Antimicrobial-modified comet assay

Abstract

The present disclosure relates to methods for assessing host DNA damage in samples exposed to infectious agents using antimicrobial-modified comet assay. The disclosure further relates to a modified comet assay configured to assess DNA damage while eliminating interference caused by infectious agents. The methods extend conventional comet assay use beyond chemical genotoxin screening to infection related damage profiling and quality controlled antimicrobial testing.

Description

[0001] ANTIMICROBIAL-MODIFIED COMET ASSAY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This PCT application claims priority to, and the benefit of, U. S. Provisional Patent Application No. 63 / 714,289, filed on October 31, 2024, which is incorporated by reference herein in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant 2R44ES030274-02 awarded by the National Institute of Environmental Health Sciences. The government has certain rights in the invention.

[0006] FIELD

[0007] Disclosed herein are methods of determining DNA damage and modifying analysis of genomic integrity in a sample using antimicrobial-modified comet assay.

[0008] BACKGROUND

[0009] Living organisms are continuously exposed to both endogenous and exogenous agents capable of damaging DNA. Such genotoxic insults can arise from metabolic byproducts, environmental chemicals, or infectious organisms. If not properly repaired, these lesions can compromise genomic integrity, leading to mutagenesis, cell death, oncogenic transformation, immunodeficiency, and accelerated aging. Although cells possess multiple DNA repair pathways, the efficiency of repair is not uniform across the genome. Actively transcribed regions are often repaired more rapidly than inactive or heterochromatic regions, resulting in strand-specific differences in lesion persistence and mutation frequency.

[0010] Various analytical methods have been developed to evaluate DNA damage and repair in different biological systems. Early approaches, such as Southern blot-based assays employing lesion-specific endonucleases, provided valuable insight into gene-specific repair but required large quantities of DNA and depended on the presence of specific restriction sites. Later, polymerase chain reaction (PCR)-based methods were introduced to detect DNA lesions that impede polymerase progression. Although these assays improved throughput, their sensitivity remained limited to detecting lesions within relatively short DNA fragments, and they required high doses of damaging agents to produce measurable signals. Consequently, existing methodologies have proven insufficient for detecting low-frequency DNA lesions occurring under biologically or environmentally relevant conditions.

[0011] In parallel, electrophoresis-based single-cell gel assays, commonly referred to as comet assays, have emerged as versatile tools for quantifying DNA strand breaks in individual cells. These assays allow visualization of DNA migration paterns reflecting the extent of genomic damage under neutral or alkaline conditions. While widely adopted in genotoxicity testing, their application to systems involving host-pathogen interactions remains technically challenging. In particular, challenges with accurate detection of host DNA damage and confounding interpretation of results still exists.

[0012] There remains a need in the art for simple, sensitive, and reproducible methods that enable evaluation of host DNA damage in the presence of infectious agents without interference from microbial contaminants. Such methods would facilitate improved study of infection-associated genomic instability, allow investigation of host-pathogen interactions at the molecular level, and support discovery of therapeutic strategies targeting DNA damage responses during infection.

[0013] SUMMARY

[0014] Disclosed herein are methods of determining DNA damage and modifying analysis of genomic integrity in a sample using antimicrobial-modified comet assay.

[0015] In one example, disclosed herein is a method of determining DNA damage in a biological sample, comprising:

[0016] obtaining a biological sample from a subject with prior exposure to an infectious agent in vivo or exposing the biological sample to an infectious agent in vitro, thereby obtaining an infected host cells;

[0017] embedding the infected host cells onto an agarose gel matrix slide; thereby obtaining a slide with embedded infected host cells;

[0018] lysing the embedded infected host cells on the slide to expose nuclear DNA; thereby obtaining a slide containing the nuclear DNA;

[0019] washing the slide containing the nuclear DNA;

[0020] applying an antimicrobial agent to the slide containing the nuclear DNA to disrupt activity of the infectious agent in the biological sample;

[0021] incubating the slide containing the nuclear DNA with the antimicrobial agent for about 5min to 120min at a temperature from 30 °C to 40 °C;

[0022] subjecting the slide containing the nuclear DNA to electrophoresis under alkaline or neutral conditions; staining the slide containing the nuclear DNA with a DNA specific fluorescent dye; capturing fluorescence microscopy images; and

[0023] analyzing the fluorescence microscopy images for tail length, tail DNA percentage, or Olive tail moment.

[0024] In some examples, the biological sample comprises tissue biopsy, blood, mucosal swab, lavage material, surgically excised tissue, or an in vitro cultured cell population.

[0025] In some examples, the antimicrobial agent halts replication, viability, or metabolic activity of the infectious agent without adding genotoxic stress to the nuclear DNA.

[0026] In some examples, the antimicrobial agent is selected from an antibacterial, an antifungal, an antiviral, or an antiparasitic compound.

[0027] In some examples, the antibacterial compound comprises ciprofloxacin, gentamicin, vancomycin, lysostaphin or chloramphenicol.

[0028] In some examples, the antifungal compound comprises amphotericin B, fluconazole, or echinocandin-class compounds.

[0029] In some examples, the antiviral compound comprises acyclovir, remdesivir, or interferon-based formulations.

[0030] In some examples, the antiparasitic compound comprises metronidazole, artemisinin, or ivermectin.

[0031] In some examples, the antimicrobial agent is lysostaphin.

[0032] In some examples, the lysostaphin comprises a concentration between 200 ng / µL and 500 ng / µL.

[0033] Some examples further comprise uninfected host cells as a negative control and host cells exposed to a known genotoxic agent as a positive control.

[0034] In some examples, the electrophoresis under alkaline conditions comprise a buffer with EDTA and a pH >12.3.

[0035] In some examples, the electrophoresis under neutral conditions comprise a buffer with a pH<10.

[0036] In one example, disclosed herein is a method of performing analysis of DNA damage in comet assays, comprising:

[0037] infecting a population of host cells with a bacteria, thereby obtaining infected host cells;

[0038] embedding the infected host cells in an agarose gel matrix; thereby obtaining embedded host cells;

[0039] lysing the embedded host cells to remove cellular membranes and soluble intracellular contents, thereby yielding nuclear DNA; terminating bacterial activity in the embedded host cells by contacting the embedded host cells with an antibacterial agent for about 5 min to 120 min at a temperature between 30 °C and 40 °C;

[0040] subjecting the embedded host cells incubated with the antibacterial agent to electrophoresis under alkaline or neutral conditions; and

[0041] staining the nuclear DNA with a fluorescent dye to visualize and analyze comet structures.

[0042] In some examples, the antibacterial agent reduces fluorescent signal derived from bacteria interfering with comet detection.

[0043] In some examples, the population of host cells comprise mammalian cell lines, human epithelial cells, macrophages, fibroblasts, endothelial cells, HeLa cell line, A549 cell line, RAW264.7 cell line, HaCat cell line or primary cell lines.

[0044] In some examples, the bacteria comprises Escherichia coli, Salmonella enterica, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Mycobacterium tuberculosis, or a genetically engineered derivative thereof.

[0045] In some examples, the antibacterial agent halts bacterial replication or metabolic activity, and preserves genomic damage state induced by infecting the population of host cells without introducing additional host DNA strand breaks.

[0046] In some examples, the antibacterial agent comprises ciprofloxacin, gentamicin, tetracycline, erythromycin, chloramphenicol, lysostaphin, bacteriophage-derived lysin, LL-37, melittin, silver ions, zinc ions, or copper ions.

[0047] In some examples, the antibacterial agent is lysostaphin. In some examples, the lysostaphin comprises a concentration between 200 ng / µL and 500 ng / µL.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several examples described below.

[0050] FIGS. 1 A and IB show that the presence of 5'. aureus creates background staining that interferes with the scoring of comets. FIG. 1 A shows the presence of S. aureus when co-cultured with HaCaT cells, creates a background “speckle.” FIG. 1B shows interference from bacteria in the background prevents quantification of DNA damage by comet assay analysis software (Comet IV). Comets were stained with SYBR Gold and imaged at 40X magnification.

[0051] FIGS. 2A and 2B show that the presence of Mycoplasma infection does not interfere with analysis of DNA damage in BE-M17 cells. FIG. 2A shows uninfected HaCaTs. FIG. 2B shows Mycoplasma infected BE-M17 show a faint background staining which did not prevent analysis by Comet IV software. Cells were stained with propidium iodide and imaged using fluorescence microscopy at 40X magnification. Use of propidium iodide has fallen out of favor due to its potential carcinogenicity and non-toxic alternatives, such as SYBR Gold are preferred.

[0052] FIGS. 3A-3D show lysostaphin treatment during the comet assay removes background staining interference from Staphylococcus aureus, allowing the scoring of DNA damage in eukaryotic cells. FIG. 3A is an illustration of the background staining caused by S. aureus in the alkaline comet assay, following infection of human keratinocytes cells; FIG. 3B is a representative image of a comet from (FIG. 3A), under analysis by Comet IV scoring software, showing that scoring is impossible due to the interference from the background staining; FIG. 3C is a representative comet assay image illustrating that treatment with 1 mg / mL lysostaphin removes the interfering background staining caused by S. aureus infected human keratinocytes; FIG. 3D is an illustration of successful scoring of the same cells as in (FIG. 3C), by Comet IV software.

[0053] FIG. 4 shows treatment with increasing concentrations of lysostaphin does not lead to the formation of artefactual DNA damage in S. aureus infected HaCaTs. HaCaT-containing comet assay gels not treated with lysostaphin (i.e., 0 ng / pL) and those treated 100 ng / pL, could not be scored due to the presence of the background fluorescence due to

[0054]

[0055] aureus. There were no significant differences in the levels of DNA damage in the HaCaT cells treated with 200-400 ng / µL lysostaphin (p > 0.05).

[0056] FIG. 5 shows treatment of HaCaTs with increasing concentrations of lysostaphin does not induce artefactual DNA damage. Artefactual DNA damage was not generated in HaCaT cells exposed to UVB (1 J / cm²), or control (CT; unirradiated) cells, irrespective of the concentration of lysostaphin used in the subsequent comet assay. None of the cells were infected with S. aureus.

[0057] FIG. 6 shows lysostaphin treatment allows scoring of S. aureus infected cells but does not increase DNA damage in untreated or heavily damaged cells. Representative images of comets taken using the Comet IV scoring software. UVB irradiated or unirradiated HaCaT cells were treated with 0 - 300 ng / pL lysostaphin and then processed according to the alkaline comet assay.

[0058] FIG. 7 shows treatment of HaCaT cells with lysostaphin does not induce artefactual damage to purines. Artefactual DNA damage to purines was not generated in HaCaT cells exposed to UVB (1 J / cm2), or control (unirradiated, 0 J / cm2) cells, as determined by the Fpg- modified alkaline comet assay. None of the cells were infected with S. aureus. The chosen lysostaphin concentration of 200 ng / pL (FIG. 6) was used for this experiment.

[0059] FIG. 8 shows overview of the steps involved in antibacterial -modified comet assay. DETAILED DESCRIPTION

[0060] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.

[0061] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0062] Terminology

[0063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific examples and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0064] The following definitions are provided for the full understanding of terms used in this specification.

[0065] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0066] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0067] Ranges can be expressed herein as from “about” one particular' value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to tire value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0068] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0069] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0070] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%’, or any amount of reduction in between as compared to native or control levels.

[0071] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0072] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0073] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0074] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0075] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder: preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0076] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Examples defined by each of these transition terms are within the scope of this disclosure.

[0077] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0078] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0079] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0080] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some examples, a desired therapeutic result is the control of type I diabetes. In some examples, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0081] As used herein, a “genotoxin” or a “genotoxic agent” is any agent that directly or indirectly damages DNA. These include compounds that arise in the cell naturally, or are directly derived from the environment, or indirectly from exposure to a agent in the environment (such as another compound or ionizing radiation). May also be called a mutagen.

[0082] “Quantitative long-DNA amplification” refers to the amplification of long (5,000 base pair) DNA templates to yield substantially only amplification products representing the intact (undamaged) complementary strands of the starting template sample.

[0083] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0084] The term “comprising”, and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various examples, the terms “consisting essentially of” and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific examples and are also disclosed.

[0085] The term “biological sample” as used herein means a sample of biological tissue or fluid. Such samples include, but are not limited to, tissue isolated from animals. Biological samples can also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin. Biological samples also include explants and primary and / or transformed cell cultures derived from patient tissues. A biological sample can be provided by removing a sample of cells from an animal but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and / or for another purpose), or by performing the methods as disclosed herein in vivo. Archival tissues, such as those having treatment or outcome history can also be used. In some examples, the sample is a genital tract sample.

[0086] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0087] The “fragments” or “functional fragments,” whether attached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.

[0088] The term “polypeptide” refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds. The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0089] In the present disclosure, “specific for” and “specificity” mean selective binding. Accordingly, an antimicrobial agent or infectious agent binds to a host cell or the respective microbe.

[0090] An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.

[0091] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd).

[0092] The term “antibodies” is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as long as they are chosen for their ability to interact with IFNT such that IFNT is inhibited from interacting with IFNT. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which the in vivo therapeutic and / or prophylactic activities are tested according to known clinical testing methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0093] “Radioimmunoassay” and “RIA” refers to in vitro assay techniques in which radioactive labelled antigen is mixed with unlabeled antigen (the test sample) and allowed to bind to an antibody or antigen binding fragment thereof. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the antibody is determined. The more antigen there is in the test sample the less radioactive antigen will bind to the antibody. A competitive binding assay may also be used with non-radioactive antigen, using antigen or an analogue linked to a reporter molecule. The reporter molecule may be a fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diamin oben zidine.

[0094] Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp.

[0095] 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991). ELISA typically uses an enzymatic reaction to convert substrates into products having a detectable signal (e.g., fluorescence). Each enzyme in the conjugate can convert hundreds of substrates into products, thereby amplifying the detectable signal and enhancing the sensitivity of the assay. ELISA assays are understood to include derivative and related methods, such as sandwich ELISA and microfluidic ELISA.

[0096] The term “mass spectroscopy” refers to a method of detecting polypeptides, peptides, or any protein fragments thereof, following digestion with trypsin or other proteolytic enzymes that cleave a protein into smaller fragments so that molecular weight can be determined and used to confirm identity of a target protein.

[0097] As used herein, “replication” means production of progeny by the infectious agent sufficient to increase infectious load in the biological sample prior to lysis. Evidence of replication is an increase over baseline in a load metric measured on an aliquot of the same sample before slide preparation, including without limitation plaque forming units per milliliter, TCID50, genome copy number by qPCR or RT qPCR, or colony forming units for non viral agents. Unless stated otherwise, an increase of at least 2 fold over a matched time zero aliquot under identical conditions indicates replication for purposes of this method.

[0098] Amplification products, as used herein are the duplicates of the template’s undamaged complementary DNA strands which are detected after the amplification process.

[0099] DNA damage, as used herein is a modification of a base or sugar moiety of DNA such that the replication of the DNA sequence is interrupted at the damage site.

[0100] As used herein, “gene-specific” means that the DNA involved is associated with a known gene.

[0101] As used herein, “genotoxin or genotoxic agent” refers to any agent that directly or indirectly damages DNA. These include compounds that arise in the cell naturally, or are directly derive from the environment, or indirectly from exposure to an agent in the environment (such as another compound or ionizing radiation). May also be called a mutagen.

[0102] As used herein, “genome integrity” means how intact and undamaged the full DNA of a cell is. High integrity means the DNA is mostly undamaged and stable. Low integrity means the DNA is nicked, cut, fragmented, oxidized, or crosslinked. Loss of genome integrity often means the cell was stressed, infected, poisoned, irradiated, or is dying.

[0103] “Host” refers to an organism or a cell in the organism being infected. For example, a human cell, a mouse cell, or a plant cell.

[0104] “Pathogen” means the infectious agent. This can be bacteria, virus, fungus, or parasite. As used herein, the “host pathogen interaction” refers to all of the biological events that happen when a pathogen infects the host cell. For example, including but not limited to a bacteria injecting toxins into the host cell, a pathogen activating inflammation, a host cell turning on defense genes, or a host cell DNA getting damaged either directly by the pathogen or indirectly by immune response such as reactive oxygen species.

[0105] “Host pathogen interaction signature” refers to a pattern of comet assay readouts obtained from host cells infected under defined conditions, optionally compared to uninfected controls. The signature can combine alkaline assay endpoints for strand breaks and alkali labile sites, enzyme modified endpoints for oxidized or alkylated bases, and neutral assay endpoints for double strand breaks. Bacterial contamination can create background fluorescence that prevents automated comet scoring. An antibacterial step such as lysostaphin treatment removes this interference while preserving host damage state.

[0106] Methods

[0107] Existing methodologies for evaluating host-pathogen interactions include numerous approaches to assess host cell responses to infection or toxin exposure. For instance, methods are known for measuring host cell DNA damage responses using immunostaining for phosphorylated histone gamma-H2AX or terminal deoxy nucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Additional techniques quantify oxidative stress or cell death markers, such as intracellular reactive oxygen species (ROS) detection by dichlorofluorescein assays, caspase activation assays, or colorimetric viability tests (e.g., MTT or resazurin reduction). These methods collectively assess cellular stress, apoptosis, or generalized DNA fragmentation after pathogen challenge.

[0108] Such art demonstrates host cellular injury in response to bacterial infection or toxin exposure. However, these established assays typically measure bulk or population-level indicators of apoptosis or oxidative stress, without providing spatially resolved, strand break-level information on individual nuclei. In contrast, the presently disclosed antibacterial-modified comet assay enables single-cell mapping of DNA strand breaks and alkali-labile sites in infected host cells. The method preserves genomic integrity readouts by controlling or halting bacterial replication before electrophoretic analysis, thereby eliminating post-harvest DNA damage artifacts and background interference from bacterial nucleic acids. This provides a direct, quantitative measure of infection-induced DNA damage within individual host cells under defined antibacterial arrest conditions.

[0109] Prior systems are primarily designed for antimicrobial efficacy testing or vaccine screening, focusing on pathogen outcome metrics rather than host cellular damage endpoints. The antibacterial-modified comet assay disclosed herein differs fundamentally from such platforms by assessing host genome integrity directly. In some examples, the assay measures both pathogen clearance and the preservation of host DNA stability, offering a dual-output approach that enables simultaneous evaluation of antibacterial efficacy and host genomic safety. This distinction positions the modified comet assay as a high-content tool for therapeutic screening, capable of identifying compounds that both eradicate infection and prevent host DNA damage

[0110] In one example, disclosed herein is a modified comet assay for assessing DNA damage in the context of host-pathogen interactions. Conventional comet assays are typically optimized for use with mammalian cells in isolation, providing a means to evaluate DNA strand breaks within a single genome system. However, infection-based systems introduce complexity due to the presence of two distinct genomic sources within the same biological mixture, namely the host ceil genome and the pathogen-derived DNA. This dual-genome environment poses specific challenges that render the standard comet assay insufficient without further adaptation.

[0111] Problems associated with the unmodified comet assay include continued bacterial replication after sample collection, which may alter the DNA damage profile and compromise result accuracy. In addition, bacterial genomic DNA can contribute to background signals or contaminate the electrophoretic readout, resulting in misinterpretation of host-specific DNA damage. For example, co-culture of Staphylococcus aureus with HaCaT cells produced extensive background fluorescence that interfered with comet quantification using Comet IV software, as observed by the formation of a granular “speckle” pattern on the gels. In contrast, infection with Mycoplasma species caused only faint background staining that did not interfere with comet analysis, consistent with its smaller genome size (-0.001 Mbp) relative to S. aureus (2.8 Mbp). Furthermore, the presence of viable S. aureus following sample harvest may continue to induce DNA lesions in host cells ex vivo, thereby confounding time-dependent analyses. In some cases, antibiotics used during sample handling, such as Gentamicin, are unsuitable because they can alter host cell metabolism or induce bystander DNA damage while failing to disrupt bacterial cell walls effectively.

[0112] In one example, disclosed herein is a method of determining DNA damage in a biological sample, comprising:

[0113] obtaining a biological sample from a subject with prior exposure to an infectious agent in vivo or exposing the biological sample to an infectious agent in vitro, thereby obtaining an infected host cells;

[0114] embedding the infected host cells onto an agarose gel matrix slide; thereby obtaining a slide with embedded infected host ceils;

[0115] lysing the embedded infected host cells on the slide to expose nuclear DNA; thereby obtaining a slide containing the nuclear DNA;

[0116] washing the slide containing the nuclear DNA;

[0117] applying an antimicrobial agent to the slide containing the nuclear DNA to disrupt activity of the infectious agent in the biological sample; incubating the slide containing the nuclear DNA with the antimicrobial agent for about 5min to 120min at a temperature between 30 °C and 40 °C;

[0118] subjecting the slide containing the nuclear DNA to electrophoresis under alkaline or neutral conditions;

[0119] staining the slide containing the nuclear DNA with a DNA specific fluorescent dye; capturing fluorescence microscopy images; and

[0120] analyzing the fluorescence microscopy images for tail length, tail DNA percentage, or Olive tail moment.

[0121] In some examples, the host cells / tissues can be obtained from a human or an animal. In some examples, the infectious agent activity can be terminated in the biological sample by contacting the biological sample with an antimicrobial agent under conditions that halt replication, viability, or metabolic activity of the infectious agent without inducing additional host DNA strand breaks, thereby preserving the genomic damage state of the host cells at the defined time point.

[0122] “Percent tail DNA”, as used herein refers to the percentage of total cellular DNA signal measured in the tail region of the comet after electrophoresis. It is widely recommended as the primary endpoint in TG 489 oriented studies and recent literature.

[0123] As used herein, the “tail length” is the distance from the head to the end of the tail along the axis of DNA migration, measured in micrometers. Tail length increases with damage until reaching a plateau at higher break frequencies. As used herein, the “tail moment” combines the extent of migration and the amount of DNA in the tail. Further, “olive tail moment” is defined as the product of the percentage of DNA in the tail and the distance between the centers of mass of the head and tail signals along the direction of electrophoresis.

[0124] In certain examples, the disclosed method detects and quantifies DNA damage in individual host cells following infection and antimicrobial treatment by visualizing electrophoretically separated nuclear DNA structures known as “comets.” The comet assay, also referred to as single-cell gel electrophoresis, enables direct detection of strand breaks and alkali-labile sites (ALS) in individual nuclei. Under alkaline conditions, the assay reveals single-strand breaks, alkali-labile sites, and incomplete excision repair sites, while under neutral conditions, the assay primarily reveals double-strand breaks.

[0125] In some examples, embedded host cells are lysed to remove cellular membranes, proteins, and cytoplasmic contents, yielding nucleoids composed of supercoiled nuclear DNA attached to the nuclear matrix. This nuclear DNA is subsequently subjected to electrophoresis in an alkaline buffer (for example, 300 mM NaOH, 1 mM Na₂EDTA, pH > 13) for a defined period, such as 20 minutes at approximately 1.19 V / cm. DNA fragments and relaxed loops migrate toward the anode, forming a characteristic comet tail extending from an intact “head.” The extent of DNA migration correlates with the number of DNA strand breaks, providing a quantitative measure of genomic integrity.

[0126] Following electrophoresis, the slides are neutralized (for example, in 0.4 M Tris-HCl, pH 7.5) and washed with distilled water to remove salts and residual detergents. In some examples, the embedded nucleoids are then stained with a fluorescent dye to visualize the comet structures. Suitable fluorescent dyes include propidium iodide (PI) at 2.5 µg / mL, SYBR Gold (1:10,000 dilution), SYBR Green I, or ethidium bromide. In one embodiment, slides are immersed in PI staining solution for approximately 20 minutes in the dark at room temperature, followed by washing in cold distilled water for 20 minutes. Stained slides are subsequently air-dried before analysis.

[0127] Visualization of comet structures is carried out using fluorescence microscopy, typically with excitation / emission filters matched to the chosen fluorophore (for example, 535 / 617 nm for propidium iodide or 495 / 537 nm for SYBR Gold). The resulting images display a circular or oval head corresponding to intact DNA and a diffuse tail representing migrated DNA fragments. Comets are analyzed quantitatively using automated image-analysis software such as Comet IV (Perceptive Instruments, UK) or comparable analytical platforms. Key parameters include tail length (the distance of DNA migration), tail intensity (the fraction of total DNA fluorescence present in the tail), and Olive tail moment (the product of tail length and tail DNA fraction). The percentage of DNA in the tail (% tail DNA) is a preferred quantitative index of strand break frequency, as it provides a direct proportional measure of DNA damage from 0% to 100%.

[0128] In further examples, slides may be processed in duplicate, with one set incubated in buffer and the other incubated with lesion-specific DNA repair enzymes, enabling the detection of oxidized purines, alkylated bases, or UV-induced photoproducts. For example, incorporation of human 8-oxoguanine DNA glycosylase (hOGGl) at 3.2 U / mL or bacterial Fpg enzyme enables detection of oxidized purines by converting these lesions into strand breaks detectable in the comet tail. Incubation may be carried out at 37 °C for 60 minutes before electrophoresis.

[0129] The fluorescent staining step also enables the differentiation of infected versus uninfected cell populations based on background signals. In the case of Mycoplasma-infected BE-M17 cells, elevated background propidium iodide staining in the gel matrix was observed, correlating with the presence of bacterial DNA fragments. Treatment of embedded gels with antimicrobial enzymes such as lysostaphin (200 ng / µL for 1 hour at 37 °C) or equivalent bactericidal reagents effectively removes bacterial interference without altering host DNA migration profiles.

[0130] In certain examples, the fluorescence-based detection step provides the endpoint for quantifying infection-induced DNA strand breaks, oxidized base lesions, or other structural DNA damage within individual host nuclei. The method enables correlation of nuclear damage parameters with infection state and antimicrobial treatment conditions, providing a single-cell level assessment of genome integrity.

[0131] In certain examples, the disclosed comet assay may be further modified by incorporating lesion-specific DNA repair enzymes that convert specific classes of base damage into detectable strand breaks. This approach, commonly referred to as the enzyme-modified comet assay, enables detection of oxidized, alkylated, or deaminated bases that do not directly produce strand scission under alkaline electrophoresis conditions.

[0132] In some examples, following lysis and optional antimicrobial treatment, the slides containing embedded nucleoids are washed in enzyme reaction buffer and incubated with a DNA glycosylase or endonuclease that recognizes and cleaves damaged bases. Non-limiting examples of suitable enzymes include formamidopyrimidine-DNA glycosylase (Fpg) for oxidized purines, human 8-oxoguanine DNA glycosylase (hOGGl) for 8-oxoguanine lesions, endonuclease III (Nth) for oxidized pyrimidines, or alkyladenine DNA glycosylase (AAG) for alkylated bases.

[0133] In one embodiment, slides are equilibrated in Fpg reaction buffer comprising 40 mM HEPES-KOH, 0.1 M KC1, 0.5 mM EDTA, and 0.2 mg / mL bovine serum albumin at pH 8.0, for 5 minutes at room temperature. The slides are then incubated with Fpg enzyme (for example, 3.2 U / mL, New England Biolabs) at 37 °C for 30 to 60 minutes in a humidified chamber. A parallel set of slides is treated with buffer alone to provide a baseline control. After incubation, the enzyme is inactivated by immersion of the slides in chilled electrophoresis buffer for 5 minutes prior to alkaline unwinding and electrophoresis.

[0134] Under these conditions, Fpg recognizes oxidized purine residues such as 8-oxoguanine and ring-opened purines (formamidopyrimidines), excises the damaged bases, and cleaves the DNA backbone at the resulting abasic site. The new strand breaks generated by this reaction are detected as increased comet tail DNA or elongated tail length relative to buffer- treated controls. The difference in % tail DNA between enzyme-treated and buffer-treated samples corresponds to the level of oxidized purine lesions in the sample.

[0135] In certain examples, the enzyme-modified comet assay is performed after antimicrobial treatment of infected host cell preparations to ensure that background bacterial DNA does not contribute to the enzymatic signal. For example, lysostaphin treatment of Staphylococcus aureus-infected HaCaT cells at 200 ng / µL for 1 hour at 37 °C effectively removes bacterial interference prior to Fpg incubation, enabling accurate quantification of host oxidative DNA damage. Experimental results show that lysostaphin treatment does not alter tail DNA percentage or oxidized purine readouts in either unirradiated or UVB-exposed control cells, confirming that antimicrobial enzyme exposure is compatible with Fpg-modified comet workflow's. In other examples, alternative lesion-specific enzymes may be used. Slides can be incubated with hOGGl to detect 8-oxoG residues, or with Nth to detect oxidized pyrimidine lesions. These enzymes are typically applied at concentrations of 1 to 4 U / mL for 30 to 60 minutes at 37 °C under gentle humidity. After incubation, electrophoresis and staining steps proceed as in the standard alkaline comet assay.

[0136] Visualization is performed by staining with a fluorescent nucleic acid dye such as SYBR Gold, SYBR Green I, or propidium iodide, followed by imaging under fluorescence microscopy. Comet scoring parameters, including % tail DNA, tail moment, and Olive tail moment, are measured using automated software (e.g., Comet IV Lite). Increases in these values after enzyme incubation relative to buffer controls indicate the presence of enzyme-recognizable lesions.

[0137] In further examples, the enzyme-modified comet assay may be used in conjunction with infection models to quantify infection-induced oxidative DNA damage. For instance, Mycoplasma-infected neuronal (BE-M17) cells have been shown to accumulate oxidative DNA lesions associated with impaired base excision repair, which are revealed by Fpg treatment as elevated % tail DNA relative to uninfected controls. The combination of antimicrobial sample control and lesion-specific enzyme detection provides a powerful method for distinguishing infection-induced oxidative injury from artifactual damage or continued bacterial metabolism post-harvest.

[0138] In certain examples, the method is further configured for quantitative correlation between enzyme-induced tail DNA increases and other vitality or metabolic metrics. For example, elevated oxidized purine levels detected by Fpg may correspond to reduced mitochondrial membrane potential or diminished proliferative recovery, linking specific oxidative genome injury to cellular vitality outcomes.

[0139] Accordingly, incorporation of lesion-specific repair enzymes into the antimicrobial-modified comet assay enables the detection of discrete classes of DNA lesions beyond strand breaks alone. The resulting data provide a high-resolution map of genome integrity and oxidative stress at the single-cell level, facilitating the identification of host damage signatures specific to bacterial infection, oxidative insult, or therapeutic intervention.

[0140] The improved staining and visualization workflow described herein ensures reproducible comet morphology, minimizes bacterial background interference, and allows reliable quantification of host genomic injury following infection and antimicrobial treatment.

[0141] In some examples, the biological sample comprises tissue biopsy, blood, mucosal swab, lavage material, surgically excised tissue, or an in vitro cultured cell population.

[0142] In some examples, the antimicrobial agent halts replication, viability, or metabolic activity of the infectious agent without adding genotoxic stress to the nuclear DNA. “Viability”, as used herein means the capacity of the infectious agent present in the biological sample to remain alive and capable of resuming growth or infectivity prior to lysis. Viability is indicated on a parallel aliquot by one or more of the following non limiting readouts: colony forming units on appropriate media, plaque formation on a permissive cell monolayer, membrane integrity by live or dead dye exclusion, or retention of infectivity following inoculation into permissive cells. Unless stated otherwise, viable means a detectable signal above vehicle control in at least one such assay, for example at least 10 percent of the untreated control level.

[0143] “Metabolic activity,” as used herein means energy dependent biochemical processes of the infectious agent present in the biological sample prior to lysis, independent of cell division. Metabolic activity is evidenced on a parallel aliquot by one or more of the following non limiting readouts: ATP or NADH levels, reduction of resazurin or tetrazolium dyes, oxygen consumption rate, extracellular acidification rate, incorporation of labeled precursors into macromolecules, or activity of an agent specific enzyme. Unless stated otherwise, metabolically active means the measured parameter is at least 20 percent of the untreated control level in a validated assay while a heat killed or chemically fixed control run in parallel is negative.

[0144] The application of the antimicrobial agent to the slide containing host nuclear DNA does not introduce additional DNA damage to the host DNA beyond that present in a matched vehicle control slide prepared from the same sample and processed in parallel. Absence of added genotoxic stress is demonstrated on the slide by no statistically significant increase in one or more primary DNA damage endpoints, including without limitation percent tail DNA, tail length, tail moment, or Olive tail moment, and by no increase in DNA damage markers measured on a matched cell aliquot prior to lysis, such as gamma H2AX or 53BP1 nuclear foci. In some examples, without adding genotoxic stress requires less than a 10 % change from vehicle control for any primary DNA damage endpoint at the antimicrobial exposure used.

[0145] For this method, disrupting activity of the infectious agent means reducing replication, viability, or metabolic activity of the infectious agent present in the sample prior to lysis such that residual components of the agent do not artificially increase host DNA damage during or after slide preparation. Disruption is evidenced by at least one of the following non limiting criteria measured on a parallel aliquot of the sample: a reduction of at least 90% in infectious load, a reduction of at least 90% in colony or plaque counts, or a reduction of at least 80 % in an accepted metabolic activity readout, while the antimicrobial agent satisfies the without adding genotoxic stress requirement on the host DNA slide processed in parallel. The following methods and assays are used to determine the viability of the host cells. In dye-exclusion assays (membrane integrity assays) the plasma membrane integrity is assessed via dyes that are excluded by intact membranes but penetrate cells with compromised membranes. For example, the classic trypan blue exclusion assay relies on manual cell counting viable cells exclude trypan blue and remain unstained, while non-viable cells take up the dye and appear blue under light microscopy. Fluorescent variants of this assay use membrane-impermeable nucleic acid stains such as Propidium iodide (PI) or Ethidium homodimer-1 (EthD-1). Cells with damaged membranes permit dye entry and become red fluorescent (for example EthD-1: Ex -528 nm, Em -617 nm) whereas live cells exclude the dye and remain unstained or fluoresce in the alternate channel. For example, using a commercial LIVE / DEAD kit (calcein AM + EthD-1), after staining at approximately 2 pM calcein AM and 4 pM EthD-1, live cells fluoresce green (calcein) and dead cells fluoresce red (EthD-1) after 30-45 minutes incubation at 20-25 °C. In the host-pathogen context, this assay permits rapid quantification of the fraction of host cells that retain membrane integrity following infection or treatment, thereby providing a measure of whether the host cell population remains viable or has lost integrity (possibly due to pathogen-induced cytotoxicity).

[0146] Fluorescence-based live / dead staining (esterase activity + membrane integrity) format builds on dye-exclusion by adding a live-cell-specific component. For example, Calcein AM is a non-fluorescent, cell-permeant ester which in viable cells is hydrolysed by intracellular esterases to produce green fluorescent calcein; because calcein is membrane-impermeant, it is retained in cells with intact membranes. The paired dye, EthD-1, enters cells with compromised membranes and binds nucleic acids to give red fluorescence. Typical parameters: e.g., 2 pM Calcein AM + 4 pM EthD-1 for 30-45 minutes incubation, then imaging under FITC (green) and RFP (red) filters. In infection models, this assay quantifies the live fraction of host cells that maintain esterase activity and membrane integrity, which complements metabolic activity and DNA damage readouts. A scenario may exist where metabolic assays show activity, but live / dead staining reveals a substantial sub-population with compromised membrane integrity (reduced vitality).

[0147] Mitochondrial membrane potential is a sensitive indicator of mitochondrial health and early apoptosis. Dyes such as JC-1 and Tetramethylrhodamine ethyl ester (TMRE) are widely used. For example, JC-1 accumulates in healthy mitochondria as aggregates with red fluorescence. When Δψm diminishes (e.g., in apoptotic or necrotic cells), JC-1 remains as monomers emitting green fluorescence. The ratio of red / green fluorescence serves as a quantitative metric of membrane potential. In one protocol, cells were stained with 2 pM JC-1 for 30 minutes at 37 °C (after e.g., 10 pg / mL CCCP for 1.5 h) and imaged using Ex 488 nm / Em 500-550 nm (green) and Ex 561 nm / Em 560-610 nm (red) channels. Within a host-pathogen assay, measuring mitochondrial membrane potential provides insight into sub-lethal injury where cells may appear alive but exhibit mitochondrial dysfunction (reduced vitality) potentially preceding DNA repair failure or apoptosis. Correlating Δψm data with comet assay results enables discrimination between cells that are functionally compromised yet still “alive” vs those with intact mitochondrial potential and genome integrity.

[0148] Vitality can also be assessed by the cell’s ability to resume proliferation or colony formation once infection or stress is resolved. In this assay format, host cell cultures are infected, then after a defined period the pathogen is cleared or neutralized (e.g., by antibacterial treatment), and the host cells are returned to normal culture conditions. The number, size, or growth rate of cell colonies or expansion of cell number over time is then measured (for example via manual counting, automated imaging, or incorporation of proliferation markers such as EdU or Ki-67). A reduced proliferative recovery indicates compromised vitality (despite possible metabolic activity) and may reflect underlying DNA damage, repair deficiency, or persistent stress. In the context of the comet assay, combining proliferation recovery measurement with DNA strand break quantification provides a longitudinal endpoint: cells that incur significant DNA damage may survive initial insult but fail to proliferate, indicating more serious functional impairment.

[0149] In certain examples, metabolic activity and vitality measurements are incorporated as complementary endpoints alongside the single-cell DNA strand break assessment provided by the antibacterial-modified comet assay. Specifically, parallel evaluation of (a) host cell metabolic activity (e.g., via MTT, resazurin reduction), (b) host cell vitality (via one or more of the assays described above), and (c) single-cell DNA strand break parameters (tail moment, percentage tail DNA) provides a multidimensional profile of infection-induced stress. After host cells are infected, at a defined time point an antibacterial arrest step is applied (to halt pathogen activity). Then, aliquots of the culture are: (i) subjected to the comet assay (to quantify strand breaks), (ii) stained for live / dead or mitochondrial potential to assess vitality, and (iii) tested for metabolic activity. The resulting datasets can then be analyzed to differentiate among: bacterially driven cytotoxicity (reduced vitality, high tail moment, low metabolic activity); energy depletion without genome fragmentation (reduced metabolic activity, normal tail moment, variable vitality); direct genotoxic injury to the host nucleus (elevated tail moment, possibly normal metabolic activity or vitality depending on repair status). Thus, vitality provides a functional link between DNA damage and the broader physiological outcome: a host cell may retain metabolic activity yet show reduced vitality if DNA repair systems are compromised or apoptosis is initiated. Quantitative linkage of vitality metrics with comet assay readouts enables discrimination between reversible DNA stress (vital but metabolically perturbed cells) and irreversible genomic injury (loss of vitality). This integrated approach enhances mechanistic interpretation of host-pathogen assays and supports screening applications where both cellular function and genomic stability are evaluated as outcomes of infection or therapeutic intervention. In some examples, the antimicrobial agent is selected from an antibacterial, an antifungal, an antiviral, or an antiparasitic compound.

[0150] In some examples, the antibacterial compound comprises ciprofloxacin, gentamicin, vancomycin, lysostaphin or chloramphenicol.

[0151] In some examples, the antifungal compound comprises amphotericin B, fluconazole, or echinocandin-class compounds.

[0152] In some examples, the antiviral compound comprises acyclovir, remdesivir, or interferon-based formulations.

[0153] In some examples, the antiparasitic compound comprises metronidazole, artemisinin, or ivermectin.

[0154] In some examples, the antimicrobial agent is lysostaphin.

[0155] In some examples, the lysostaphin comprises a concentration between 200 ng / µL and 500 ng / µL.

[0156] Some examples, further comprises uninfected host cells as a negative control and host cells exposed to a known genotoxic agent as a positive control.

[0157] In some examples, the electrophoresis under alkaline conditions comprise a buffer with EDTA and a pH >12.3.

[0158] In some examples, the electrophoresis under neutral conditions comprise a buffer with a pH<10.

[0159] In one example, disclosed herein is a method of performing analysis of DNA damage in comet assays, comprising:

[0160] infecting a population of host cells with a bacteria, thereby obtaining infected host cells,

[0161] embedding the infected host cells in an agarose gel matrix; thereby obtaining embedded host cells;

[0162] lysing the embedded host cells to remove cellular membranes and soluble intracellular contents, thereby yielding nuclear DNA;

[0163] terminating bacterial activity in the embedded host cells by contacting the embedded host cells with an antibacterial agent for about 5 min to 120 min at a temperature between 30 °C and 40 °C;

[0164] subjecting the embedded host cells incubated with the antibacterial agent to electrophoresis under alkaline or neutral conditions; and

[0165] staining the nuclear DNA with a fluorescent dye to visualize and analyze comet structures. In certain examples, an antibacterial agent is added to the infected culture immediately following harvest, thereby halting further bacterial replication or host-pathogen interaction. The antibacterial treatment effectively “freezes” the biological state of infection without introducing additional strand breaks or alkali-labile sites in host DNA. The antibacterial treatment step may be followed by one or more washing and embedding steps designed to minimize the carryover of pathogen-derived DNA into the electrophoresis matrix. The result is a preparation in which the electrophoretic comet signal reflects host genome integrity exclusively.

[0166] In some examples, the antibacterial reagent is selected from antibiotics that arrest bacterial replication without inducing secondary DNA damage in host cells. Non-limiting examples include gentamicin, ciprofloxacin at sublethal bacteriostatic concentrations, or polymyxin B. In other examples, the reagent comprises bacteriophages, bactericidal peptides such as lysostaphin or defensin analogs, or silver ion-releasing nanoformulations. The timing and sequence of reagent addition can be defined so as to achieve controlled bacterial inactivation. For example, treatment of an infected culture with a bacteriostatic compound for approximately ten minutes at 37 °C immediately prior to electrophoresis setup provides a time-locked snapshot of infection-induced DNA damage.

[0167] In some examples, the antibacterial agent reduces fluorescent signal derived from bacteria interfering with comet detection.

[0168] In some examples, the population of host cells comprise mammalian cell lines, human epithelial cells, macrophages, fibroblasts, endothelial cells, HeLa cell line, A549 cell line, RAW264.7 cell line, HaCat cell line or primary cell lines.

[0169] In some examples, the bacteria comprises Escherichia coli, Salmonella enterica. Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Mycobacterium tuberculosis, or a genetically engineered derivative thereof.

[0170] In some examples, the antibacterial agent halts bacterial replication or metabolic activity, and preserves genomic damage state induced by infecting the population of host cells without introducing additional host DNA strand breaks.

[0171] In some examples, the antibacterial agent comprises ciprofloxacin, gentamicin, tetracycline, erythromycin, chloramphenicol, lysostaphin, bacteriophage-derived lysin, LL-37, melittin, silver ions, zinc ions, or copper ions.

[0172] In some examples, an antibacterial -modified comet assay is provided to enable accurate evaluation of host genomic integrity following bacterial infection. The modified assay comprises at least one antibacterial handling, chemical, or timing step incorporated into the assay workflow to control, eliminate, standardize, or account for bacterial interference. In some examples, the modification may include chemical treatment to inhibit bacterial replication prior to lysis, a timed sampling approach to capture a defined infection stage, or the inclusion of a bactericidal or bacteriolytic reagent that halts pathogen activity without inducing host DNA damage. In particular examples, treatment of S. aureus -infected HaCaT cells with lysostaphin at 200 ng / pL to 500 ng / pL for about 1 hour to 24 hours at 30°C to 40° C effectively removed bacterial background fluorescence while preserving host DNA integrity. Lysostaphin, a Staphylococcus-specific endopeptidase that cleaves pentaglycine cross-bridges in peptidoglycan, selectively degrades Gram-positive bacterial cell walls without damaging mammalian cells. The EDTA present in the electrophoresis buffer deactivates lysostaphin after the post-lysis incubation step, preventing carryover effects during electrophoresis. Subsequent evaluation by both the alkaline comet assay and the Fpg-modified comet assay confirmed that lysostaphin did not induce artefactual DNA damage or oxidized purine formation in either control or UVB -irradiated HaCaT cells (FIG. 6).

[0173] In certain examples, the disclosed antibacterial-modified comet assay comprises contacting a population of host cells infected with a bacterial pathogen with an antibacterial reagent under conditions sufficient to inhibit, lyse, or otherwise neutralize the bacterial component prior to electrophoretic analysis. The antibacterial reagent is selected or formulated such that it halts bacterial activity without inducing detectable artifactual DNA damage in the host genome.

[0174] In some examples, the antibacterial reagent comprises a bacteriolytic enzyme such as lysostaphin, which specifically cleaves pentaglycine cross-bridges in the peptidoglycan of Grampositive bacterial cell walls. When applied to co-cultures of Staphylococcus aureus and human keratinocyte (HaCaT) cells following the lysis wash step of a standard alkaline comet assay, lysostaphin treatment at a concentration of approximately 200 ng / pL for one hour at 37 °C effectively removes bacterial background fluorescence without increasing host DNA strand break frequency.

[0175] In certain examples, the electrophoresis buffer of the comet assay comprises ethylenediaminetetraacetic acid (EDTA) at a concentration sufficient to deactivate residual lysostaphin or other enzymatic agents following antibacterial treatment. This configuration ensures that no further antibacterial activity occurs during electrophoresis or subsequent staining steps.

[0176] In some examples, the antibacterial -modified comet assay may further include an enzyme-modified detection step, such as treatment with formamidopyrimidine DNA glycosylase (Fpg), to reveal oxidized purine lesions. Application of the Fpg-modified comet assay following antibacterial treatment can confirm that the antibacterial agent does not introduce additional oxidative modifications in host genomic DNA. In certain examples, the host cells are mammalian cells selected from human keratinocytes, epithelial cells, fibroblasts, macrophages, or neuronal cells. In particular examples, HaCaT keratinocytes infected with S. aureus serve as a model for epithelial infection. In further examples, dopaminergic BE-M17 neuronal cells infected with Mycoplasma species may be used to assess infection-associated oxidative stress and impaired base excision repair activity.

[0177] In some examples, the antibacterial-modified comet assay comprises the following sequential steps:

[0178] (a) embedding host-pathogen co-culture cells in low-melting point agarose on microscope slides;

[0179] (b) performing cell lysis under alkaline and high- salt conditions;

[0180] (c) washing the slides and applying an antibacterial reagent to the post-lysis gels under controlled temperature and humidity for a predetermined incubation period;

[0181] (d) deactivating the antibacterial reagent through exposure to electrophoresis buffer containing EDTA;

[0182] (e) conducting electrophoresis under alkaline conditions to separate DNA fragments; (f) neutralizing and staining the slides with a nucleic acid dye such as SYBR Gold; and (g ) imaging and quantifying DNA migration using a comet assay scoring platform.

[0183] In certain examples, the antibacterial reagent may alternatively be selected from ciprofloxacin, gentamicin, polymyxin B, silver ion-releasing nanoformulations, bacteriophage preparations, or antimicrobial peptides, each chosen to achieve bacterial inactivation without detectable host genomic damage. The incorporation of known antibacterial compositions into the workflow allows flexible adaptation for various host-pathogen systems while maintaining assay integrity.

[0184] In additional examples, the modified comet assay may be performed in conjunction with therapeutic screening workflows. For example, a test compound may be applied to infected host cell cultures before the antibacterial modification step, and the resulting DNA damage profile may be used to assess both antibacterial efficacy and host genome protection. The ability to obtain quantitative, single-cell strand break data under controlled antibacterial arrest distinguishes the disclosed assay from conventional cytotoxicity or pathogen survival assays, which provide only population-level metrics of stress or viability.

[0185] In certain examples, data generated from the antibacterial-modified comet assay can be used to generate a genome integrity map for individual host cells following pathogen exposure. The resulting data may correlate with measures of oxidative stress, repair enzyme activity, or apoptosis to provide a comprehensive profile of infection-induced genomic injury. In further examples, the disclosed assay enables simultaneous evaluation of infection-induced DNA damage and antibacterial agent safety. By maintaining defined antibacterial control during sample handling, the assay yields reproducible, interpretable data reflecting host-specific DNA damage rather than artifacts from continued bacterial metabolism or antibiotic cytotoxicity.

[0186] Accordingly, the antibacterial-modified comet assay provides a versatile, single-cell platform for quantifying host genomic integrity under infectious or antimicrobial stress conditions, for screening therapeutic candidates that preserve host DNA stability, and for mechanistic studies of bacterial genotoxicity.

[0187] Comet Assay

[0188] The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic / apyrimidinic sites), alkylated and oxidized nucleobases, DNA DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Among various applications of the comet assay one includes an in vivo test for genotoxicity in animal organs. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

[0189] Exposure of cells to compounds inducing DNA damage may produce single or double strand breaks in genomic DNA as well as chemical modifications. Comet assay treated cells are embedded in agarose, lysed, and treated with alkali to denature DNA. Subsequent electrophoresis leads to migration of fragmented DNA and / or relaxation of chromatin away from the nucleus, thereby producing the appearance of a comet (from which the assay is named) which is imaged by fluorescence microscopy. The amount of DNA liberated from the head of the comet into the tail is quantified as a measure of DNA damage.

[0190] The comet assay has become widely used for assessing genotoxicity (DNA damage with the potential to lead to a heritable deletion or mutation) as it is very sensitive, provides single cell statistics and requires only small samples of cells. The assay is most commonly carried out using lymphocytes, but recent developments have focused on the use of cells which may be obtained using minimally invasive procedures such as buccal cells from mouth swabs (Szeto, Y. T., et.al., Mutat. Res., (2005), 578(1-2), 371 -81 ). The comet assay detects DNA damage by electrophoresis of cellular DNA. Cells to be tested are harvested by centrifugation and resuspended in buffer solution. Cells are combined with a molten solution of low melting point agarose and spread on a microscope slide. The agarose suspension of cells is then cooled and allowed to set. The slide is then immersed in a lysis solution to break open the cells, and the cellular DNA is denatured by immersion of the slide in an alkaline solution. This lysis and denaturation procedure removes most of the cellular components except nuclear- structural proteins and DNA which remain as discrete nucleoids. Following DNA lysis, the slide is washed several times with electrophoresis buffer and the slide transferred to a horizontal electrophoresis apparatus and subjected to an electric current. At this stage, any small DNA fragments resulting from damage to the cell DNA migrate into the electric field away from the bulk DNA which is retained in the nucleoids. Following electrophoresis, the slide is fixed in ethanol, stained for DNA using a fluorescent DNA binding dye and visualized by microscopy. Cells in which DNA damage has occurred show a staining pattern of a bright nucleoid head with a tail of DNA fragments trailing in the direction of electrophoresis.

[0191] US 4,695,548 application incorporated herein by reference, describes the use of gel inserts comprising a solidified liquid such as agarose suitable for use in an electrophoretic method. Lysed cells entrapped within a matrix formed by the solidified liquid and macromolecules such as DNA or intact chromosomes derived from the lysed cells may be advantageously used in electrophoretic separations. The gel inserts are placed directly in a suitable support medium and subjected to one or more electric fields to separate the macromolecules. However this method is intended for use with DNA separations of genomic DNA specifically for the purpose of avoiding physical damage to the DNA by shearing caused by pipetting of DNA solutions, and does not relate to handling of samples for measurement of DNA damage, nor to high-throughput analysis of DNA.

[0192] Long-term exposure to genotoxic agents has been identified as a cause of chronic disease and cancer. A primary health concern, although not normally associated with direct DNA damage, has been bacterial infection. With the microbiome increasingly associated with colon cancer, irritable bowel syndrome, and other chronic conditions, elucidation of the potential DNA damage mechanisms of bacterial species has been considered paramount. This urgent need to study bacteria induced DNA damage has been supported by several studies that have demonstrated the presence of DNA damage induced by bacterial infections, as well as by specific bacterial species found in the microbiome. Classically, this DNA damage has been considered a consequence of activation of the host immune system or bacterial metabolic waste, unrelated to virulence, that has also induced DNA damage. However, the coexistence of bacteria with mammalian cells has induced DNA damage in the latter through mechanisms that have remained unclear. Therefore, to understand bacterial pathogenesis, it has been considered vital to determine how bacteria and mammalian cells interact during infection. To achieve this, co-culture of bacteria and mammalian cells has been regarded as a prerequisite. Previous studies have shown increased DNA damage in HeLa cells infected with the Gram-positive bacterium Staphylococcus aureus, but these studies have used less sensitive methods, such as staining for gamma H2AX foci. Singlecell gel electrophoresis (the comet assay) has been a widely used method for studying DNA damage in eukaryotic cells, and the assay has been successfully utilized herein to demonstrate the induction of DNA damage by Mycoplasma bacteria. However, the presence of bacteria alongside eukaryotic cells has interfered with the comet assay. Indeed, it has been shown that methicillin-resistant S. aureus alongside eukaryotic cells has created a background signal (FIG. 1A), preventing the quantification of damage by comet scoring software (FIG. 1B).

[0193] As used herein, “alkaline comet assay” (single-cell gel electrophoresis) is a sensitive method that detects DNA strand breaks (SBs) and alkali-labile sites (ALS) in the nucleus of virtually all types of eukaryotic cells. ALS are not well defined but, as the name suggests, are essentially any DNA modification that becomes an SB under alkaline conditions, e.g., apurinic / apyrimidinic (AP) sites. The principle of the comet assay relies on the spatial organization of DNA in the nucleus, namely loops of DNA formed by attachment of the linear- molecule at intervals to the nuclear matrix, and additional winding of the double helix around protein cores to form nucleosomes. This organization means that, when the proteins are removed during the lysis step of the assay, the DNA remains in a compact supercoiled state. However, if a DNA SB is present, the supercoiling of the loops relaxes. As a result of this relaxation, these loops, which are still attached to the nuclear matrix, are drawn towards the anode, forming the characteristic ‘comet tail’, seen under a fluorescence microscope. The relative amount of total DNA in the tail reflects the frequency of breaks.

[0194] Antimicrobial agents

[0195] Gram-positive bacteria consist of cytoplasmic membrane surrounded by a tough and rigid mesh called cell wall. As used herein, “Gram-negative bacteria” consist of thin cell wall that is surrounded by second lipid membrane called outer membrane (OM). The space between the OM and cytoplasmic membrane is referred as periplasm. The OM is an additional protective layer in Gram-negative bacteria and prevents many substances from entering into the bacterium. However, this membrane contains channels called porins, which allow' the entry of various molecules such as drugs. The cell wall is a tough layer that gives bacterium a characteristic shape and prevents it from osmotic and mechanical stresses. The cytoplasmic membrane prevents ions from flowing into or out of the cell and maintains the cytoplasmic and bacterial components in a defined space. There are different types of antibiotics, as described herein with different modes of action.

[0196] Antibiotics targeting cell wall- Bacterial cells are surrounded by a cell wall made of peptidoglycan, which consists of long sugar polymers. The peptidoglycan undergoes cross-linking of the glycan strands by the action of transglycosidases, and the peptide chains extend from the sugars in the polymers and form cross links, one peptide to another. The D-alanyl-alanine portion of peptide chain is cross linked by glycine residues in the presence of penicillin binding proteins (PBPs). This cross-linking strengthens the cell wall, p-lactams and the glycopeptides inhibit cell wall synthesis.

[0197] The primary targets of the p-lactam agents are the PBPs. It has been hypothesized that the p-lactam ring mimics the D-alanyl D-alanine portion of peptide chain that is normally bound by PBP. The PBP interacts with P-lactam ring and are not available for the synthesis of new peptidoglycan. The disruption of peptidoglycan layer leads to the lysis of bacterium.

[0198] The glycopeptides binds to D-alanyl D-alanine portion of peptide side chain of the precursor peptidoglycan subunit. The large drug molecule vancomycin prevents bi nding of this D-alanyl subunit with the PBP, and hence inhibits cell wall synthesis.

[0199] Inhibitors of protein biosynthesis-The bacterial 70S ribosome is composed of two ribonucleoprotein subunits, the 30S and 50S subunits. Antimicrobials inhibit protein biosynthesis by targeting the 30S or 5 OS subunit of the bacterial ribosome.

[0200] The aminoglycosides (AG's) are positively-charged molecules which attach to the OM which is negatively charged leading to formation of large pores, and thus allow antibiotic penetration inside the bacterium. The main target of action is bacterial ribosome; to enter, there it must pass through cytoplasmic membrane requiring energy dependent active bacterial transport mechanism, which requires oxygen and an active proton motive force. For these reasons, AG work in aerobic conditions and have poor activity against anaerobic bacteria. These AG have synergism with those antibiotics, which inhibit cell wall synthesis (such as P-lactam and glycopeptides) as it allows greater penetration of AG within the cell and at low dosages. AG's interact with the 16S r-RNA of the 30S subunit near the A site through hydrogen bonds. They cause misreading and premature termination of translation of mRNA.

[0201] Tetracyclines, such as tetracycline, chlortetracycline, doxycycline, or minocycline, act upon the conserved sequences of the 16S r-RNA of the 30S ribosomal subunit to prevent binding of t-RNA to the A site.

[0202] It interacts with the conserved sequences of the peptidyl transferase cavity of the 23 S r-RNA of the 50S subunit. Hence, it inhibits the protein synthesis by preventing binding of t-RNA to the A site of the ribosome. These affect the early stage of protein synthesis, namely translocation, by targeting the conserved sequences of the peptidyl transferase center of the 23S r-RNA of the 50S ribosomal subunit. This results in a premature detachment of incomplete peptide chains. Macrolides, lincosamides, and streptogramins B show a similar mechanism of action.

[0203] Linezolid is a recently approved member of novel class of antibiotic of this group which is completely synthetic. Oxazolidinones interfere with protein synthesis at several stages: (i) inhibit protein synthesis by binding to 23Sr RNA of the 50S subunit and (ii) suppress 70S inhibition and interact with peptidyl-t-RNA.

[0204] Inhibitors of DNA replication- The fluoroquinolones (FQ) inhibit the enzyme bacterial DNA gyrase, which nicks the double-stranded DNA, introduces negative supercoils and then reseals the nicked ends. This is necessary to prevent excessive positive supercoiling of the strands when they separate to permit replication or transcription. The DNA gyrase consists of two A subunits and two B subunits. A subunit carries out the nicking of DNA, B subunit introduces negative supercoils, and then A subunit reseal the strands. The FQ's bind to A subunit with high affinity and interfere with its strand cutting and resealing function. In Gram-positive bacteria, the major target of action is topoisomerase IV which nicks and separates daughter DNA strand after DNA replication. Greater affinity for this enzyme may confer higher potency against Grampositive bacteria. In place of DNA gyrase or topoisomerase IV, mammalian cells possess topoisomerase II, which has very low affinity for FQ-hence low toxicity to cells.

[0205] Folic acid metabolism inhibitors- Each of these drugs inhibits distinct steps in folic acid metabolism. A combination of sulpha drugs and trimethoprim acting at distinct steps on the same biosynthetic pathway shows synergy and a reduced mutation rate for resistance. Sulfonamides inhibit dihydropteroate synthase in a competitive manner with higher affinity for the enzyme than the natural substrate, p-amino benzoic acid. Agents such as trimethoprim act at a later stage of folic acid synthesis and inhibit the enzyme dihydrofolate reductase.

[0206] In certain examples, an antibacterial agent is added to the infected culture immediately following harvest, thereby halting further bacterial replication or host-pathogen interaction. The antibacterial treatment effectively “freezes” the biological state of infection without introducing additional strand breaks or alkali-labile sites in host DNA. The antibacterial treatment step may be followed by one or more washing and embedding steps designed to minimize the carryover of pathogen-derived DNA into the electrophoresis matrix. The result is a preparation in which the electrophoretic comet signal reflects host genome integrity exclusively.

[0207] In some examples, the antibacterial reagent is selected from antibiotics that arrest bacterial replication without inducing secondary DNA damage in host cells. Non-limiting examples include gentamicin, ciprofloxacin at sublethal bacteriostatic concentrations, or polymyxin B. In other examples, the reagent comprises bacteriophages, bactericidal peptides such as lysostaphin or defensin analogs, or silver ion-releasing nanoformulations. The timing and sequence of reagent addition can be defined so as to achieve controlled bacterial inactivation. For example, treatment of an infected culture with a bacteriostatic compound for approximately ten minutes at 37 °C immediately prior to electrophoresis setup provides a time-locked snapshot of infection-induced DNA damage.

[0208] In some examples, the antibacterial agent reduces fluorescent signal derived from bacteria interfering with comet detection.

[0209] In some examples, the population of host cells comprise mammalian cell lines, human epithelial cells, macrophages, fibroblasts, endothelial cells, HeLa cell line, A549 cell line, RAW264.7 cell line, HaCat cell line or primary cell lines.

[0210] In some examples, the bacteria comprises Escherichia coli, Salmonella enterica, Staphylococcus aureus, Listeria monocytogenes. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Mycobacterium tuberculosis, or a genetically engineered derivative thereof.

[0211] In some examples, the antibacterial agent halts bacterial replication or metabolic activity, and preserves genomic damage state induced by infecting the population of host cells without introducing additional host DNA strand breaks.

[0212] In some examples, the antibacterial agent comprises ciprofloxacin, gentamicin, tetracycline, erythromycin, chloramphenicol, lysostaphin, bacteriophage-derived lysin, LL-37, melittin, silver ions, zinc ions, or copper ions.

[0213] In some examples, the antibacterial agent includes but is not limited to antibiotics, such as enedine antibiotics (e.g. calicheamicin, especially calicheamicin yl, 51, al or pi (e.g. J. Med. Chem., 39 (11), 2103-2117 (1996), Angew Chem Inti. Ed. Engl. 33:183-186 (1994)]; dynemycins, including dynemycin A and deoxydynemycin; esperamicin, kedarcidine, C-1027, maduropeptin as well as the neocarzinostatin chromophore and the related chromoprotein enedine antibiotic chromophore), aclasinomycin, actinomycin, othramycin, azaserine, bleomycin, cactinomycin, and cara. Vicin, caminomycin, carzinophylline; Chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, nitomycin, mycophenolic acid, nogalamycin, olibomycin, pephlomycin, popperomycin, puromycin, and quelamycin, rhodolubicin, streptonigrin, streptozocin, tubercidin, ubenimex, gynostatin, zorubicin; i) Others: such as polyketides (acetogenin), especially bullatacin and bullatacinone, gemcitabine, epoxomicin (e.g. carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zibrestat, PLX4032, STA-9090, Stimuvax, Allovectin-7, Xegeva, Provenge, Yervoy, proteinase inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1 -methyl -4-phenylpyridinium ion), cell cycle inhibitors (e.g., staurosporine), actinomycin (e.g., actinomycin D, dactinomycin), bleomycin (e.g., bleomycin A2, bleomycin B2, pephlomycin), anthracyclines (e.g., daunorubicin, doxorubicin (Adriamycin), idarubicin, epirubicin, eribulin, pyrarubicin, zorubicin, emtoxantrone, MDR inhibitors (e.g., verapamil), Ca2+ ATPase inhibitors (e.g., thapsigargin), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat (MGCD0103), Vorinostat, PCT-24781, entinostat, SB939, resminostat, zibinostat, AR-42, CUDC-101, sulforaphane, trichostatin A); thapsigargin, celecoxib, glitazone, Epigallocatechin gallate, di sulfiram, salinosporamide A; anti-adrenergics such as aminoglutethimide, mitotane, trilostane; aceglatone; aldophosphamide glycoside; aminolevulinic acid; Anisacrine; Arabinoside, Vestrabucil; Bisantrene; Edatraxate; Depopamine; Demecolcin; Diaziquone; Eflonithine (DEMO), Elfomitine; Elliptinium acetate, Etogluside; Gallium nitrate, gancytosine, hydroxyurea; ibandronate, lentinan; ronidamine; mitoguazone; mitoxantrone; furidamole; nitracrine; pentostatin; penamet; pyrarubicin; podophyllic acid; 2- Ethylhydrazide; Procarbazine; PSK®; Razoxan; Rhizoxin; Sizopyran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, loridin A, and anguidine); urethanes, siRNA, antisense drugs; and nucleolytic enzymes.

[0214] In some examples, the antimicrobial agents include, but are not limited to: a) Aminoglycosides: amikacin, astromycin, gentamicin (netilmycin, sisomicin, isefamicin), hygromycin B, Kanamycin (amikacin, arbecacin, becanamycin, dibecacin, tobramycin), neomycin (pramycetin, paromomycin, ribostamycin), netilmicin, spectinomycin, streptomycin, tobra mycin, verdamicin; b) Amphenicol: azidaphenicol, chloramphenicol, flophenicol, thiamphenicol; c) Ansamycins: geldanamycin, herbimycin; d) Carbapenems: biapenem, doripenem, ertapenem, imipenem / cilastatin, meropenem, panipenem; e) Cefem: Carbacepem (loracarbef), cefacetril, cefaclor, cephaladin, cefadroxil, cephalonium, cephaloridine, cephalothin or cephalocin, cephalexin, cephaloglycine, Cefamandole, cefaphyrin, cephatrizine, cefazaflu, cefazedone, cefazolin, cefbuperazone, cefcapen, cefdaloxime, cefepime, cefminox, cefoxitin, cefprozil, cefroxadine, cef Tezole, cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozofran, cephalexin, cefpimizole, cefpyramide, cefpyrome, cefpodoxime, cefprozil, cefquinome, cefsulodine, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime, cef Tobiprole, ceftriaxone, cefuroxime, cefuzonam, cefamycin (cefoxitin, cefoctetan, cefmetazole), oxacepem (flomoxef, latamoxef); f) Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin), teicoplanin (dalbavancin), ramoplanin; g) Glycylcyclines: for example tigecycline; g) P-lactamase inhibitors: penam (sulbactam, tazobactam), clavam (clavulan acid) i) lincosamides: clindamycin, lincomycin; j) Lipopeptides: daptomycin, A54145, calcium-dependent antibody (CDA); k) Macrolides: azithromycin, cetromycin, clarithromycin, diristromycin, erythromycin, fluritromycin, yosamycin, ketolides (telithromycin, cetromycin), midecamicin, myo. Carmycin, oleandomycin, rifamycin (rifampicin, rifampin, rifabutin, rifapeptin), lokitamycin, roxithromycin, spectinomycin, spiramycin, tacrolimus (FK5O6), troleandomycin, telithroma Isin; 1) Monobactam: aztreonam, tigemonam; m) Oxazolidinone: Linezolid; n) Penicillin: amoxicillin, ampicillin (pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathine benzylpenicillin, benzathine phenoxymethylpenicillin., Clomethocillin, Procaine, Benzylpenicillin, Carbenicillin (Carindacillin), Cioxacillin, Dicloxacillin, Epicillin, Flucioxacillin, Mecillinam (Pivmesillinam), Mezlocillin, Methicillin, nafcillin, oxacillin, phenamecillin, penicillin, peneticillin, phenoxyniethylpenicillin, piperacillin, propicillin, sulbenicillin, temocillin, ticarcillin; o) Polypeptides: bacitracin, colistin, polymyxin B; p) Quinolones: alatrofloxacin, valofloxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, floxacin, garenoxacin, gatifloxacin, gemifloxacin, grepafloxacin, trovafloxacin, levofloxacin, lomefloxacin, mabofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxacin, trovafloxacin., grepafloxacin, sitafloxacin, spafloxacin, temafloxacin, tosufloxacin, trovafloxacin; q) Streptogramins: pristinamycin, quinupristin / dalfopristin; r) Sulfonamides: Mafenide, Protosil, Sulfacetamide, Sulfamethizole, Sulfanylimide, Sulfasalazine, Sulfisoxazole, Trimethoprim, Trimethoprim-sulfamethoxazole (co-trimoxazole); s) Steroid antibacterial agents: fusidic acid; t) Tetracyclines: doxycycline, chlortetracycline, chlormocycline, demeclocycline, lymecycline, meclocy cline, metacycline, minocycline, oxytetracycline, phenimepicycline, lolitetracycline, tetracycline, glycylcycline ( Examples include tigecy cline); u) Other types of antibiotics: annonacin, asphenamine, bactoprenol inhibitor (bacitracin), DADAL / AR inhibitor (cycloserine), dictiostatin, discodermolide, eleuterobine, epothilone, ethambutol, Etoposide, faropenem, fusidic acid, furazolidone, isoniazid, laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesis inhibitors (e.g., fosfomycin), nitrofurantoin, paclitaxel, flavonoids. Tensimycin, pyrazinamide, quinupristin / dalfopristin, rifampicin (rifampin), tazobactam tinidazole, uvaricin.

[0215] In some examples, the antiviral agents include, but are not limited to: a) Entry / fusion inhibitors: apraviroc, maraviroc, vicriviroc, gp41 (enfuvirocide), PRO 140, CD4 (ibalijunap); b) Integrase inhibitors: raltegravir, elvitegravir, globoidan A; c) Maturation inhibitors: Bevirimat, Vibecon; d) Neuraminidase inhibitors: oseltamivir, zanamivir, peramivir; e) Nucleosides and nucleotides: abacavir, acyclovir, adefovir, amdoxovir, apricitabine, bribudine, cidofovir, clevudine, dexelbucitabine, didanosine (ddl), elbusitabine, M. Tricitabine (PTC), entecavir, famcyclovir, fluorouracil (5-FU), 3'-fluoro-substituted 2’,3'-dideoxynucleoside analogs (e.g., 3'- Fluoro-2’,3'-dideoxythymidine (FLTj and 3'-fluoro-2',3’-dideoxyguanosine (FLG)), fomivirsen, ganciclovir, Idoxuridine, lamivudine (3TC), 1 -nucleosides (p-1 -thymidine and p-1,2'-deoxycytidine), penciclovir, lacivir, ribavirin, stampidine, stavudine (d4T) ), taribavirin (viramidine), telbivudine, tenofovir, trifluridine, valacyclovir, valganciclovir, zalcitabine (ddC), zidovudine (AZT); f) Non-nucleosides: amantadine, ateviridine, capravirine, diaryl pyrimidines (etravirine, rilpivirine), delavirdine, docosanol, emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod, interferon alpha, loviride, rodenosine, metisazone, nevirapine, NOV-205, peginterferon alpha, podophyllotoxin, rifampicin, rimantadine, resiquimod (R-848), Tromantadine; g) Protease inhibitors: amprenavir, atazanavir, boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h) Other types of anti-viral drugs: Abzyme, Arbidol, calanolide a, seragenin, cyanovirin-n, diarylpyrimidine, epigallocatechin gallate (EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea, KP-1461, miltefosine, pleconaril, portmanteau inhibitor, ribavirin, seliciclib.

[0216] Infectious agents

[0217] In some examples, disclosed herein are methods to determine DNA damage in a biological sample, wherein the biological sample from the subject exhibits prior exposure to an infectious agent in vivo or the biological sample is exposed to an infectious agent in vitro, such as, for example, the infectious agent is selected from, including but not limited to Acinetobacter baumannii, Actinomyces israelii, and Actinomyces gerencseriae. ) and Propionibacterium propionicus, Trypanosoma brucei, HIV (human immunodeficiency virus), Entamoeba histolytica, Anaplasma genus, Bacillus anthracis anthracis), hemolytic Arcanobacterium haemolyticum, Junin virus, Ascaris lumbricoides, Aspergillus genus, Astroviridae family, Babesia genus ( Babesia genus, Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli, Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis, Blastomyces dermatitides, Machupo virus, Borrelia genus, Clostri Clostridium botulinum, Sabia, Brucella genus, usually Burkholderia. cepacia and other Burkholderia. species, Mycobacterium ulcerans, Cali Caliciviridae family, Campylobacter genus, commonly Candida albicans and other Candida species, Bartonella henselae, Group A Streptococcus and Staphylococcus cits, Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV), Chlamydia trachomatis, Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile, Coccidioides immilis and Coccidioi Coccidioides posadasii, Colorado tick fever virus, rhinovirus, coronavirus, CJD prion, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Ancylostoma braziliense; Multiple parasites, Cyclospora cayetanensis, Taenia solium, cytomegalovirus, dengue virus (DEN- 1, DEN -2, DEN -3 and DEN -4) - flaviviruses (Flavivirus), Dientamoeba fragilis, Corynebacterium diphtheriae, Diphyllobothrium, Dracunculus medinensis, Ebola virus, Echinococcus genus (Echinococcus genus), Ehrlichia genus, Enterobius vermicularis, Enterococcus genus, Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, human herpesvirus 6 and human herpesvirus 7, Fasciolopsis buski, Fasciola hepatica and Fasciola gigantica, FFI prion, Philariode super Family (Filarioidea superfamily), Clostridium perfringens, Fusobacterium genus, Clostridium perfringens; Other Clostridium species, Geotrichum candidum, GSS prion, Giardia intestinalis, Burkholderia mallei, Gnathostoma spinigerum and Gnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis, Streptococcus pyogene, Streptococcus agalactiae agalacliae), Haemophilus influenzae, enteroviruses, mainly Coxsackie A virus and Enterovirus 71, Sin Nombre virus, Helicobacter pylori, Escherichia coli O157:H7, Bunyaviridae family, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, herpes simplex virus 1, herpes simplex virus 2, histoplasma Histoplasma capsulatum, Ancylostoma duodenale and Necator americanus, Hemophilus influenzae, Human bocavirus, Ehrlichia ewingii, Anaplasma phagocytophilum (Anaplasma phagocytophilum), human metapneumovirus, Ehrlichia chaffeensis, human papillomavirus, human parainfluenza virus, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Viruses, Orthomy-xoviridae family, Isospora belli, Kingella kingae, Klebsiella pneumoniae, Klebsiella ozaenae Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion, Lassa virus, Legionella pneumophila, Legionella pneumophila, Leish Leishmania genus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospira genus, Listeria monocytogenes, Borrelia burg Borrelia burgdorferi and other Borrelia species, Wuchereria bancrofti and Brugia malayi, Lymphocytic choriomeningitis virus (LCMV), Plasmodium genus ), Marburg virus, Measles virus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimus yokagawai, Mycoplasma phylum (Microsporidia phylum), Molluscum contagiosum virus (MCV), Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, and numerous species of bacteria (Actinomycetoma) and fungi. (Eumycetoma), parasitic dipterous fly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion, Nocardia asteroides and other Nocardia Nocardia, species, Onchocerca volvulus, Paracoccidioides brasiliensis, Paragonimus westermani and other lung fluke species, Pasteurella genus, Pediculus humanus capitis ), body lice (Pediculus humanus corporis), pubic lice (Phthiru pubis), Bordetella pertussis, Eirsinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii), Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophila psittaci, Coxiella burnetii, rabies virus, Streptobacillus moniliformis, and Spirillum minus, respiratory’ syncytial virus, Rhinosporidium seeberi, rhinovirus, Rickettsia genus, Rickettsia Rickettsia akari, Rift Valley fever virus, Rickettsia rickettsii, rotavirus, Rubella virus, Salmonella genus, SARS coronavirus, Sarcoptes scabiei (Sarcoptes scabiei), Schistosoma genus, Shigella genus, Varicella zoster virus, Variola major or Variola minor, Sporo Sporothrix schenckii, Staphylococcus genus, Staphylococcus aureus, Streptococcus pyogenes, Strongyloides stercoralis, Syphilis Treponema pallidum, Taenia genus, Clostridium tetani, Trichophyton genus, Trichophyton tonsurans, Trichophyton genus, Epidermophyton floccosum Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes, Trichophyton rubrum, Hortaea werneckii, Trichophyton genus genus), Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasma gondii, Trichinella spiralis, Trichomonas tenax. Trichomonas vaginalis, Trichuris trichiura, Mycobacterium tuberculosis, Francisella tularensis, Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibrio cholera, Guanarito virus, West Nile virus, Trichosporon beigelii, Yersinia pseudotuberculosis (Yersinia pseudotuberculosis, Yersinia enterocolitica, yellow fever virus, Mucorales order (Mucormycosis) and Entomophthorales order (Entomophthorales order) Entomophthoramycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis, Shigella Shigella dysenteriae, Shigella, flexneri, Shigella sonnei, Salmonella typhimurium, Treponema, pertenue, Treponema carateum Treponema carateum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohaemorrhagiae, Pneumocystis carinii, Brucella abortus (Brucella abortus), Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia Clamydia spp.; Pathogenic fungi (Aspergillus fumigatus, Candida albicans, Histoplasma capsulatum); Protozoa (Shigella ameba, Trichomonas tenas) Trichomonas tenas, Trichomonas hominis, Trypanosoma gambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmania tropica. ), Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminth (including, but not limited to, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, or hookworm.

[0218] In some examples, the infectious agent is a virus, comprising pathogenic viruses, including but not limited to: Poxviridae, Herpesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Leoviridae (Reoviridae), Retroviridae, influenza virus, parainfluenza virus, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, non-A / non-B viruses, Rhinoviridae, Coronaviridae, Rotoviridae, oncoviruses (e.g. HBV (hepatocellular carcinoma), HPV (cervical cancer, anal cancer), Kaposi’s sarcoma-associated herpes virus (Kaposi's sarcoma), Epstein-Barr virus (nasopharyngeal carcinoma, Burkitt's lymphoma, primary central nervous system lymphoma), MCPyV (Merkel cell carcinoma), SV40 (Simian virus 40), HCV (hepatocellular- carcinoma), HTLV-I (adult T-cell leukemia / lymphoma)], viruses causing immune disorders: [e.g., human immunodeficiency virus (AIDS)]; Central nervous system viruses: [e.g., progressive multifocal leukoencephalopathy (JCV), subacute sclerosing panencephalitis (MeV), lymphocytic choriomeningitis (LCMV), arbovirus encephalitis, Orthomyxoviridae (possible) ( encephalitis lethargic), RV (rabies), herpesvirus meningitis, Ramsay Hunt syndrome type II; Poliovirus (polio, post-polio syndrome), HTLV-I (tropical paralysis)]; Cytomegalovirus (cytomegalovirus retinitis, HSV (herpetic keratitis)); Cardiovascular viruses (e.g., CBV (pericarditis, myocarditis)); Respiratory / acute viral nasopharyngitis / viral pneumonia: [Epstein-Barr virus (EBV infection / infectious mononucleosis), cytomegalo virus; SARS Coronavirus (Severe Acute Respiratory Syndrome), Orthomyxoviridae: Influenza A / B / C (Influenza / avian influenza), Paramyxovirus: Human Parainfluenza Virus (Parainfluenza), RSV (Human Respiratory Syncytial Virus), hMPV); digestive system viruses [MuV (mumps), cytomegalovirus (cytomegalovirus esophagitis); adenovirus (adenovirus infection); rotavirus, novovirus, astrovirus, coronavirus; HBV (hepatitis B virus), CBV, HAV (hepatitis A virus), HCV (hepatitis C virus), HDV (hepatitis D virus), HEV (hepatitis E virus), HGV (hepatitis G virus) ]; genitourinary viruses [e.g., BK virus, MuV (mumps)].

[0219] Immunoassays and fluorochromes

[0220] The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Maggio et al., Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook of Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety and specifically for its teaching regarding immunodetection methods. Immunoassays, in their most simple and direct sense, are binding assays involving binding between antibodies and antigen. Many types and formats of immunoassays are known and all are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmune precipitation assays (RIPA), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery / localization after photobleaching (FRAP / FLAP).

[0221] In general, immunoassays involve contacting a sample suspected of containing a molecule of interest (such as the disclosed biomarkers) with an antibody to the molecule of interest or contacting an antibody to a molecule of interest (such as antibodies to the disclosed biomarkers) with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immunocomplexes. Contacting a sample with the antibody to the molecule of interest or with the molecule that can be bound by an antibody to the molecule of interest under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply bringing into contact the molecule or antibody and the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any molecules (e.g., antigens) present to which the antibodies can bind. In many forms of immunoassay, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, can then be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0222] Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest (such as the disclosed biomarkers or their antibodies) in a sample, which methods generally involve the detection or quantitation of any immune complexes formed during the binding process. In general, the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or any other known label.

[0223] Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; -Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5 -Carboxyte tramethylrhodamine (5 -TAMR A); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy -X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anitin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAG™ CBQCA; ATI’O-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAG 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFPblue shifted GFP (Y66H); Blue Fluorescent Protein; BFP / GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bis- BTC; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™-3; Bodipy492 / 515; Bodipy493 / 503; Bodipy500 / 510; Bodipy; 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy 1’MR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson; Calcium Green; Calcium Green-1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP / YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelen terazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.18; Cy3.5™; Cy3™; Cy5.18; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3’DCFDA; DCFII (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DilC 18(5)); DIDS; Dihydrorhodamine 123 (DHR); Dil (DiiC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC 18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer- 1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxy stilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™ / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type’ non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxy coumarin; Hydroxy stilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; lndo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; lissamine Rhodamine B; Calcein / Ethidium homodimer; LOID-1; LO-PRO-1;; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow'; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO- 1; POPO-3; PO-PRO-1; PO- 1 PRO-3; Primuline; Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; P yrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™ (super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARE calcein; SNARF1; Sodium Green; Spectrum Aqua; SpectruniGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhod amine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YEP; YO-PRO-1; YO- PRO 3; YOYO- 1; YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof.

[0224] The detection of patterns of labelled DNA fragments may be accomplished using an image analyzer. The DNA is stained with a suitable fluorescent stain (e.g. Acridine Orange, Ethidium Bromide, DAPI, TOTOO-3, or Y0Y0®-3) and the plate is imaged using fluorescence excitation and emission filters suitable for the DNA stain used. Acquired images are analyzed using suitable image analysis software (e.g. IN Cell Investigator, GE Healthcare) to segment images, identify regions of DNA staining and make appropriate quantitative measurements (e.g. comet tail intensity, length and moment) to determine the amount of DNA damage in each sample under test.

[0225] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

[0226] A number of examples of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0227] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0228] EXAMPLES

[0229] The following examples are set forth below to illustrate the compounds, systems, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all examples of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0230] Example 1: Antimicrobial agents for removing the background in comet assay.

[0231] The general understanding is that small genomes, such as those of bacteria and mitochondria, are not detectable in the comet assay because these lower molecular weight genomes are lost during either the lysis or electrophoresis steps of the assay due to the presence of high pH and salt. Although earlier study results suggest that this assumption may not be correct, as background staining can be observed with Mycoplasma (FIG. 2B), in that instance the background presence of Mycoplasma is faint and does not interfere with the comet assay analysis software. In contrast, the background speckle caused by S. aureus (FIG. 1) interferes with the imaging and scoring of HaCaT comets by the comet IV Scoring program. It is concluded that this interference results from the size of the S. aureus genome (2.8 Mbp vs. approximately 0.001 Mbp in Mycoplasma) and / or possibly from the presence of the exposed S. aureus peptidoglycan cell wall, which is unique to Gram-positive bacteria. The lysis step does not remove the peplidoglycan-containing cell wall of 5. aureus, allowing the DNA stain (SYBR Gold) to bind non-specifically to the peptidoglycan of the cell wall.

[0232] Physical separation of S. aureus and HaCaT cells by centrifugation is considered but proves unsuccessful. The use of antibiotics such as Gentamicin is excluded as impractical for the experimental design because that may cause bystander effects on mammalian cells and because Gentamicin kills bacteria by targeting the ribosome rather than disrupting the cell wall.

[0233] This consideration leads to exploration of how the peptidoglycan can be removed at some stage in the experimental procedure. As the high salt and pH conditions of the comet assay prove unsuccessful, a commonly used antimicrobial peptide, lysostaphin, is considered (FIG. 6). Lysostaphin targets Staphylococcus-specific peptidoglycan cross-bridges, which are not found in other bacteria. Due to this specificity, lysostaphin is able to remove the background by eliminating S. aureus. Lysostaphin when introduced as a step during the comet assay did not lead to the formation of artefactual damage in the mammalian cells of interest.

[0234] The application of lysostaphin to the cell (mammalian and bacteriaj-containing gels is proposed after they undergo the wash following the lysis step of the comet assay. Initially, a relatively high concentration of 1 pg / pL is used to demonstrate proof-of-principle that lysostaphin can remove S. aureus from the background, allowing the infected HaCaT cells to be scored (FIGS.

[0235] 3A-3D). 1 mg / mL is an extremely high concentration of lysostaphin, and much smaller concentrations are used in most protocols, albeit none in the context of the comet assay. Therefore, the efficacy of lower concentrations is examined.

[0236] Initially, a range of concentrations from 10 ng / pL to 100 ng / pL is tested, but these are unable to fully clear the presence of S. aureus from the background. Based on this, lysostaphin concentrations between 100 ng / pL and 500 ng / pL are examined. All these concentrations are successful in removing the 5'. aureus background fluorescence and, importantly, do not show any sign of the induction of artefactual DNA damage (FIG. 4). Example 2: Lysostaphin -antimicrobial agent does not induce artefactual DNA damage. To confirm that lysostaphin does not induce artefactual DNA damage, damaged and undamaged HaCaT cells are exposed to increasing concentrations of lysostaphin. At no concentration of lysostaphin used is artefactual DNA damage induced in either irradiated or unirradiated cells (FIG. 5). These results confirm the ability of lysostaphin to remove background fluorescence caused by S', aureus without inducing artefactual DNA damage in the host cells, irrespective of whether these cells possess control or induced levels of DNA damage.

[0237] Various images from the comet IV scoring box are compared to illustrate how treatment with lysostaphin allows comets to be scored by the removal of S. aureus staining (FIG. 6). These data, together with the results described above, lead to the conclusion that a concentration of 200 ng / L is optimal. This concentration successfully removes the background staining due to the presence of the bacteria, and no significant increase in DNA damage is observed at any lysostaphin concentration. A concentration of 100 ng / pL lysostaphin is not sufficient to remove bacterial background staining.

[0238] The enzyme-modified comet assay is widely used to provide additional specificity to the types of DNA damage detected. Formamidopyrimidine DNA glycosylase (New England Biolabs Inc.) is commonly used to detect modified purines. Therefore, the potential for the optimal lysostaphin concentration to induce modified purines is examined. Similar to the previous experiment, HaCaT cells are either exposed or unexposed to 1 J / cm2UVB and then incubated first with lysostaphin (200 ng / pL), followed by processing according to the Fpg-modified alkaline comet assay. The use of the Fpg-modified comet assay does not reveal any induction of modified purines by lysostaphin in HaCaT cells, irrespective of whether or not they are pre-exposed to UVB (FIG. 7). The alkaline comet assay was performed essentially as described in FIG. 8. The comet assay slides were prepared and processed according to the traditional alkaline comet assay protocol until the post-lysis wash step (step IV). After washing, microscope slides were placed horizontally on a tray, similar to that used for the enzyme-modified comet assay. Approximately, 80 pL (200 ng / pL) of lysostaphin (AMBI Products LLC) was added to each individual gel, and a cover slip is placed on top to spread out the lysostaphin evenly over the gel. The slides were then placed in a humidified incubator at 37 °C for 1 h. Slides were then removed from the incubator, and the coverslips removed. The slides were then processed according to the remainder of the comet assay protocol i.e., transferred into the vertical slide rack, and placed into the electrophoresis tank (step VIII). The EDTA present in the electrophoresis buffer deacti vates lysostaphin, so no further effects of lysostaphin occur after this step. The gels were stained with SYBR Gold, and imaged with the comet IV lite software. Scoring data was analyzed using the student t-test to compare DNA damage between doses. Lysostaphin represents a simple and cost-effective means to modify both the alkaline and enzyme-modified comet assays to facilitate the analysis of Gram-positive bacteria, such as S. aureus. These findings lead to the conclusion that certain anti-microbial agents, when applied under appropriate conditions, can be readily incorporated into the comet assay protocol. This incorporation effectively removes pathogen interference, enables accurate analysis of DNA damage in cells, and expands this important line of investigation into a public health issue that affects billions of individuals worldwide.

[0239] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred examples of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

[0240] Throughout this application, various publications are referenced. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

Claims

CLAIMSWhat is claimed is:

1. A method of determining DNA damage in a biological sample, comprising:obtaining a biological sample from a subject with prior exposure to an infectious agent in vivo or exposing the biological sample to an infectious agent in vitro, thereby obtaining infected host cells;embedding the infected host cells onto an agarose gel matrix slide; thereby obtaining a slide with embedded infected host cells;lysing the embedded infected host cells on the slide to expose nuclear DNA; thereby obtaining a slide containing the nuclear DNA;washing the slide containing the nuclear DNA;applying an antimicrobial agent to the slide containing the nuclear DNA to disrupt activity of the infectious agent in the biological sample;incubating the slide containing the nuclear DNA with the antimicrobial agent for about 5 min to 120 min at a temperature from 30 °C to 40 °C;subjecting the slide containing the nuclear DNA to electrophoresis under alkaline or neutral conditions;staining the slide containing the nuclear DNA with a DNA specific fluorescent dye; capturing fluorescence microscopy images; andanalyzing the fluorescence microscopy images for tail length, tail DNA percentage, or Olive tail moment.

2. The method of claim 1, wherein the biological sample comprises tissue biopsy, blood, mucosal swab, lavage material, surgically excised tissue, or an in vitro cultured cell population.

3. The method of claim 1, wherein the antimicrobial agent halts replication, viability, or metabolic activity of the infectious agent without adding genotoxic stress to the nuclear DNA.

4. The method of any one of claims 1 to 3, wherein the antimicrobial agent is selected from an antibacterial, an antifungal, an antiviral, or an antiparasitic compound.

5. The method of claim 4, wherein the antibacterial compound comprises ciprofloxacin, gentamicin, vancomycin, lysostaphin or chloramphenicol.

6. The method of claim 4, wherein the antifungal compound comprises amphotericin B, fluconazole, or echinocandin-class compounds.

7. The method of claim 4, wherein the antiviral compound comprises acyclovir, remdesivir, or interferon -based formulations.

8. The method of claim 4, wherein the antiparasitic compound comprises metronidazole, artemisinin, or ivermectin.

9. The method of any one of claims 1 to 8, wherein the antimicrobial agent is lysostaphin.

10. The method of claim 9, wherein the lysostaphin comprises a concentration between 200 ng / uL and 500 ng / pL.

11. The method of any one of claims 1 to 10, further comprises uninfected host cells as a negative control and host cells exposed to a known genotoxic agent as a positive control.

12. The method of any one of claims 1 to 11, wherein the electrophoresis under alkaline conditions comprise a buffer with EDl’A and a pH >12.3.

13. The method of any one of claims 1 to 11, wherein the electrophoresis under neutral conditions comprise a buffer with apHclO.

14. A method of performing analysis of DNA damage in comet assays, comprising:infecting a population of host cells with a bacteria, thereby obtaining infected host cells,embedding the infected host cells in an agarose gel matrix; thereby obtaining embedded host cells;lysing the embedded host cells to remove cellular' membranes and soluble intracellular contents, thereby yielding nuclear DNA;terminating bacterial activity in the embedded host cells by contacting the embedded host cells with an antibacterial agent for about 5 min to 120 min at a temperature between 30 °C and 40 °C;subjecting the embedded host cells incubated with the antibacterial agent to electrophoresis under alkaline or neutral conditions; andstaining the nuclear DNA with a fluorescent dye to visualize and analyze comet structures.

15. The method of claim 14, wherein the antibacterial agent reduces fluorescent signal derived from bacteria interfering with comet detection.

16. The method of claim 14, wherein the population of host cells comprise mammalian cell lines, human epithelial cells, macrophages, fibroblasts, endothelial cells, HeLa cell line, A549 cell line, RAW264.7 cell line, HaCat cell line or primary cell lines.

17. The method of claim 14, wherein the bacteria comprises Escherichia coli, Salmonella enterica, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Mycobacterium tuberculosis, or a genetically engineered derivative thereof.

18. The method of any one of claims 14 to 17, wherein the antibacterial agent halts bacterial replication or metabolic activity, and preserves genomic damage state induced by infecting the population of host cells without introducing additional host DNA strand breaks.

19. The method of any one of claims 14 to 18, wherein the antibacterial agent comprises ciprofloxacin, gentamicin, tetracycline, erythromycin, chloramphenicol, lysostaphin, bacteriophage-derived lysin, LL-37, melittin, silver ions, zinc ions, or copper ions.

20. The method of claim 19, wherein the antibacterial agent is lysostaphin.

21. The method of claim 20, wherein the lysostaphin comprises a concentration between 200 ng / uL and 500 ng / pL.

22. The method of any one of claims 14 to 21, wherein the electrophoresis under alkaline conditions comprise a buffer with EDTA and a pH >12.3.

23. The method of any one of claims 14 to 21, wherein the electrophoresis under neutral conditions comprise a buffer with a pH<10.

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