Systems and methods for using ultraviolet light to enhance microbial detection and identification

Abstract

A system and method for enhanced detection and identification of microbes and microbe-associated substances, includes a pre-treatment device comprising one or more compartments configured to expose portions of a sample to different levels of ultraviolet (UV) light radiation; and a detection and identification module configured to identify at least one of microbes and microbe-associated substances that are present in the sample using one or more analytical techniques. The microbe-associated substances include at least one of genes, proteins, and chemical substances associated with microbes.

Description

Atty. Dock. No. 009040-001 US PCT PatentSYSTEMS AND METHODS FOR USING ULTRAVIOLET LIGHT TO ENHANCE MICROBIAL DETECTION AND IDENTIFICATIONCROSS REFERENCE TO RELATED DOCUMENTS

[0001] The present application claims benefit of priority to U.S. Provisional Patent Application Ser. No. 63 / 652,631 of WATERHOUSE, entitled "SYSTEMS AND METHODS FOR USING ULTRAVIOLET LIGHTTO ENHANCE MICROBIAL DETECTION AND IDENTIFICATION," filed on 28 May 2024, now pending, the entire disclosure of which is hereby incorporated by reference herein.BACKGROUND OF THE DISCLOSUREFIELD OF THE DISCLOSURE

[0002] The present disclosure generally relates to systems and methods for detection and identification of microbes, and more particularly to systems and methods for enhanced detection and identification of microbes, and the like, by employing ultraviolet (UV) light radiation, and the like.DISCUSSION OF THE BACKGROUND

[0003] In recent years, systems and methods for discovery and identification of microorganisms, and the like, have been developed. However, such systems and methods typically are lacking in effective detection and identification of a subset of the vast numbers of microbes present in samples, and the like, in an efficient and cost-effective manner.SUMMARY OF THE DISCLOSURE

[0004] Therefore, there is a need for a method and system that addresses the above and other problems. The above and other problems are addressed by the illustrative embodiments of the present disclosure, which provide systems and methods for enhanced detection and identification of microbes, and the like, by employing ultraviolet (UV) light radiation, and the like. In the illustrative embodiments of the present disclosure, an improved system and method for microbial detection, identification, and the like, includes recognition that ultraviolet light typically is known to decrease many microbes, and the like. By contrast, the novel system and method of the present disclosure advantageously employs UV radiation to increase the ability to detect and identify microbes, and the like, in a sample, and the like. Advantageously, both UV light pretreated and non-pre-treated portions of a sample are analyzed by various means (e.g., sequencing, polymerase chain reaction (PCR), spectroscopy), wherein results are combined toAtty. Dock. No. 009040-001 US PCT Patent enhance overall microbial detection from the sample. Such a novel system and method allows for certain UV radiation resistant microbes, and the like, to be detected, which are generally missed otherwise, particularly when they are present at low levels. Thus, the novel system and method is advantageous because radiation resistant microbes, and the like, are increasingly being found to be abundant and important in many contexts. Another advantage of such an approach is that new taxa can be detected and found to be relevant in a variety of situations. Thus, the libraries of genetic sequences can be advantageously expanded to more completely represent microbes present in living beings, the environment, and the like, leading to advances in detection, monitoring, and the like, of microbes for a variety of purposes, including the diagnosis, treatment of diseases, and the like.

[0005] Accordingly, in illustrative aspects of the present disclosure there is provided a system and method for enhanced detection and identification of microbes and microbe- associated substances, including a pre-treatment device comprising one or more compartments configured to expose portions of a sample to different levels of ultraviolet (UV) light radiation; and a detection and identification module configured to identify at least one of microbes and microbe-associated substances that are present in the sample using one or more analytical techniques. The microbe-associated substances include at least one of genes, proteins, and chemical substances associated with microbes.

[0006] The system and method further include a sample preparation module configured to prepare the sample for analysis; and an automated system configured to integrate the sample preparation module, the pre-treatment device, and the detection and identification module into a single device. The automated system is configured to combine portions of the sample that have undergone different pre-treatments into a single sample.

[0007] The system and method further include an automated cleaning and sterilization system configured to clean the compartments of the pre-treatment device between samples.

[0008] The detection and identification module is configured to perform one or more identification techniques including at least one of nucleic acid sequencing, polymerase chain reaction (PCR), and spectroscopic analysis on the sample, and the detection and identification module is configured to detect antibiotic resistance genes in the microbes present in the sample.Atty. Dock. No. 009040-001 US PCT Patent

[0009] The pre-treatment device is configured to emit UV light at multiple wavelengths, and the pre-treatment device is configured to set parameters of the UV light exposure in the one or more compartments, including at least one of exposure time, light intensity, and wavelength.

[0010] The system and method further include a culturing module configured to increase an abundance of microbes in the sample.

[0011] The pre-treatment device comprises a chamber configured to receive a test tube and to rotate the test tube during UV light exposure.

[0012] The test tube is rotated about a longitudinal axis thereof.

[0013] The test tube is rotated in an orbital path within the chamber.

[0014] The chamber comprises ultraviolet (UV) light sources mounted on two or more interior walls of the chamber.

[0015] Still other aspects, features, and advantages of the present disclosure are readily apparent from the following detailed description, by illustrating a number of illustrative embodiments and implementations, including the best mode, contemplated for carrying out the present disclosure. The present disclosure is also capable of other and different embodiments, and its several details can be modified in various respects, all without departing from the spirit and scope of the present disclosure. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The embodiments of the present disclosure are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which like reference numerals refer to similar elements and in which:

[0017] FIG. 1 is a diagram for illustrating systems and methods for enhanced detection and identification of microbes, and the like, by employing ultraviolet (UV) light radiation, and the like;

[0018] FIG. 2 is a diagram for illustrating detection and identification of microbes from a blood sample from a human to determine treatment employing the systems and methods of FIG. 1;Atty. Dock. No. 009040-001 US PCT Patent

[0019] FIG. 3 is a diagram for illustrating a device including a UV light section and a sample plate section that can be employed in the systems and methods of FIGs. 1-2;

[0020] FIG. 4 is a diagram for illustrating a muti-compartment device based on the device of FIG. 3;

[0021] FIG. 5 is a diagram for illustrating a side view of a device having a hinge and two compartments based on the devices of FIGs. 3-4 and 7-9;

[0022] FIG. 6 is a diagram for illustrating a microbe detection and analysis system employing the systems and methods of FIGs. 1-5 and 7-9;

[0023] FIG. 7 is a diagram for illustrating another muti-compartment device based on the devices of FIGs. 3-4;

[0024] FIG. 8 is a diagram for illustrating compartment device usable alone or in the embodiments of FIGs. 1-7; and

[0025] FIG. 9 is a diagram for illustrating another compartment device usable alone or in the embodiments of FIGs. 1-7.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present disclosure includes recognition that ever since the discovery of microorganisms, after the invention of the microscope, the perfection of the culturing, detection and identification of microbes has been pursued. The present disclosure recognizes that despite continual advances in methodologies, such as culturing approaches, DNA and / or RNA sequencing, spectroscopy, and the like, it is still desirable to improve methodologies for detection, identification, and the like, of a vast numbers of microbes present in samples, particularly when the microbes are slow growing, low in abundance or when there is some characteristic of the sample that impedes their detection, identification, and the like (e.g., presence of high levels of host nucleic acids, due to other characteristics of the sample, etc.).

[0027] Although microbial detection and identification has greatly improved and is satisfactory in many circumstances, there have been long-standing problems with detecting slower growing and / or lower abundance microbes accurately and efficiently. For example, a recent study (see, e.g., Isola et al. Hydrocarbon-Contaminated Sites: Is There Something More Than Exophiala xenobiotica? New Insights into Black Fungal Diversity Using the Long ColdAtty. Dock. No. 009040-001 US PCT PatentIncubation Method. J. Fungi 2021, 7, 817 available on the world wide web at doi.org / 10.3390 / jof7100817) compared different methods of detecting black fungi and compared several different culturing methods using different culturing media and different environmental conditions, and the method that was determined to be most effective involved prolonged incubation to detect the fungi. Waiting weeks to months for results would be impractical for most use cases, so improved methods would be very useful. In some cases, the presence of faster growing species can be detrimental to the ability to detect slower growing species, especially if they are low in abundance.

[0028] The present disclosure includes recognition that microbes can be particularly hard to detect when they are present at low levels, and the results of identification techniques are often less certain. The present disclosure includes recognition that distinguishing microbial sequences from the background of animal or human nucleic acid sequences is problematic and complicates such a process. The present disclosure includes recognition of the difficulty in distinguishing low levels of microbes present in a sample versus contaminant microbes introduced during the collection, sample preparation, DNA and / or RNA extraction, identification steps, and the like. The above and other problems are advantageously addressed by the methods and systems of the present disclosure, and as will be further described.

[0029] Referring now to the drawings, wherein like reference numerals designate identical or corresponding parts throughout the several views, and more particularly to FIG. 1 thereof, there is shown a diagram for illustrating systems and methods 100 for enhanced detection and identification of microbes, and the like, by employing ultraviolet (UV) light radiation, and the like.

[0030] In FIG. 1, the systems and methods 100 can include an entity 102 (e.g., an animal, plant, human being, fungi, etc.) for which microbial identification process is to be applied. A sample 104 is obtained and then prepared at 106 in any suitable manner or left as is, as appropriate for the sample type, as known to those of ordinary skill in the relevant art(s) (e.g., filtering, adding sterile water or anticoagulant, centrifuging, etc.). Advantageously, a pretreatment step 108 is performed in which the sample 104 or portions thereof are exposed to ultraviolet (UV) light radiation of one or more wavelengths for a given length of time. An optional step 110 can include allowing any microbes in the sample 104 time to increase by employing culturing techniques, and the like, with orwithout adding culture media, various other substancesAtty. Dock. No. 009040-001 US PCT Patent or changing the condition of the sample in a suitable way (e.g., changing the temperature of the sample), as known to those of ordinary skill in the relevant art(s). At step 112, detection, identification, and the like, of any microbes present is performed. Such detection, identification, and the like, at step 112, for example, can include DNA and / or RNA extraction (e.g., done manually with a kit or through an automated method), specific procedures performed to prepare the sample for spectroscopic analysis through various methods, such as matrix assisted laser desorption / ionization - time of flight (MALD-TOF), Raman spectroscopy, and the like, as known to those of ordinary skill in the relevant art(s). Next, appropriate action(s) are taken at step 114 upon the identification, such as diagnosis, treatment, and the like.

[0031] FIG. 2 is a diagram for illustrating detection and identification of microbes from a blood sample from a human to determine treatment employing the systems and methods 100 of FIG. 1. In FIG. 2, beginning with a human being 202 for which an analysis of blood microbes is desired, a blood sample in a test tube 204 is obtained via venipuncture, and the like, and then prepared by adding an anticoagulant at step 206. The blood sample with the added anticoagulant 206 or a portion thereof is placed in a device 208 that accomplishes a pre-treatment with ultraviolet (UV) radiation, and the like. An optional step 210 is to allow any microbes in the prepared blood sample after it has gone through step 208 time to increase by culturing, and the like, in an incubator 210, and the like, with or without providing other substances, such as culture media, as known by persons skilled in the art. At step 212, detection, identification, and the like, of any microbes present in the blood sample taken from the previous step 208 or 210 is performed. Such detection, identification, and the like, at step 212, for example, can include DNA and / or RNA extraction (e.g., done manually with a kit or through an automated method), specific procedures performed to prepare the sample for spectroscopic analysis through various methods, such as matrix assisted laser desorption / ionization - time of flight (MALD-TOF), Raman spectroscopy, and the like, as known to those of ordinary skill in the relevant art(s). At step 214, depending on the results of step 212, antimicrobial treatment, and the like, can be instituted.

[0032] FIG. 3 is a diagram for illustrating a device 108 including a UV light section 302 and a sample plate section 304 that can be employed in the systems and methods of FIGs. 1-2. In FIG. 3, the device 108, including the UV light / cover section 302, can be configured to have one or more compartments in the sample plate section 304 into which one or more blood samples, andAtty. Dock. No. 009040-001 US PCT Patent the like, can be placed. The top part of the device 108 includes the UV light 302 placed over the plate 304 section on which the samples are placed.

[0033] FIG. 4 is a diagram for illustrating a multi-compartment device 108 based on the device of FIG. 3. In FIG. 4, the multi-compartment device 108 includes the top part 302 having one or more UV light generation components 402, 406 and 408 (e.g., UV1, UV2 ... UVn) of the types known to those of ordinary skill in the relevant art(s) that can generate ultraviolet light of various wavelengths, and the like. One or more blank components 404 that include no UV components or a component that generates ordinary visible light can be provided. Walls or dividers 416 extending from the top part 302 to the bottom part 304 form compartments 410 (e.g., Cl, C2, C4 ... Cn) over the sample section 412.

[0034] The top section 302 can include light sources of various types (e.g., ultraviolet, visible light, etc.) with options configured to adjust settings so that light is not turned on during a pre-treatment phase, and the like, so as to allow inclusion of a portion of the sample that is not pre-treated with UV light to be analyzed, and to provide flexibility in exposure time used for different compartments 410. Advantageously, such a configuration can be used to maximize the detection of taxa, and the like, with a low level of UV tolerance, as well as taxa with higher levels of UV tolerance. The wavelengths employed can be configured to vary throughout an entire UV range, with a UV light with about a 240 to 280 nm wavelength being advantageous. The length of time for the pre-treatment and the distance of the light from the sample 412 can be configured to vary depending on the application, and the like, advantageously, with about a 10 second pretreatment with a UVC light of about 250 nm wavelength at about a distance of 1 inch from the sample 412, and the like.

[0035] Just below the light section 302, the section 410 includes the series of compartments Cl to Cn created by the dividers 416. Advantageously, the dividers 416 can fit into slots 418 to help reduce the intrusion of light from one compartment Cl to Cn into another. Advantageously, pre-treatment types can be kept separate, thus increasing the ability to detect more microbes. Wells 414 are provided on the sample plate 412 for holding the samples (e.g., blood, plant / animal tissue, water, etc.). Advantageously, the wells 414 maintain the samples in one area, so that the samples do not contaminate the dividers, other parts of the device 108, and the like. Advantageously, the device can be made of any suitable material (e.g., plastic, metal, ceramic, etc.). Parts that border the compartments Cl to Cn preferably allow little to noAtty. Dock. No. 009040-001 US PCT Patent transmission of light through (e.g., metals such as stainless steel, aluminum or ceramic, opaque glass or opaque plastic, etc.). Parts that do not border the compartments can be of made of many suitable materials (e.g., plastic, metal, glass, ceramic, etc.), and need not to be limited to those that do not transmit light therethrough. Parts that could potentially come into contact with the sample, can preferably be of the type that are able to be sterilized with known methods (e.g., autoclaving, sterilizing chemicals, etc.).

[0036] FIG. 5 is a diagram for illustrating a side view of a device 108 having a hinge 502 and two compartments 504 and 506 based on the devices of FIGs. 3-4 and 7-9. In FIG. 5, the UV light section 302 is shown in a vertical orientation, and the sample plate 304 is shown in a horizontal orientation with the hinge 502 connecting the sections 302 and 304. The divider 416 creates two compartments when the top part 302 is lowered to a horizontal position over the sample plate 304. The sample plate 412 can include the wells 414 for the samples, as shown in FIG. 4. The hinge 502 can be of any suitable configuration, as is known to those of ordinary skill in the relevant art(s).

[0037] FIG. 6 is a diagram for illustrating a microbe detection and analysis system 600 employing the systems and methods of FIGs. 1-5 and 7-9. In FIG. 6, the sample plate 304 can be slid at 604 into a microbe detection and analysis sub-system 602. The UV light section 302 can be formed in a top part of detection and analysis sub-system 602 that allows the sample plate 304 having a sample to be detected slid into the detection and analysis sub-system 602 with UV light selectively controlled by settings in the UV light section 302 to emit UV light or not emit UV light. Dividers (not shown) inside the sub-system 602 can be configured to form one or more separate compartments, as described in FIG. 4. A controlling switch (not shown) can be included in the sub-system 602 to cause the dividers to move downward to form the one or more compartments, after the sample plate 304 is slid in, as described in FIG. 4. The slots 414, as described in FIG. 4, can also be included to minimize light from one compartment affecting adjacent compartments, as previously described.

[0038] FIG. 7 is a diagram for illustrating another muti-compartment device 108 based on the devices of FIGs. 3-4. In FIG. 7, the multi-compartment device 108 functions in a in a similar manner as the device shown and described in FIG. 4. As with the earlier embodiment, the device includes a top portion 302 and a bottom sample plate portion 304. The top portion 302 comprises a series of compartments Cl through Cn, each of which is associated with a different lightAtty. Dock. No. 009040-001 US PCT Patent treatment condition, such as UV1 402, a blank or control 404, UV2 406, and Uvn 408. Adjacent compartments are separated by divider walls 416 that reduce or eliminate light bleed between zones. In this embodiment, the sample plate 304 is adapted to accommodate horizontally oriented test tubes instead of flat-bottomed sample wells. Specifically, the surface 412 of the plate includes a series of circular indentations 702, each aligned with a corresponding compartment (Cl through Cn), and configured to receive and stabilize a test tube laid horizontally. The indentations 702 replace or supplement the individual wells 414 of the previous embodiment. Divider elements 418 between adjacent indentations assist in maintaining spatial isolation and physical stabilization. Such configuration advantageously allows for test tubes to be inserted directly into the plate and exposed to compartment-specific UV or control conditions.

[0039] FIG. 8 is a diagram for illustrating compartment device Cl-Cn usable alone or in the embodiments of FIGs. 1-7. In FIG. 8, a test tube treatment chamber is provided that can correspond to, for example, boxed-shaped compartments Cl-Cn, as shown in FIGs. 4 and 7. The embodiment of FIG. 8 includes a motorized rotation mechanism for uniformly exposing a test tube 808 to ultraviolet (UV) radiation from multiple sides. A motor 802 is mounted to the exterior of a wall 810 of the chamber and is operatively connected to an axle 804 that extends into the interior of the chamber. The axle 804 is coupled to a test tube holder 806 configured to support and rotate the test tube 808 along its longitudinal axis. The box-shaped chamber is defined by a set of walls including a left wall 810, right wall 812, top wall 814, bottom wall 816, front wall 818, and back wall 820. One or more of these walls can be removable or hinged to allow insertion and removal of the test tube 808. In the illustrated embodiment, each of the walls is optionally equipped with one or more ultraviolet (UV) light sources. The UV light sources may be implemented using either conventional UV emitters or ultraviolet light-emitting diodes (UV LEDs), and the like. For example, a UV light source 822 is mounted on the left wall 810; a UV light source 824 on the right wall 812; a UV light source 826 on the top wall 814; and a UV light source 828 on the bottom wall 816. Additional UV light sources can include a UV light source 830 on the front wall 818 and a UV light source 832 on the back wall 820.

[0040] During operation, the motor 802 rotates the test tube 808 via the axle 804 and holder 806, thereby enabling even exposure of the tube's contents to UV radiation. Such configuration advantageously enhances irradiation efficacy, particularly when sample opacity or blood density might otherwise limit light penetration. Any of the walls 810, 812, 814, 816, 818,Atty. Dock. No. 009040-001 US PCT Patent or 820 can include one or more UV light sources and can optionally be configured as removable lids or panels. The embodiment of FIG. 8 can be implemented as a standalone device or as part of a multi-compartment system such as that shown in FIGs. 4 and 7.

[0041] FIG. 9 is a diagram for illustrating another compartment device Cl-Cn usable alone or in the embodiments of FIGs. 1-7. In FIG. 9, another embodiment of a test tube treatment chamber shares structural elements with the embodiment shown in FIG. 8, but differs in that the test tube 808 is rotated axially, for example, about a longitudinal axis thereof. The chamber again includes a motor 802 mounted to the exterior of a wall 810, with an axle 804 extending into the interior of the chamber. In this embodiment, the axle 804 is connected to an axial test tube holder 906, which is configured to securely engage and rotate the test tube 808 about its longitudinal axis, rather than orbitally. As with FIG. 8, the chamber is defined by a left wall 810, right wall 812, top wall 814, bottom wall 816, front wall 818, and back wall 820. One or more of these walls can be removable or hinged to allow access to the interior. Each wall can include one or more ultraviolet (UV) light sources, which can be implemented using either conventional UV lamps or UV light-emitting diodes (LEDs), and the like. Specifically, the chamber includes a UV light source 822 on the left wall 810, a UV light source 824 on the right wall 812, a UV light source 826 on the top wall 814, a UV light source 828 on the bottom wall 816, a UV light source 830 on the front wall 818, and a UV light source 832 on the back wall 820.

[0042] During operation, the motor 802 drives rotation of the test tube 808 along its longitudinal axis via the axial holder 906 and axle 804. Such axial rotation enhances uniform UV exposure of the test tube's contents, particularly in applications where even irradiation of the entire volume is desired. The embodiment of FIG. 9 can be implemented as a single-chamber device or as a component of a multi-compartment system such as the one shown in FIGs. 4 and 7.

[0043] In some embodiments, the rotation mechanism of FIGs. 8-9 can be configured for manual operation rather than motorized control. For example, a hand-powered or mechanically actuated crank system can be employed to rotate the sample holder in environments where electrical power is unavailable or unreliable. Such configurations can be advantageous for field use or in low-resource settings, and the like.Atty. Dock. No. 009040-001 US PCT Patent

[0044] In certain embodiments, the treatment chamber need not be fully enclosed, particularly when operated in controlled environments, such as unoccupied rooms or with shielding protocols, and the like. However, when ultraviolet (UV) radiation is used, it may be preferable to implement shielding or compartmentalization to prevent human exposure and to avoid unintended irradiation of adjacent samples, and the like.

[0045] The length of ultraviolet (UV) light exposure in the embodiments of FIGs. 1-9 can vary depending on various factors, for example, including the intensity and wavelength of the UV source, the distance between the light and the test tube, and the optical properties of the test tube material, and the like. In some applications, longer exposure times may be desirable when samples are enclosed in test tubes, to advantageously penetrate the tube walls. Suitable exposure durations can range from approximately 20 seconds to five minutes, and the like, although other durations can be used as appropriate for a given configuration. Such values are non-limiting and can be adjusted based on system parameters and desired outcomes, and the like.

[0046] Although the present disclosure can be described in terms of microbial identification, and the like, microbial detection can be of most interest, wherein identification of the microbes need not be performed (e.g., monitoring for contamination from sewage or manufacturing activities), wherein any microbes being present or being present above a certain threshold level would be of interest. Accordingly, the present disclosure includes systems and methods designed for microbial detection, with or without quantification or identification and the like.

[0047] Table 1 shows illustrative results for two human beings, using the systems and methods described herein. For half of the sample a mercury vapor UVC device (e.g., Green Piece Germreaper, etc.) emitting light with a wavelength of approximately 250 nm was turned on when it was held above the sample for 10 seconds at a distance of about 1 inch and for the other half of the sample, the light was held above it but was not turned on. The DNA and / or RNA extraction was performed manually, in this case, with a Qiagen kit. The procedures were performed under biosafety level 2 conditions under aseptic conditions. The analysis and identification were performed via an Illumina sequencer and using a QIIME2 bioinformatics program.Atty. Dock. No. 009040-001 US PCT Patent

[0048] Table 1. Number of reads of a subset of the fungal taxa detected when the blood samples were UVC pre-treated for 10 seconds versus not UVC pre-treated, for two individuals, a 64-year-old female and a blood donor of unknown age and gender.

[0049] It is expected that UV light will kill many microbes. However, the use of UV light pre-treatment as part of systems and methods to increase overall microbial detection is novel and consequential. The discovery that came out of this innovative step of the UV light pretreatment being included is that some microbes will not be detected without the UV pretreatment, at least in some circumstances (see, e.g., Table 1). The data of Table 1 illustrates how a device using the systems and methods of this disclosure, which combines UV and non-UV treated portions of the sample, can yield more information about the microbes present than would have been revealed otherwise.

[0050] The reason it is advantageous to use UV light pre-treatment for the detection of some microbes could be related to something else present in the sample that is affected by the UV light. One possibility is that UV light sensitive microbes and / or immune system components (e.g., antibodies, complement, T-cells, phagosomes, etc.) are somehow interfering with the detection of some UV light resistant microbes. By adding the UV light pre-treatment, such interference is advantageously removed, thus leading to relatively improved detection of certain UVC resistant microbes. Notably, the data of Table 1 was obtained without a culturing step. Adding the culturing step would likely lead to the detection of more microbes, since very lowAtty. Dock. No. 009040-001 US PCT Patent abundance microbes would have had the chance to increase to levels that allowed the determination of their identity with certainty.

[0051] In further illustrative embodiments, additional nucleic acid sequencing, and the like, can be employed, advantageously, to allow the detection of antibiotic resistance genes, and the like, in the microbes, and the like, detected by the systems and methods described herein, as will be appreciated by those of ordinary skill in the relevant art(s) based on the teachings of the present disclosure. Another embodiment employs the option to combine parts of samples that have undergone different pre-treatments (e.g., different UV exposure time, intensities and wavelengths or other variations of sample preparation or culturing, etc.) into one sample for analysis to advantageously save time, resources, and the like. Another embodiment employs the ability to alter settings in a single device to achieve different sample treatments that vary in ways described previously (e.g., exposure time, intensities, wavelengths, sample preparation, culturing, etc.), which are determined to be most advantageous for different purposes. This disclosure also includes the incorporation of other technologies as part of the methods and systems described herein, such as microfluidics, nanopore sequencing and long read sequencing, and the like, as known to persons skilled in the relevant art(s).

[0052] In some embodiments, the detection and identification of microbes may further include detection and identification of microbe-associated substances, such as microbial genes, proteins, enzymes, toxins, transfer RNA (tRNA), messenger RNA (mRNA), micro RNA (miRNA), or other chemical substances produced directly or indirectly as a result of microbial presence. For instance, in certain scenarios, the presence of a microbe may result in the production of a protein that catalyzes a reaction converting one substance into another, and such a conversion product may itself be of interest. Accordingly, the systems and methods described herein are not limited to detecting the microbes themselves but may also encompass the identification of microbial activity or influence based on associated molecular products or reaction intermediates. In further embodiments, the microbes may advantageously be inferred rather than detected and identified by the presence of one or more substances that are associated with microbes.

[0053] All the features of the illustrative systems and methods of FIGs. 1-9, as described herein, can be employed alone or in any suitable combination, and include automation of various steps, including cleaning, sterilization between samples, and the like, through a variety of suitableAtty. Dock. No. 009040-001 US PCT Patent technologies (e.g., robotics, Artificial Intelligence, etc.), as will be appreciated by those of ordinary skill in the relevant art(s).

[0054] All the features of the illustrative systems and methods of FIGs. 1-9, as described herein, can be employed alone or in any suitable combination, and include computer automation, as will be appreciated by those of ordinary skill in the relevant art(s).

[0055] Accordingly, the above-described devices and subsystems of the illustrative embodiments can include, for example, any suitable servers, workstations, PCs, laptop computers, PDAs, Internet appliances, handheld devices, cellular telephones, wireless devices, other devices, and the like, capable of performing the processes of the illustrative embodiments. The devices and subsystems of the illustrative embodiments can communicate with each other using any suitable protocol and can be implemented using one or more programmed computer systems or devices.

[0056] One or more interface mechanisms can be used with the illustrative embodiments, including, for example, Internet access, telecommunications in any suitable form (e.g., voice, modem, and the like), wireless communications media, and the like. For example, employed communications networks or links can include one or more wireless communications networks, cellular communications networks, G3 communications networks, Public Switched Telephone Network (PSTNs), Packet Data Networks (PDNs), the Internet, intranets, a combination thereof, and the like.

[0057] It is to be understood that the devices and subsystems of the illustrative embodiments are for illustrative purposes, as many variations of the specific hardware used to implement the illustrative embodiments are possible, as will be appreciated by those skilled in the relevant art(s). For example, the functionality of one or more of the devices and subsystems of the illustrative embodiments can be implemented via one or more programmed computer systems or devices.

[0058] To implement such variations as well as other variations, a single computer system can be programmed to perform the special purpose functions of one or more of the devices and subsystems of the illustrative embodiments. On the other hand, two or more programmed computer systems or devices can be substituted for any one of the devices and subsystems of the illustrative embodiments. Accordingly, principles and advantages of distributed processing, suchAtty. Dock. No. 009040-001 US PCT Patent as redundancy, replication, and the like, also can be implemented, as desired, to increase the robustness and performance of the devices and subsystems of the illustrative embodiments.

[0059] The devices and subsystems of the illustrative embodiments can store information relating to various processes described herein. This information can be stored in one or more memories, such as a hard disk, optical disk, magneto-optical disk, RAM, and the like, of the devices and subsystems of the illustrative embodiments. One or more databases of the devices and subsystems of the illustrative embodiments can store the information used to implement the illustrative embodiments of the present disclosure. The databases can be organized using data structures (e.g., records, tables, arrays, fields, graphs, trees, lists, and the like) included in one or more memories or storage devices listed herein. The processes described with respect to the illustrative embodiments can include appropriate data structures for storing data collected and / orgenerated by the processes of the devices and subsystems of the illustrative embodiments in one or more databases thereof.

[0060] All or a portion of the devices and subsystems of the illustrative embodiments can be conveniently implemented using one or more general purpose computer systems, microprocessors, digital signal processors, micro-controllers, and the like, programmed according to the teachings of the illustrative embodiments of the present disclosure, as will be appreciated by those skilled in the computer and software arts. Appropriate software can be readily prepared by programmers of ordinary skill based on the teachings of the illustrative embodiments, as will be appreciated by those skilled in the software art. Further, the devices and subsystems of the illustrative embodiments can be implemented on the World Wide Web. In addition, the devices and subsystems of the illustrative embodiments can be implemented by the preparation of application-specific integrated circuits or by interconnecting an appropriate network of conventional component circuits, as will be appreciated by those skilled in the electrical art(s). Thus, the illustrative embodiments are not limited to any specific combination of hardware circuitry and / or software.

[0061] Stored on any one or on a combination of computer readable media, the illustrative embodiments of the present disclosure can include software for controlling the devices and subsystems of the illustrative embodiments, for driving the devices and subsystems of the illustrative embodiments, for enabling the devices and subsystems of the illustrative embodiments to interact with a human user, and the like. Such software can include, but is notAtty. Dock. No. 009040-001 US PCT Patent limited to, device drivers, firmware, operating systems, development tools, applications software, and the like. Such computer readable media further can include the computer program product of an embodiment of the present disclosure for performing all or a portion (if processing is distributed) of the processing performed in implementing the disclosure. Computer code devices of the illustrative embodiments of the present disclosure can include any suitable interpretable or executable code mechanism, including but not limited to scripts, interpretable programs, dynamic link libraries (DLLs), Java classes and applets, complete executable programs, Common Object Request Broker Architecture (CORBA) objects, and the like. Moreover, parts of the processing of the illustrative embodiments of the present disclosure can be distributed for better performance, reliability, cost, and the like.

[0062] As stated above, the devices and subsystems of the illustrative embodiments can include computer readable medium or memories for holding instructions programmed according to the teachings of the present disclosure and for holding data structures, tables, records, and / or other data described herein. Computer readable medium can include any suitable medium that participates in providing instructions to a processor for execution. Such a medium can take many forms, including but not limited to, non-volatile media, volatile media, transmission media, and the like. Non-volatile media can include, for example, optical or magnetic disks, magneto-optical disks, and the like. Volatile media can include dynamic memories, and the like. Transmission media can include coaxial cables, copper wire, fiber optics, and the like. Transmission media also can take the form of acoustic, optical, electromagnetic waves, and the like, such as those generated during radio frequency (RF) communications, infrared (IR) data communications, and the like. Common forms of computer-readable media can include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, any other suitable magnetic medium, a CD-ROM, CDRW, DVD, any other suitable optical medium, punch cards, paper tape, optical mark sheets, any other suitable physical medium with patterns of holes or other optically recognizable indicia, a RAM, a PROM, an EPROM, a FLASH-EPROM, any other suitable memory chip or cartridge, a carrier wave or any other suitable medium from which a computer can read.

[0063] While the present disclosure has been described in connection with a number of illustrative embodiments, and implementations, the present disclosure is not so limited, but rather covers various modifications, and equivalent arrangements, which fall within the purview of the appended claims.

Claims

Atty. Dock. No. 009040-001 US PCT PatentWHAT IS CLAIMED IS:

1. A system for enhanced detection and identification of microbes and microbe-associated substances, the system comprising: a pre-treatment device comprising one or more compartments configured to expose portions of a sample to different levels of ultraviolet (UV) light radiation; and a detection and identification module configured to identify at least one of microbes and microbe-associated substances that are present in the sample using one or more analytical techniques, wherein the microbe-associated substances include at least one of genes, proteins, and chemical substances associated with microbes.

2. The system of claim 1, further comprising: a sample preparation module configured to prepare the sample for analysis; and an automated system configured to integrate the sample preparation module, the pretreatment device, and the detection and identification module into a single device, wherein the automated system is configured to combine portions of the sample that have undergone different pre-treatments into a single sample.

3. The system of claim 1, further comprising an automated cleaning and sterilization system configured to clean the compartments of the pre-treatment device between samples.

4. The system of claim 1, wherein the detection and identification module is configured to perform one or more identification techniques including at least one of nucleic acid sequencing, polymerase chain reaction (PCR), and spectroscopic analysis on the sample, and the detection and identification module is configured to detect antibiotic resistance genes in the microbes present in the sample.

5. The system of claim 1, wherein the pre-treatment device is configured to emit UV light at multiple wavelengths, and the pre-treatment device is configured to set parameters of the UV light exposure in the one or more compartments, including at least one of exposure time, light intensity, and wavelength.

6. The system of claim 1, further comprising a culturing module configured to increase an abundance of microbes in the sample.Atty. Dock. No. 009040-001 US PCT Patent7. The system of claim 1, wherein the pre-treatment device comprises a chamber configured to receive a test tube and to rotate the test tube during UV light exposure.

8. The system of claim 7, wherein the test tube is rotated about a longitudinal axis thereof.

9. The system of claim 7, wherein the test tube is rotated in an orbital path within the chamber.

10. The system of claim 7, wherein the chamber comprises ultraviolet (UV) light sources mounted on two or more interior walls of the chamber.

11. A method for enhanced detection and identification of microbes and microbe- associated substances, the method comprising: exposing portions of a sample to different levels of ultraviolet (UV) light radiation using a pre-treatment device comprising one or more compartments; and identifying at least one of microbes and microbe-associated substances that are present in the sample using a detection and identification module configured to perform one or more analytical techniques, wherein the microbe-associated substances include at least one of genes, proteins, and chemical substances associated with microbes.

12. The method of claim 11, further comprising: preparing the sample for analysis using a sample preparation module before exposing the sample to UV light radiation; integrating the sample preparation module, the pre-treatment device, and the detection and identification module into a single automated system; and combining with the automated system portions of the sample that have undergone different pre-treatments into a single sample.

13. The method of claim 11, further comprising cleaning and sterilizing the compartments of the pre-treatment device between samples using an automated cleaning and sterilization system.

14. The method of claim 11, further comprising: performing with the detection and identification module one or more identification techniques including at least one of nucleic acid sequencing, polymerase chain reaction (PCR), and spectroscopic analysis on the sample; andAtty. Dock. No. 009040-001 US PCT Patent detecting antibiotic resistance genes in the microbes present in the sample using the detection and identification module.

15. The method of claim 11, wherein the UV light pre-treatment includes exposing the sample to UV light at multiple wavelengths using the pre-treatment device, and further comprising setting with the pre-treatment device parameters of the UV light exposure in the one or more compartments, including at least one of exposure time, light intensity, and wavelength.

16. The method of claim 11, further comprising increasing an abundance of microbes in the sample using a culturing module before the step of identifying microbes.

17. The method of claim 11, wherein the pre-treatment comprises placing a portion of the sample in a test tube and rotating the test tube during ultraviolet (UV) light exposure within a chamber.

18. The method of claim 17, wherein the rotating comprises rotating the test tube about a longitudinal axis thereof.

19. The method of claim 17, wherein the rotating comprises moving the test tube in an orbital path within the chamber.

20. The method of claim 17, wherein the UV light is emitted from ultraviolet (UV) light sources mounted on two or more interior walls of the chamber.

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