacting the sample with the biosensor, contacting the biosensor-bound Staphylococcus aureus with a targeting molecule attached to a detectable label, such that the targeting molecule binds to the biosensor-bound Staphylococcus aureus, and detecting the signals of the detectable label generated by binding of the targeting molecule to the biosensor-bound Staphylococcus aureus. In specific embodiments, the targeting molecule is LysGH15. In specific embodiments, the detectable label is a fluorescent label.

[0010] In embodiments, the biosensor is a bio-layer interferometry (BLI) biosensor. In some embodiments, the signals are indicative of spectral shift measured using BLI. In some embodiments, a positive spectral shift indicates binding of Staphylococcus aureus to the biosensor.

[0011] In further embodiments, the signals in a plurality of samples are detected simultaneously, for example using BLI. In certain embodiments, the plurality of samples are each placed in a plurality of wells in a plate, and the signals in the plurality of wells are detected in plate reader format.

[0012] In some embodiments, the method further includes quantitating the amount of Staphylococcus aureus in the sample. The amount of Staphylococcus aureus in the sample is quantitated based on the signals generated by binding of Staphylococcus aureus to the biosensor and a calibration curve of association between known amounts of Staphylococcus aureus and signals generated by each known amount of Staphylococcus aureus.

[0013] In embodiments of the methods provided herein, the presence of Staphylococcus aureus in the sample is detected within 30 minutes of contacting the sample with the biosensor. In further embodiments of the methods provided herein, the amount of Staphylococcus aureus in the sample is quantitated within 30 minutes of contacting the sample with the biosensor.

[0014] In certain embodiments, the sample is a biological sample obtained from a subject.

[0015] In some embodiments, the method further includes selecting the subject as a candidate for, or administering to the subject, treatment or procedure directed to Staphylococcus aureus infection. In embodiments, the treatment or the procedure directed to Staphylococcus aureus infection includes administering to the subject an antibiotic effective for reducing the amount of Staphylococcus aureus in the subject (antibiotics treatment).

[0016] In certain embodiments, the sample is a biological sample obtained from a subject, and the method further includes selecting the subject as a candidate for, or administering to the subject, treatment or procedure directed to MRSA infection. In embodiments, the treatment or the procedure directed to MRSA infection comprises one or more of administering to the subject an antibiotic effective for reducing the amount of MRSA in the subject (antibiotics treatment for MRSA) and implementing a procedure to prevent spread of MRSA infection to another subject(infection prevention).

[0017] In some embodiments of the present disclosure, a biosensor for detecting MRSA in a sample is provided. The biosensor contains a core component and an anti-PBP2a antibody immobilized thereon, configured to detect MRSA in the sample. In embodiments, the biosensor is a BLI biosensor.

[0018] In certain embodiments of the present disclosure, a biosensor for detecting MSSA in a sample is provided. The biosensor contains a core component and an anti-PBP2 antibody immobilized thereon, configured to detect MSSA in the sample. In embodiments, the biosensor is a BLI biosensor.Brief Description of the Drawings

[0019] Various aspects of the present disclosure are described hereinbelow with reference to the drawings, which are incorporated in and constitute a part of this specification.

[0020] FIG. 1 is a schematic representation of an example biosensor for detecting Staphylococcus aureus in a sample according to the embodiments of the present disclosure.

[0021] FIG. 2 is a flowchart with schematic representations of a process of detecting the presence or amount of Staphylococcus aureus in a sample according to embodiments of the present disclosure.

[0022] FIG. 3 is a schematic representation of a process of detecting the presence or amount of MRSA in a sample according to embodiments of the present disclosure.Detailed Description

[0023] The present disclosure will now be described more fully hereinafter with reference to example embodiments thereof and with reference to the drawings. These example embodimentsare described so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Features from one embodiment or aspect can be combined with features from any other embodiment or aspect in any appropriate combination. For example, any individual or collective features of method aspects or embodiments can be applied to apparatus, product, or component aspects or embodiments and vice versa. The disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0024] As used herein, the term “biosensor” refers to a device that detects the presence, physical characteristics, or amount of a substance of interest (an analyte) using a biological molecule (such as an enzyme, an antibody, an aptamer, DNA), a living organism, or a cell or tissue thereof. In embodiments, the biosensor has a distal end and a proximal end. The proximal end of the biosensor can have a surface coated with a thin layer of analyte-binding molecules (such as an antibody). The distal end of the biosensor can be connected to a transducer that converts biorecognition events into a measurable signal and / or a display that displays the signals.

[0025] As used herein, the term “proximal” refers to the portion of the device or component thereof that is closer to the light source in the device, and the term “distal” refers to the portion of the device or component thereof that is farther from the light source in the device and closer to the subject sample.

[0026] An “analyte-binding” molecule refers to any molecule or ligand capable of participating in a specific binding reaction with an analyte molecule. Examples include, but are not limited to, antibody-antigen binding reactions.

[0027] As used herein, the term “bio-layer interferometry (BLI) biosensor” refers to a biosensor that uses BLI for detection of the presence, physical characteristics, or amount of a substance of interest (an analyte). BLI is an optical technique for measuring biomolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. BLI allows for label-free optical analysis for real-time monitoring of biomolecular interactions.

[0028] An “antibody” refers to a peptide or polypeptide derived from, modeled after, or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. An antibody includes an immunoglobulin molecule having two heavy chains and two light chains prepared by any method known in the art or later developed. Antibodies can be produced by immunizing mice, rats, or rabbits or by genetic engineering methods such as cloning of native immunoglobulin genes (or humanized immunoglobulin genes) in mammalian plasmid vectors and then expressing them in mammalian cell lines. An antibody also refers to and includes an antibody fragment, such as an antigen binding site (e.g., a fragment, a subsequence, a complementarity determining region (CDR)) that retains capacity to bind antigen, including: a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment, which consists of a VH domain; and an isolated complementarity determining region (CDR). For example, a fragment of an antibody molecule, produced by chemical cleavage or genetic engineering techniques, is within the scope of antibodies. Single chain variablefragments (SCFvs) such as those produced using combinatorial genetic libraries and phage display technologies are within the scope of antibodies.

[0029] Antibodies include polyclonal antibodies and monoclonal antibodies. A “polyclonal antibody” refers to a heterologous mixture of immunoglobulins against an antigen, and can be produced by multiple cells by inoculating a mammal such as a goat, a mouse, and a rabbit with an immunogen. A “monoclonal antibody” refers to an antibody produced by identical immune cells which are clones of a single cell, for example using the well-known Kohler Milstein hybridoma fusion technique. A polyclonal antibody can bind to different epitopes of the same antigen, whereas a monoclonal antibody binds to one epitope of the antigen.

[0030] A “subject sample” as used herein refers to a sample with an unknown presence or amount of Staphylococcus aureus (such as MRS A or MS SA) to be detected. A “reference sample” as used herein refers to a sample with a known presence or amount of Staphylococcus aureus (such as MRSA or MSSA).

[0031] A “subject” refers to an animal, such as a mammal, including a primate (such as a human, a non-human primate, such as a monkey) and a non-primate (such as a mouse). In some aspects of the disclosure, the subject is a human. In some aspects, the subject is a pediatric subject, such as a neonate, an infant, or a child. In other aspects, the subject is an adult subject.

[0032] A “patient” refers to a subject who shows symptoms and / or signs of a disease, is under treatment for disease, has been diagnosed with a disease, and / or is at risk of developing a disease. A “patient” can be a human or veterinary subject. Any reference to subjects in the present disclosure should be understood to include the possibility that the subject is a “patient” unless clearly dictated otherwise by context. More specifically, the subject in certain aspects is a patientwho has, is suspected to have, or is in need of diagnosis for Staphylococcus aureus infection (such as MRSA or MSSA infection).

[0033] As used herein, the singular forms “a,” “an,” “the,” and the like include plural referents unless the context clearly dictates otherwise. In addition, while reference may be made herein to quantitative measures, values, geometric relationships or the like, unless otherwise stated, any one or more if not all of these may be absolute or approximate to account for acceptable variations that may occur, such as those due to manufacturing or engineering tolerances or the like. Reference to a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se with in a range suitable in the context, for example ± 10%. For example, description referring to “X” includes description of “X” and extends to a suitable range.

[0034] In certain aspects of the present disclosure, a biosensor for detecting Staphylococcus aureus in a sample is provided. The biosensor contains a core component and an anti-penicillin- binding protein (PBP) antibody immobilized thereon, and is configured to detect Staphylococcus aureus in the sample. Any suitable material can be used for the core component. For example, the core component can comprise a silicon substrate, activated with silane group, such as aminopropylsilane (APS) and epoxypropylsilane (EPS).

[0035] Staphylococcus aureus expresses PBPs on the cell surface. PBPs polymerize and modify peptidoglycan, the stress-bearing component of the bacterial cell wall. MSSA express PBP1, PBP2, PBP3, and PBP4. PBP1-4 of Staphylococcus aureus provide both transglycosylase and transpeptidase activity. MRSA express PBP2a instead of PBP2. PBP2a expression is specific to MRSA. PBP2a confers cross-resistance to most P-lactam antibiotics (including methicillin), and is a major cause of resistance of MRSA to P-lactams. Without wishing to be bound by theory,antimicrobial resistance in MRSA occurs due to the mutation or modification of antibiotic targets, inactivation of P-lactam antibiotics by P-lactamase, a reduction in membrane permeability, or increased activity of efflux pumps.

[0036] Thus, in certain aspects of the present disclosure, a biosensor for detecting MRSA in a sample provided herein contains a core component and an anti-PBP2a antibody immobilized thereon configured to detect MRSA in a sample. The anti-PBP2a antibody can be a monoclonal anti-PBP2a antibody. The biosensor having an anti-PBP2a antibody (such as a monoclonal anti- PBP2a antibody) immobilized thereon is configured to specifically detect MRSA such that it can specifically bind and detect MRSA, but does not bind or detect MS SA. In particular, the monoclonal anti-PBP2a antibody of the biosensor can allow for specific binding and detection of MRSA. Further, in certain aspects of the present disclosure, a biosensor for detecting MSSA in a sample provided herein contains a core component and an anti-PBP2 antibody immobilized thereon configured to detect MSSA in a sample. The anti-PBP2 antibody can be a monoclonal anti-PBP2 antibody. The biosensor having an anti-PBP2 antibody (such as a monoclonal anti- PBP2 antibody) immobilized thereon is configured to specifically detect MSSA such that it can specifically bind and detect MSSA, but does not bind or detect MRSA. In particular, the monoclonal anti-PBP2 antibody of the biosensor can allow for specific binding and detection of MSSA.

[0037] The term “immobilize” or “immobilized” or “immobilizing” in the context of immobilizing an analyte-binding molecule (such as an antibody) onto a biosensor or a structure thereof (such as a core component) refers to the process of attaching or binding the analyte-binding molecule to the biosensor or the structure thereof (such as a core component). Analyte-binding molecules (such as antibodies) may be immobilized (attached, bound) to specific zones of thebiosensor either by conjugating directly to the biosensor surface, or by indirect binding. In an example of indirect binding, analyte-binding molecules (such as antibodies) may be immobilized on particles or other solid supports, and the solid supports may be immobilized onto the biosensor surface. Solid supports that may be used to immobilize an analyte-binding molecule (such as an antibody) include membrane filters, cellulose-based papers, beads (including polymeric, latex, and paramagnetic particles), silicon wafers, nanoparticles, gels, and multi-well plates. Immobilization of an analyte-binding molecule (such as an antibody) on a biosensor can be done by any methods known in the art, for example by binding a biotinylated analyte-binding molecule (such as a biotinylated antibody) to streptavidin bound to the core component of the biosensor. An analytebinding molecule (such as an antibody) can also be immobilized on a biosensor by passive adsorption of the analyte-binding molecule onto the core component of the biosensor, for example by incubating the biosensor with a solution containing the analyte-binding molecule (such as the antibody). An analyte-binding molecule (such as an antibody) can also be immobilized on a biosensor by using a crosslinker. Conventional immobilization chemistries can be used for chemically (such as covalently) attaching a layer of the analyte-binding molecule to the biosensor. For example, a variety of bifunctional reagents containing a siloxane group can be used for chemical attachment to SiCh, and a hydroxyl, amine, carboxyl or other reaction group can be used for attachment of biological molecules, such as proteins (such as antigens, antibodies). It is also well known to etch or otherwise treat glass or glass surfaces to increase the density of hydroxyl groups by which analyte-binding molecules (such as antibodies) can be bound. Where the core element of the biosensor is formed of a polymer, such as polystyrene, a variety of methods are available for exposing available chemically active surface groups, such as amine, hydroxyl, and carboxyl groups, for binding with analyte-binding molecules (such as antibodies).

[0038] FIG. 1 schematically depicts an embodiment of a biosensor for detecting Staphylococcus aureus in a sample. The biosensor 102 includes a core component 104 and an anti-PBP antibody 106 immobilized thereon. In embodiments, the anti-PBP antibody 106 is an anti-PBP2a antibody, such as a monoclonal anti-PBP2a antibody, such that the biosensor specifically binds and detects MRSA in a sample. In other embodiments, the anti-PBP antibody 106 is an anti-PBP2 antibody, such as a monoclonal anti-PBP2 antibody, such that the biosensor specifically binds and detects MSSA in a sample.

[0039] In embodiments, the biosensor is a BLI biosensor. The BLI biosensor uses BLI to measure the signal / wavelength / spectral shift (nm) over time at the biosensor generated by the biosensor and an analyte (such as Staphylococcus aureus), with a positive shift indicating binding of Staphylococcus aureus (specifically MRSA or MSSA) to the biosensor.

[0040] A method of the present disclosure for detecting the presence or amount of Staphylococcus aureus in a sample can include the steps of contacting the sample with a biosensor, and detecting signals generated by binding of Staphylococcus aureus to the biosensor, thereby detecting Staphylococcus aureus in the sample. The biosensor contains a core component and an anti-penicillin-binding protein (PBP) antibody immobilized thereon, such that Staphylococcus aureus in the sample binds the anti-PBP antibody of the biosensor.

[0041] In embodiments, the anti-PBP antibody is an anti-PBP2a antibody, such that MRSA in the sample binds to the anti-PBP2a antibody of the biosensor. In these embodiments, the method includes detecting signals generated by binding of MRSA to the biosensor, thereby detecting the presence or amount of MRSA in the sample. In some embodiments, the anti-PBP2a antibody is a monoclonal anti-PBP2a antibody to allow for specific binding and detection of MRSA. Thebiosensor having an anti-PBP2a antibody immobilized thereon detects the presence or amount of MRS A, but not MS SA.

[0042] In other embodiments, the anti-PBP antibody is an anti-PBP2 antibody, such that MS SA in the sample binds to the anti-PBP2 antibody of the biosensor. In these embodiments, the method includes detecting signals generated by binding of MSSA to the biosensor, thereby detecting the presence or amount of MSSA in the sample. In some embodiments, the anti-PBP2 antibody is a monoclonal anti-PBP2 antibody to allow for specific binding and detection of MSSA. The biosensor having an anti-PBP2 antibody immobilized thereon detects the presence or amount of MSSA, but not MRSA.

[0043] In embodiments, the biosensor is a BLI biosensor. In some embodiments, the signals are indicative of signal / wavelength / spectral (nm) shift, which can be measured using BLI. The signal / wavelength / spectral (nm) shift over time can indicate binding of an analyte (such as Staphylococcus aureus') to the BLI biosensor. In some embodiments, a positive signal / wavelength / spectral shift indicates binding of Staphylococcus aureus to the biosensor, which can be measured using BLI.

[0044] In some embodiments, the method further includes, after contacting the sample with the biosensor, contacting the biosensor-bound Staphylococcus aureus with a targeting molecule attached to a detectable label, such that the targeting molecule binds to the biosensor-bound Staphylococcus aureus, and detecting the signals of the detectable label generated by binding of the targeting molecule to the biosensor-bound Staphylococcus aureus. In specific embodiments, the targeting molecule is LysGH15. LysGH15 is a lysin derived from the staphylococcal phage GH15, and specifically binds to Staphylococcus aureus, including MRSA and MSSA. Anydetectable label may be attached to the targeting molecule (such as LysGH1 ) to allow for detection of binding of the targeting molecule to the target (such as Staphylococcus aureus). In specific embodiments, the detectable label is a fluorescent label, and the fluorescent signals of fluorescent labeled LysGH15 bound to Staphylococcus aureus (MRSA or MSSA) that is bound to the biosensor is measured, to detect the presence or amount of Staphylococcus aureus (MRSA or MSSA) in the sample. Fluorescence can be measured using any methods known in the art, such as using a fluorometer. The methods of detecting Staphylococcus aureus using a targeting molecule (such as LysGH15) attached to a detectable label (such as a fluorescent label) can be used independently from, or in conjunction with, the label free methods of detecting Staphylococcus aureus based on the signals generated by binding of Staphylococcus aureus to the biosensor (such as by using BLI).

[0045] The signals in a plurality of samples can be detected simultaneously, for example using BLI and / or a targeting molecule attached to a detectable label. To facilitate simultaneous or high throughput detection, the plurality of samples can be each placed in a plurality of wells in a plate, and the signals in the plurality of wells can be detected in plate reader format. Any other sample placement format can be used to facilitate simultaneous or high throughput detection.

[0046] In some embodiments, the method further includes quantitating the amount of Staphylococcus aureus in the sample. The amount of Staphylococcus aureus in the sample (subj ect sample) can be quantitated based on the signals generated by binding of the Staphylococcus aureus in the sample (subject sample) to the biosensor and a calibration curve (or a standard curve). The calibration curve can represent an association between known amounts of Staphylococcus aureus and signals generated by each known amount of Staphylococcus aureus obtained for example by using reference samples.

[0047] The method provided herein can provide fast and accurate detection of the presence or amount of Staphylococcus aureus in a sample. For example, the presence of Staphylococcus aureus in the sample can be detected within 1 hour or 30 minutes of contacting the sample with the biosensor. Likewise, the amount of Staphylococcus aureus in the sample can be quantitated within 1 hour or 30 minutes of contacting the sample with the biosensor. Such short time until diagnosis (detection of the presence or amount of Staphylococcus aureus in a sample) and the ability to differentially detect MRSA and MSSA of the methods provided herein can offer advantages over currently available methods of detecting Staphylococcus aureus. For example, a treatment or a procedure directed to MRSA or MSSA infection can be selected and started sooner to improve treatment outcome and / or prevent spread of the infection.

[0048] In certain embodiments, the sample is a biological sample. A “biological sample” as used herein refers to any sample obtained from a living organism. A biological sample can be a tissue sample, a swab containing cells (such as nasal or throat swab), or a body fluid sample. A “body fluid sample” as used herein refers to a sample of bodily fluid obtained from a subject, such as a patient. A body fluid sample can be blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions obtained from a subject, such as a patient, for example who has or is suspected to have Staphylococcus aureus infection. In addition, one of skill in the art would realize that certain body fluid samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

[0049] FIG. 2 schematically represents an embodiment of a process of detecting the presence or amount of Staphylococcus aureus in a sample, such as a biological sample. Process 200, for detecting the presence or amount of Staphylococcus aureus in a sample, begins with an optionalstep of immobilizing an anti-PBP antibody onto the biosensor core component. The anti-PBP antibody can be an anti-PBP2a antibody, for specific binding and detection of MRSA. The anti- PBP2a antibody can be a monoclonal anti-PBP2a antibody. Additionally or alternatively, the anti- PBP antibody can be an anti-PBP2 antibody, for specific binding and detection of MSSA. The anti-PBP2 antibody can be a monoclonal anti-PBP2 antibody. Any suitable method of immobilization can be used, including biotinylation of the antibody and binding of the biotinylated antibody to the biosensor coated with streptavidin, passive adsorption, or attachment using a crosslinker. Process 200 continues to step 204, to provide the biosensor having an anti-PBP antibody (such as an anti-PBP2a antibody for specific detection of MRSA, or an anti-PBP antibody for specific detection of MSSA) immobilized thereon. Process 200 continues to step 206, to contact the sample with the biosensor. Contacting the sample with the anti-PBP antibody bound biosensor allows for binding of Staphylococcus aureus to the biosensor. Specifically, contacting the sample with the anti-PBP2a antibody bound biosensor allows for binding of MRSA to the biosensor, and contacting the sample with the anti-PBP2 antibody bound biosensor allows for binding of MSSA to the biosensor. Process 200 continues to step 208 to detect signals generated by binding of Staphylococcus aureus to the biosensor, thereby detecting the presence or amount of Staphylococcus aureus in the sample. In embodiments, the biosensor is a BLI biosensor. In some embodiments, the signals are generated by binding of a material (such as Staphylococcus aureus) to the BLI biosensor, and are indicative of signal / wavelength / spectral (nm) shift, which can be measured using BLI. In some embodiments, a positive signal / wavelength / spectral shift can indicate binding of Staphylococcus aureus to the biosensor, which can be measured using BLI. In this step, use of calibration curve (or standard curve) may facilitate accurate quantitation of the amount of Staphylococcus aureus in the sample. The calibration curve (or standard curve) canrepresent an association between known amounts of Staphylococcus aureus and signals generated by each known amount of Staphylococcus aureus obtained for example by using reference samples. The presence or amount of Staphylococcus aureus in each subject sample can be determined based on the signals generated by binding of Staphylococcus aureus in the sample to the biosensor, and the calibration curve.

[0050] From step 206, process 200 can alternatively or additionally continue to step 210, to contact the biosensor-bound Staphylococcus aureus with a targeting molecule attached to a detectable label, such that the targeting molecule binds to the biosensor-bound Staphylococcus aureus. The targeting molecule can be LysGH15 and the detectable label can be a fluorescent label. Process 200 continues to step 212 to detect the signals of the detectable label generated by binding of the targeting molecule to the biosensor-bound Staphylococcus aureus, thereby detecting the presence or amount of Staphylococcus aureus. Steps 210 and 212 for detecting Staphylococcus aureus using a targeting molecule (such as LysGH15) attached to a detectable label (such as a fluorescent label) can be used independently from, or in conjunction with, step 208 for detecting Staphylococcus aureus based on the signals generated by binding of Staphylococcus aureus to the biosensor without using labels, for example by using BLI.

[0051] FIG. 3 schematically represents an embodiment of a process of detecting the presence or amount of MRSA in a sample. Process 300, a method of detecting the presence or amount MRSA in a sample, begins with an optional step 302 of immobilizing (or loading) an anti-PBP2a antibody onto the biosensor core component. The anti-PBP2a antibody can be a monoclonal anti-PBP2a antibody, for specific binding and detection of MRSA. Any suitable method of immobilization can be used, including biotinylation of the antibody and binding of the biotinylated antibody to the biosensor coated with streptavidin, passive adsorption, or attachment using acrosslinker. Process 300 continues to step 304, to provide the biosensor having an anti-PBP2a antibody immobilized thereon. Process 300 continues to step 306, to contact the sample with the biosensor. Contacting the sample with the anti-PBP2a antibody bound biosensor allows for binding of MRSA in the sample to the biosensor, but MSSA in the sample does not bind to the biosensor. Process 300 continues to step 308 to transduce and detect signals generated by binding of MRSA to the biosensor, thereby detecting the presence or amount of Staphylococcus aureus in the sample. In specific embodiments, the distal end of the biosensor is connected to a transducer (such as one shown in step 308) that converts bio-recognition events into a measurable signal and further to a display that displays signals (such as those displayed in steps 310 and 312). In embodiments, the biosensor is a BLI biosensor, and as shown in step 310, the signals generated by binding of an analyte (such as Staphylococcus aureus) to the BLI biosensor are indicative of signal / wavelength / spectral (nm) shift, which can be measured using BLI. As shown in step 312, a positive signal / wavelength / spectral shift can indicate binding of MRSA to the biosensor, which can be measured using BLI. On the other hand, MSSA contained in the sample does not generate a positive signal / wavelength / spectral shift, thus differentially detecting MRSA in the sample. In steps 308, 310, and 312, use of a calibration curve (or a standard curve) may facilitate accurate quantitation of the amount of Staphylococcus aureus in the sample. The calibration curve (or standard curve) can represent an association between known amounts of MRSA and signals generated by each known amount of MRSA obtained for example by using reference samples. The presence or amount of MRSA in each subject sample can be determined based on the signals generated by binding of MRSA in the sample to the biosensor as shown in steps 310 and 312, and the calibration curve.

[0052] In some embodiments, the biosensor provided herein comprises an optical fiber having a proximal end portion and a distal end portion, the proximal end portion configured to receive light from a light source and configured to deliver reflected light to a detector. The distal end portion configured to have analytes bind thereto such that light reflected from the distal end portion is phase shifted based on a thickness of analytes bound to the distal end portion. In some embodiments, the biosensor further comprises an optical resonator at a distal end portion of the optical fiber, the optical resonator including a first reflective surface and a second reflective surface, the first reflective surface configured to reflect light with a first phase and the second reflective surface configured to reflect light with a second phase which is phase shifted based on a thickness of analytes bound to the optical resonator.

[0053] In some embodiments of the methods provided herein, the signals such as those generated by binding of Staphylococcus aureus (such has MRSA or MSSA) to the biosensor, or binding of a targeting molecule (such as LysGH15) are measured by a detector. The signals can be detected based on any label-free technique for detecting a change in a property of a sensor surface, such as BLI, Surface Plasmon Resonance (SPR), Surface Acoustic Wave (SAW), Quartz Crystal Microbalance (QCM), and Refl ectometric Interference Spectroscopy (RIfS). Alternatively, the signals of the detectable label, such as fluorescent label, can be detected by a detector. In specific embodiments, the signals are measured by an interferometer. The interferometer can comprise the biosensor, and can constitute a BLI sensor. In some embodiments, the interferometer further comprises: a first optical waveguide configured to receive light from a light source; a second optical waveguide configured to deliver reflected light to a detector; and an optical coupler spatially separates a distal portion of the first optical waveguide from a distal portion of the second optical waveguide, wherein the biosensor is attached to the optical coupler.In some embodiments, the interferometer further comprises a light source that is in optical communication with the first optical waveguide and configured to provide light to the first optical waveguide. In some embodiments, the interferometer further comprises a detector configured to receive light from the second optical waveguide. In some embodiments, the first optical waveguide and the second optical waveguide are disposed in a fiber optic bundle.

[0054] For example, an interferometer can include a light source, an optical assembly, and a detector unit. The BLI sensor or optical assembly functions as a sensing element or detector tip to detect analytes attached to an end thereof. The detector unit detects interference signals produced by interfering light waves reflected from the optical assembly. The light source directs light into the optical assembly, which is reflected back to the detector unit through an optical coupling assembly. The coupling assembly includes a first optical waveguide or fiber that extends from the light source to the optical assembly, a second optical waveguide or fiber which carry reflected light from the optical assembly to the detector, and an optical coupler which optically couples the first optical waveguide and the second optical waveguide. In some embodiments, the coupling assembly includes a lens system constructed to focus a light beam on an upper surface of the optical assembly and to direct reflected interfering light from the optical assembly to the detector.

[0055] The light source can be a white light source, such as a light emitting diode (LED), that produces light over a broad spectrum, e.g., 400 nm or less to 700 nm or greater, typically over a spectral range of at least 100 nm. In some embodiments, the light source can be a plurality of sources each having a different characteristic wavelength, such as LEDs designed for light emission at different selected wavelengths in the visible light range. The same function can be achieved by a single light source, such as, white light source, with suitable filters for directing light with different selected wavelengths onto the optical assembly.

[0056] The detector may be a spectrometer, such as charge-coupled device (CCD), capable of recording the spectrum of the reflected interfering light from the optical assembly. In some embodiments, where the light source operates to direct different selected wavelengths onto the optical assembly, the detector may be a simple photodetector for recording light intensity at each of the different irradiating wavelengths. In certain embodiments, the detector may include one or more filters which allows detection of light intensity, for instance from a white-light source, at each of a plurality of selected wavelengths of the interference reflectance wave.

[0057] The first optical waveguide and / or the second optical waveguide may be in the form of a fiber optic bundle (FOB). As shown, the first optical waveguide includes several fiber optic elements surrounding a single fiber optic element of the second optical waveguide. This arrangement separates delivery of light from the light source from delivery of the reflected light from the optical assembly to the detector. It will be appreciated that other arrangements of the first optical waveguide and the second optical waveguide may allow for spatial separation of the light from the light source and the reflected light from the optical assembly. The separation of the light from the light source and the reflected light from the optical assembly may improve a signal to noise ratio (SNR) of the apparatus. In some embodiments, the first optical waveguide is a single fiber and the second fiber optical waveguide is formed of a plurality of fibers.

[0058] The distal tip of the fiber optic bundle can be aligned with a proximal end portion of the optical fiber when the BLI sensor is attached to the optical coupler. The BLI sensor may be fixedly attached to the optical coupler to align and maintain a position of the proximal end portion with respect to the tip.

[0059] The BLI sensor can include the optical fiber having a proximal end and a distal end. The proximal end and / or the distal end of the optical fiber may be polished ends. The BLI sensor can have an optical resonator having a first reflecting surface and a second reflecting surface distal of the first reflecting surface. The optical fiber is substantially transparent between the proximal end and distal end thereof. The optical resonator may be transparent between the first and second reflecting surfaces. The distance between the first and second reflecting surfaces defines a thickness of the optical resonator. The thickness of the optical resonator may be in a range of 50 nm to 5,000 nm, such as between 400 nm and 1,000 nm.

[0060] The second reflecting surface is formed of a layer of analyte-binding molecules which are effective to bind analyte molecules specifically and with high affinity. That is, the analyte and anti-analyte molecules are opposite members of a binding pair which can include, without limitations, antigen-antibody pairs, complementary nucleic acids, and receptor-binding agent pairs. In specific embodiments, the analyte and anti-analyte molecules are antigen-antibody pairs. For example, the analyte and anti-analyte molecules are antigens (such as PBP2a or PBP2) and antibodies (such as an anti-PBP2 antibody or an anti-PBP2a antibody), respectively.

[0061] The index of refraction of the optical fiber may be similar to that of the second reflecting surface so that light reflected from the second reflecting surface occurs predominantly from the layer formed by the analyte-binding molecules, rather than from the interface between the optical fiber and the analyte-binding molecules. Similarly, as analyte molecules bind to distal end portion of the optical assembly, light reflected from the distal end portion of the assembly occurs predominantly from the layer formed by the analyte-binding molecules and bound analyte, rather than from the interface region.

[0062] The first reflecting surface of the optical assembly is formed as a layer of transparent material having an index of refraction that is substantially different from that of the optical fiber, such that this layer functions to reflect a portion of the light directed onto the optical assembly.

[0063] The thickness of an analyte-binding layer disposed in the distal end portion of the optical element may be designed to optimize the overall sensitivity based on specific hardware and optical components. Conventional immobilization chemistries are used in chemically, such as covalently, attaching a layer of analyte-binding molecules to the lower surface of the optical element. For example, a variety of bifunctional reagents containing a siloxane group for chemical attachment to SiCh, and a hydroxyl, amine, carboxyl or other reaction group for attachment of biological molecules, such as proteins (such as antigens, antibodies), or nucleic acids. It is also well known to etch or otherwise treat glass or glass surfaces to increase the density of hydroxyl groups by which analyte-binding molecules can be bound. Where the optical fiber is formed of a polymer, such as polystyrene, a variety of methods are available for exposing available chemically active surface groups, such as amine, hydroxyl, and carboxyl groups.

[0064] In certain embodiments, the analyte-binding layer is formed under conditions in which a distal end surface of the optical fiber is densely coated, so that binding of analyte molecules to the layer forces a change in the thickness of the layer, rather than filling in the layer. The analytebinding layer can be either a monolayer or a multi-layer matrix.

[0065] The measurement of the presence or amount of analyte (such as Staphylococcus aureus,MRSA, or MSSA) to the optical assembly is enabled by the interference of reflected light beams from the two reflecting surfaces in the optical assembly. Specifically, as analyte molecules attach to or detach from the surface, the average thickness of the second reflecting surfaces changesaccordingly. Because the thickness of all other layers remains the same, the interference wave formed by the light waves reflected from the two surfaces is phase shifted in accordance with this thickness change.

[0066] Assuming that there are two reflected beams, the first beam is reflected from the first reflecting surface and the second beam is reflected from the analyte-binding molecules and bound analyte and the surrounding medium at the second reflecting surface. The conversion of the phase shifting to a thickness change of the bound analytes is well known in the art.

[0067] Once the presence or amount of Staphylococcus aureus (such as MRS A or MS SA) is detected in a sample obtained from a subject (for example by using the biosensor provided herein to which an anti-PBP antibody is immobilized thereon), one of ordinary skill in the art (such as a clinician) can readily select a procedure or a treatment regimen that is compatible with the detection results. For example, the method provided herein can further include selecting the subject as a candidate for treatment or procedure directed to Staphylococcus aureus infection, or administering to the subject such treatment or procedure. The treatment or the procedure directed to Staphylococcus aureus infection can include administering to the subject an antibiotic effective for reducing the amount of Staphylococcus aureus in the subject. The treatment or the procedure directed to Staphylococcus aureus infection can also include administering to the subject a medication or therapy for reducing symptoms or signs associated with Staphylococcus aureus infection, such as non-steroid anti-inflammatory drugs. Specifically, upon detection of the positive presence or positive amount of MRSA in a sample obtained from a subject (for example by using the biosensor provided herein to which an anti-PBP2a antibody is immobilized thereon), the method provided herein can further include selecting the subject as a candidate for treatment or procedure directed to MRSA infection, or administering to the subject such treatment or procedure.The treatment or the procedure directed to MRSA infection comprises one or more of administering an antibiotic effective for reducing the amount of MRSA in the subject (such as vancomycin, daptomycin, linezolid, sulfamethoxazole and trimethoprim, quinupristin- dalfopristin, clindamycin, and tigecycline) and implementing a procedure to prevent spread of MRSA infection to another subject (such as isolation of the subject and gown changing techniques). On the other hand, when MSSA is detected (for example by using the biosensor provided herein to which an anti-PBP2 antibody is immobilized thereon) but MRSA is not detected (for example by using the biosensor provided herein to which an anti-PBP2a antibody is immobilized thereon) in the sample, the method provided herein can include selecting the subject as a candidate for treatment or procedure directed to MSSA infection but not to MRSA infection, or administering to the subject such treatment or procedure (such as administering to the subject a P-lactam antibiotic such as nafcillin, oxacillin, flucloxacillin, cefazolin, or benzylpenicillin). One of ordinary skill in the art is aware of appropriate treatments for conditions discussed in relation the methods of detection described herein. In addition, because the methods and apparatus described herein can detect the amount of Staphylococcus aureus (such as MRSA or MSSA) in a sample, the methods and apparatus provided herein can be used to monitor a course of treatment and adjust the treatment accordingly. For example, reduced amount of Staphylococcus aureus (such as MRSA or MSSA) in samples obtained from the subject over time may indicate that the particular treatment is effective and Staphylococcus aureus (such as MRSA or MSSA) infection is improving, and the clinician may taper the treatment. On the other hand, increased amount of Staphylococcus aureus (such as MRSA or MSSA) in samples obtained from the subject over time may indicate that the particular treatment is not effective and Staphylococcus aureus (such as MRSA or MSSA) infection is worsening, and the clinician may consider modification or changeof the treatment regimen. The positive presence or amount of MRSA in samples from the subject in whom only MSSA was previously detected may indicate the acquisition of methicillin resistance of the MSSA or new infection with MRSA, and the clinician may initiate appropriate procedures and treatment for MRSA infection and prevention of spread of MRSA infection.

[0068] While several embodiments of the disclosure have been shown in the drawings, it is not intended that the disclosure be limited thereto, as it is intended that the disclosure be as broad in scope as the art will allow and that the specification be read likewise. Any combination of the above embodiments is also envisioned and is within the scope of the appended claims. Therefore, the above description should not be construed as limiting, but merely as exemplifications of particular embodiments. Those skilled in the art will envision other modifications within the scope of the claims appended hereto.

Claims

What is Claimed:

1. A method for detecting the presence or amount of Staphylococcus aureus in a sample, the method comprising: contacting the sample with a biosensor, the biosensor comprising a core component and an anti-penicillin-binding protein (PBP) antibody immobilized thereon, such that the Staphylococcus aureus in the sample binds the anti-PBP antibody of the biosensor; and detecting signals generated by binding of the Staphylococcus aureus to the biosensor, thereby detecting the presence or amount of Staphylococcus aureus in the sample.

2. The method of claim 1, wherein the anti-PBP antibody is an anti -penicillin-binding protein 2a (PBP2a) antibody, such that methicillin resistant Staphylococcus aureus (MRSA) in the sample binds to the anti-PBP2a antibody of the biosensor, and wherein the method comprises detecting signals generated by binding of the MRSA to the biosensor, thereby detecting the presence or amount of MRSA in the sample.

3. The method of claim 1, wherein the anti-PBP antibody is an anti -penicillin-binding protein 2 (PBP2) antibody, such that methicillin sensitive Staphylococcus aureus (MSSA) in the sample binds to the anti-PBP2 antibody of the biosensor, and wherein the method comprises detecting signals generated by binding of the MSSA to the biosensor, thereby detecting the presence or amount of MSSA in the sample.

4. The method of claim 2, wherein the anti-PBP2a antibody is a monoclonal anti-PBP2a antibody.

5. The method of claim 3, wherein the anti-PBP2 antibody is a monoclonal anti-PBP2 antibody.

6. The method of claim 1, wherein the method further comprises: contacting the biosensor-bound Staphylococcus aureus with a targeting molecule attached to a detectable label after contacting the sample with the biosensor, such that the targeting molecule binds to the biosensor-bound Staphylococcus aureus,' and detecting the signals, wherein the signals are signals of the detectable label generated by binding of the targeting molecule to the biosensor-bound Staphylococcus aureus.

7. The method of claim 6, wherein the targeting molecule is LysGH15.

8. The method of claim 6, wherein the detectable label is a fluorescent label.

9. The method of claim 1, wherein the biosensor is a bio-layer interferometry (BLI) biosensor.

10. The method of claim 1, wherein the signals are indicative of spectral shift measured using bio-layer interferometry (BLI).

11. The method of claim 10, wherein a positive spectral shift indicates binding of the Staphylococcus aureus to the biosensor.

12. The method of claim 1, wherein the signals in a plurality of samples are detected simultaneously.

13. The method of claim 12, wherein the plurality of samples are each placed in a plurality of wells in a plate, and the signals in the plurality of wells are detected in plate reader format.

14. The method of claim 1, wherein the method further comprises: detecting the amount of Staphylococcus aureus in the sample based on the signals generated by binding of the Staphylococcus aureus in the sample to the biosensor and a calibration curve of association between known amounts of Staphylococcus aureus and signals generated by each known amount of Staphylococcus aureus.

15. The method of claim 1, wherein the presence or amount of Staphylococcus aureus in the sample is detected within 30 minutes of contacting the sample with the biosensor.

16. The method of claim 1, wherein the sample is a biological sample obtained from a subject.

17. The method of claim 16, wherein the method further comprises selecting the subject as a candidate for, or administering to the subject, treatment or procedure directed to Staphylococcus aureus infection.

18. The method of claim 17, wherein the treatment or the procedure directed to Staphylococcus aureus infection comprises administering to the subject an antibiotic effective for reducing the amount of Staphylococcus aureus in the subj ect.

19. The method of claim 2, wherein the sample is a biological sample obtained from a subject, and wherein the method further comprises selecting the subject as a candidate for, or administeringto the subject, treatment or procedure directed to MRSA infection.

20. The method of claim 19, wherein the treatment or the procedure directed to MRSA infection comprises one or more of administering to the subject an antibiotic effective for reducing the amount of MRSA in the subject and implementing a procedure to prevent spread of MRSA infection to another subject.

21. A biosensor for detecting methicillin resistant Staphylococcus aureus (MRSA) in a sample, the biosensor comprising: a core component; and an anti-penicillin-binding protein 2a (PBP2a) antibody immobilized thereon, and the biosensor being configured to detect MRSA in the sample.

22. The biosensor of claim 21, wherein the biosensor is a bio-layer interferometry (BLI) biosensor.

23. A biosensor for detecting methicillin sensitive Staphylococcus aureus (MSSA) in a sample, the biosensor comprising: a core component; and an anti-penicillin-binding protein 2 (PBP2) antibody immobilized thereon, and the biosensor being configured to detect MSSA in the sample.

24. The biosensor of claim 23, wherein the biosensor is a bio-layer interferometry (BLI) biosensor.

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