USUN 20200237786A1 IN (19 ) United States (12 ) Patent Application Publication (10 ) Pub . No .: US 2020/0237786 A1 Hunt (43 ) Pub . Date : Jul. 30 , 2020

( 54 ) DIAGNOSIS AND TREATMENT OF Publication Classification ENDOMETRIOSIS BY SCREENING FOR ( 51 ) Int. Ci. L - FORM BACTERIA A61K 31/65 (2006.01 ) C12Q 1/04 (2006.01 ) (71 ) Applicant: Soft Cell Biological Research , LLC , A61K 31/407 (2006.01 ) St. George , UT ( US ) A61K 31/426 (2006.01 ) A61K 9/00 ( 2006.01 ) (72 ) Inventor: John Brent Hunt, St. George , UT (US ) A61K 31/424 (2006.01 ) A61P 31/04 (2006.01 ) (52 ) U.S. CI. ( 73 ) Assignee: Soft Cell Biological Research , LLC , CPC A61K 31/65 (2013.01 ) ; C12Q 1/04 St. George , UT (US ) (2013.01 ) ; A61K 31/407 ( 2013.01 ) ; A61P 31/04 (2018.01 ) ; A61K 9/0019 ( 2013.01) ; (21 ) Appl. No .: A61K 9/0053 (2013.01 ) ; A61K 31/424 16 /635,608 ( 2013.01 ) ; A61K 31/426 (2013.01 ) ( 22) PCT Filed : Aug. 1, 2018 (57 ) ABSTRACT In some aspects , the disclosure relates to methods for ( 86 ) PCT No.: PCT /US2018 / 044885 screening clinical samples for the presence of L - form bac teria associated with endometriosis and / or ovarian fibroid $ 371 ( c ) ( 1 ) , tumors . The disclosure is based , in part , on screening (2 ) Date : Jan. 31 , 2020 methods used to diagnose a subject as likely having or likely not having endometriosis and /or an ovarian fibroid tumor. In some embodiments , the disclosure relates to methods of Related U.S. Application Data treating endometriosis by administering an antibiotic to a ( 60 ) Provisional application No. 62 / 539,688 , filed on Aug. subject that has been determined to be infected by one or 1, 2017 . more strains of L - form bacteria . Patent Application Publication Jul. 30 , 2020 Sheet 1 of 6 US 2020/0237786 A1

Collect Sample

Contact Sample To A First Growth Medium -120

bodo

Incubate First Growth Medium Under A 130 First Set Of Incubation Conditions

Monitor First Growth Medium For The Presence Of L - Form Bacteria

Transfer A Portion of Bacteria From The First - 150 Growth Medium To A Second Growth Medium

Incubate Second Growth Medium Under 160 A Second Set Of Incubation Conditions

Monitor Second Growth Medium For The 170 Presence Of Bacteria

Isolate Bacteria Grown On Second Growth Medium Patent Application Publication Jul. 30 , 2020 Sheet 2 of 6 US 2020/0237786 A1

210

220

230

240

250

260

270

280

FIG . 2 Patent Application Publication Jul. 30 , 2020 Sheet 3 of 6 US 2020/0237786 A1

300 Collect Sample 310

Contact Sample TO A FirstGrowih Medium 320 Within A Comminuting Container

Comminute Sample 322

Incubate First Growth Medium under A First Set Of Incubation Conditions

Monitor First Growth Medium For The Presence 340 Of L - Form Bacteria

Transfer A Portion of Bacteria From The First 350 Growth Medium To A Second Growth Medium

Incubate Second Growth Medium Under -360 A Second Set Of incubation Conditions

Monitor Second Growth Medium For The -370 Presence Of Bacteria

Isolaie Bacteria Grown On Second Growth Medium Patent Application Publication Jul. 30 , 2020 Sheet 4 of 6 US 2020/0237786 A1

400

Withdraw inoculaniFrom Liquid Medium 410

Contact Inoculant To An Exposed 420 Surface Of A Solid Medium

Cover looculant To Maintain 430 Hydration Of Inoculate

Position Solid Medium For Incubation 440 With Inoculant Side Facing Down

Incubate For A First Solid - Phase Incubation Time Period

Reposition Solid Medium For -460 Incubation With InoculantSide Facing Up

Incubate For A Second Solid Medium Incubation Time Period Mooddobb Patent Application Publication Jul. 30 , 2020 Sheet 5 of 6 US 2020/0237786 A1

dark color indicative of production of enzyme or other bioactive agent

FIG . 5 Patent Application Publication Jul. 30 , 2020 Sheet 6 of 6 US 2020/0237786 A1

Microbacterium maritypicum

Staphylococcus epidermidis

FIG . 6 US 2020/0237786 A1 Jul. 30 , 2020

DIAGNOSIS AND TREATMENT OF [0006 ] Determining the presence in a patient of an L - form ENDOMETRIOSIS BY SCREENING FOR bacteria associated with endometriosis and /or ovarian L - FORM BACTERIA fibroid tumors can eliminate the need for such patients to undergo other more invasive and more costly diagnostic RELATED APPLICATIONS procedures . Methods described herein enable the culturing and detection of such L - form bacteria within a sample from [ 0001 ] This application is a national stage filing under 35 a subject having or suspected of potentially having endo U.S.C. § 371 of international PCT application , PCT/ metriosis ( e.g. , patients experiencing infertility , patients US2018 /044885 , filed Aug. 1 , 2018, which claims priority exhibiting one or more signs or symptoms or risk factors for under 35 U.S.C. $ 119 (e ) to U.S. provisional patent appli endometriosis , etc.) , providing a means for more rapidly and cation , U.S. Ser . No. 62/ 539,688 , filed Aug. 1 , 2017, the less invasively determining the likelihood of endometriosis entire contents of each of which are incorporated herein by before proceeding with more intensive and more costly reference . diagnostic procedures. In addition , faster diagnoses of endo metriosis can ease anxiety in patients suffering from the BACKGROUND effects of endometriosis , particularly where the condition [0002 ] Endometriosis is a condition in which endome has proven difficult to diagnose using standard clinical trium tissue abnormally grows at locations outside the measures known in the art . uterus. Because such tissue growth often takes place on the [0007 ] Under culture conditions and process steps ovaries , fallopian tubes , and surrounding tissues, the condi described herein , L -form bacteria that are associated with tion can cause infertility and chronic pelvic pain . Endome endometriosis and symptoms thereof can be successfully trial tissue will bleed as part of the menstrual cycle , and cultured and isolated , even in circumstances in which the areas of endometriosis can become inflamed and scarred as sample from which the L - form bacteria are cultured is a result . It is estimated that endometriosis affects 6 to 10 % unable to produce any detectable growth of cell wall suffi of women . Among women with endometriosis , about 40 % cient bacteria using conventional bacterial culturing tech niques. In addition , embodiments of the disclosure have will have infertility issues. been used to culture bacteria for which no previous reports [ 0003 ] Diagnosis of endometriosis can be challenging . of successful culture or isolation have been made . In some Although a physician may be able to feel endometrial embodiments , L - form bacteria having no known culturable , growth during a pelvic exam , proper diagnosis cannot be cell- wall -sufficient / cell- wall -including form in nature have confirmed by exam only . Pelvic ultrasound may identify been transformed into culturable , cell- wall - sufficient form large endometriotic cysts but will not be able visualize using methods described by the disclosure . and detect smaller regions of endometriosis . Laparoscopy [0008 ] Certain embodiments relate to methods for cultur and biopsy are presently the only methods for fully diag ing L -form bacteria , methods of isolating a bacterial strain nosing endometriosis . These methods, however, are rela from a sample containing L - form bacteria , methods of tively invasive and expensive . transforming an unculturable L - form bacteria into a cultur able cell -wall - sufficient form , and methods of analyzing a BRIEF SUMMARY bacterial strain cultured or isolated from a sample in order to [0004 ] In some aspects , the disclosure relates to methods identify the bacterial strain , and /or propagate or harvest the for culturing and identifying L - form bacteria within a sub bacterial strain . ject's sample (e.g. , blood, saliva, etc. ) in order to diagnose [0009 ] In some aspects , the disclosure relates to methods the subject as having endometriosis and /or an ovarian fibroid for treating a subject having or suspected of having endo tumor. Such methods beneficially enable the detection of metriosis . The disclosure is based , in part, on administration such conditions that would otherwise potentially go unde of one or more antibiotic agents to a subject that has been tected or would require lengthier, costlier, and /or more determined to be infected by one or more L - form bacteria . invasive diagnosis . The methods described herein may Accordingly , in some embodiments , the disclosure provides therefore provide, in some embodiments , time savings, cost a method for treating endometriosis in a subject comprising savings , as well as reductions in patient/ subject anxiety obtaining (or having obtained ) a biological sample from the associated with diagnostic uncertainty surrounding the con subject ; detecting ( or having detected ) in the biological dition . sample one or more bacteria , each bacteria having a genus [ 0005 ] Endometriosis may often go undetected until pro selected from Microbacterium , Bacillus, Micrococcus, and longed periods of infertility indicate that it may be an issue . Staphylococcus; and administering to the subject an antibi Even then , an official diagnosis may require invasive lapa otic agent based upon the presence of the one or more roscopy and/ or biopsy procedures . The disclosure is based , bacteria in the sample . in part , on methods for detecting the presence of one or more [ 0010 ] In some embodiments , a subject has or is suspected types ( e.g., species ) of L - form bacterial infection within a of having endometriosis . In some embodiments , a subject is subject that has or is suspected of having endometriosis characterized by one or more signs, symptoms , or risk and / or ovarian fibroid tumors . In some embodiments , the factors associated with endometriosis . In some embodi methods comprise detecting in biological samples ( blood ments , a subject is a human (e.g. , a female human ). samples , etc. ) taken from patients known to have endo [0011 ] In some embodiments , a biological sample is metriosis a particular subset of L - form bacteria . In some blood . embodiments , the L -form bacteria are incapable of being [0012 ] In some embodiments , detecting comprises cultur cultured , and thereby detected , using standard clinical cul ing at least one L - form bacteria from a biological sample turing protocols ( e.g., standard culturing procedures for cell using a method described by the disclosure . In some wall sufficient bacteria ) . embodiments , an L - form bacteria detected by a method US 2020/0237786 A1 Jul. 30 , 2020 2 described by the disclosure is not able to be cultured by [0018 ] In some embodiments , a broad - spectrum antibiotic conventional bacterial culture techniques. In some embodi is a carbapenem . In some embodiments , a carbapenem is ments , an L - form bacteria detected by a method described Imipenem or Meropenem . by the disclosure is absent from a subject that does not have [0019 ] In some embodiments , a broad -spectrum antibiotic endometriosis or an ovarian fibroid tumor ( e.g., a healthy is a tetracycline . In some embodiments , a tetracycline is subject) . Doxycycline . [0020 ] In some embodiments , a broad - spectrum antibiotic [ 0013 ] In some embodiments , at least one of the L - form is Amoxiclav . bacteria is of a species set forth in Table 2. In some [0021 ] In some embodiments , an antibiotic agent is embodiments , a Micrococcus bacteria is Micrococcus luteus . administered to a subject intravenously . In some embodi In some embodiments , a Bacillus bacteria is Bacillus safen ments , an antibiotic agent is administered to a subject orally . sis, Bacillus simplex , Bacillus licheniformis . Bacillus strato In some embodiments , an antibiotic agent is administered to sphericus, Bacillus dretensis , Bacillus subtillis, Bacillus the subject during menstruation of the subject. velezensis . Bacillus cereus, Bacillus subterraneus, Bacillus [0022 ] Additional features and advantages will be set forth persicus, or Bacillus thuringiensis . In some embodiments , a in part in the description that follows, and in part will be Microbacterium bacteria is Microbacterium maritypicum . obvious from the description , or may be learned by practice [0014 ] In some embodiments , a Staphylococcus bacterium of the embodiments disclosed herein . The objects and is Staphylococcus epidermis , Staphylococcus petrasii, advantages of the embodiments disclosed herein will be Staphylococcus equorum , Staphylococcus pasteuri , Staphy realized and attained by means of the elements and combi lococcus speibonae, and Staphylococcus warneri. nations particularly pointed out in the appended claims. It is [ 0015 ] In some embodiments , detecting comprises detect to be understood that both the foregoing brief summary and ing or having detected one or more additional L - form the following detailed description are exemplary and bacteria (e.g. , one or more additional genus of L - form explanatory only and are not restrictive of the embodiments bacteria ) in the biological sample . Examples of one or more disclosed herein or as claimed . additional L - form bacterial genera include Brachybacte rium , Paenibacillus, Planococcus, Pseudomonas, Kocuria , BRIEF DESCRIPTION OF THE DRAWINGS Streptomyces , Dietzia , and Amnibacterium . [0023 ] In order to describe various features and concepts [ 0016 ] In some aspects , the disclosure relates to non of the present disclosure , a more particular description of culture -based methods for detecting L - form bacteria ( e.g. , certain subject matter will be rendered by reference to L - form bacteria associated with endometriosis ) in a biologi specific embodiments which are illustrated in the appended cal sample obtained from a subject . For example , in some drawings . Understanding that these figures depict just some embodiments , one or more L - form bacteria are detected in example embodiments and are not to be considered to be a sample using species -specific markers ( e.g., identification limiting in scope , various embodiments will be described of one or more genes , proteins, metabolites , signaling mol and explained with additional specificity and detail through ecules , etc. that are unique to a given type, genus , and / or the use of the accompanying drawings in which : species of bacteria ) or a panel of species -specific markers [0024 ] FIG . 1 illustrates one embodiment of a method for ( e.g. , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 20 , or more markers ) . Markers screening a sample for the presence of L - form bacteria ; or other biological, chemical or genetic characteristics could [ 0025 ] FIG . 2 illustrates progression of an aging cell be assayed for example using hybridization assays ( e.g. , infected with L - form bacteria ; nucleic acid sequencing, such as DNA and RNA sequencing , [0026 ] FIG . 3 illustrates one embodiment of a method for sequence - specific binding assays, sequence - specific ampli screening for L - form bacteria including comminution of the fication , etc. ) or protein binding assays , for example using sample ; antibodies ( e.g., polyclonal antibodies , labeled polyclonal [ 0027 ] FIG . 4 illustrates one embodiment of a method for antibodies , monoclonal antibodies, labeled antibodies , transferring an L -form inoculant from a liquid growth labeled monoclonal antibodies, etc. ) . In some embodiments , medium to a solid growth medium ; the presence and /or identity of L - form bacteria in a biologi [0028 ] FIG . 5 is a photograph of a mixed culture of cal sample are determined using nucleic acid sequencing Microbacterium and Staphylococcus bacteria which have ( e.g., sanger sequencing, Illumina sequencing , nanopore been converted from an L - form morphology to a cell -wall sequencing, single molecule sequencing , etc. ). In some sufficient morphology , the mixed culture showing produc embodiments , the presence and / or identity of L - form bac tion of a purple / violet pigment indicative of production of a teria in a biological sample are determined using protein biologically active agent; and binding assays , for example an immunoassay (ELISA , [ 0029 ] FIG . 6 is a photograph showing the individual radioimmunoassay, enzyme immunoassay , counting immu bacterial strains of the culture of FIG . 5 after isolation into noassay , chemiluminescence immunoassay, fluoroimmuno separate cultures, showing that production of the pigment assay , etc.) . In some embodiments , the presence and / or has stopped . identity of L - form bacteria in a biological sample are determined using an analytical method, for example mass DETAILED DESCRIPTION spectrometry (MS / MS , MALDI- TOF, HPLC -MS , LC -MS , ICP -MS , Ion Mobility Spectroscopy , SELDI- TOF , etc. ), a Introduction colorimetric assay, a fluorescence assay, etc. [0030 ] L -form bacteria have been found to reside in the [0017 ] In some embodiments , an antibiotic agent is a blood and other tissues of some individuals , including broad - spectrum antibiotic . In some embodiments , a broad individuals otherwise appearing healthy and not otherwise spectrum antibiotic is a beta - lactam antibiotic . showing symptoms of infection . In some circumstances , US 2020/0237786 A1 Jul. 30 , 2020 3

such L - form bacteria may transition from a latent/ asymp the term refers to L - form bacteria that are associated with tomatic form to a symptomatic form . The disclosure is endometriosis and /or ovarian fibroid tumors. based , in part, on detecting certain L - form bacterial infec [0034 ] As used throughout this disclosure, the terms " cell tions in subjects having or suspected of having endometrio wall- sufficient bacteria ” (“ CWS bacteria ” ) or “ classic - form sis . In some embodiments , L - form bacterial infections may bacteria ” refer to strains of bacteria having a morphology go undiagnosed because standard laboratory testing proce with an identifiable and recognizable cell wall structure , dures fail to identify L - form bacteria and fail to diagnose the such as the thick peptidoglycan layer of Gram positive subject as having an infection . Further, diagnosis of endo bacteria and the thin peptidoglycan layer positioned between metriosis is often not even sought until prolonged periods of the cell membrane and the outer membrane ( lipopolysac infertility suggest it may be an issue . charide layer) ofGram negative bacteria . As used herein , the [0031 ] Aspects of the disclosure relate to methods for term CWS bacteria also refers to mycobacteria , bacteria diagnosing and / or treating a subject having or suspected of within the archaea domain , and other forms of bacteria having ( e.g. , having a higher risk of developing relative to known to those of skill in the art to typically exhibit a cell the normal female population ) endometriosis . In some wall structure , even if not necessarily easily categorized as embodiments , a sample is obtained from a subject and Gram positive or Gram negative bacteria . evaluated for L - form bacteria . In some embodiments , the [ 0035 ] The terms “ L - form bacteria ," “ pleomorphic bacte presence, absence and /or identity of one or more L -form ria , " " hidden bacteria , ” “ intracellular bacteria , " " fastidious bacteria in the sample is determined , for example by a bacteria , ” and the like generally do not have standard culture -based method or a non -culture based method as definitions . The terms are often used synonymously , but in described herein . In some embodiments , the one or more some instances, for example, the term “ intracellular bacte L - form bacteria of the sample are tested for antibiotic ria ” may refer to bacteria residing within a host cell regard sensitivity . In some embodiments , the subject from which less of level of cell wall formation of the bacteria . the biological sample was obtained is diagnosed as having or [0036 ] As used herein , the term “ L - form bacteria ” refers to likely having ( e.g. , having an increased risk of developing ) strains of bacteria often found to reside intracellularly within endometriosis and /or ovarian fibroid tumors . In some a host cell and which do not exhibit a full cell wall structure . embodiments , the subject is administered a therapeutic ( e.g. , Such bacteria are distinguished from typical cell -wall - suf one or more antibiotic agents ) based on the presence of the ficient bacteria for which traditional culturing and detection L - form bacteria and / or based on the results of antibiotic methods are directed . “ L - form bacteria ” include bacterial sensitivity testing. In some embodiments, a subject is treated strains with morphologies lacking any identifiable cell wall with an antibiotic agent ( e.g. , is administered an antibiotic structure or cell wall components , and include strains agent) that is cytostatic or cytotoxic to L - form bacteria that including an undeveloped or incomplete cell wall structure , are present in the subject ( e.g. , based on detection of the such as strains containing some cell wall components but L - form bacteria in the subject using one or more assays or lacking sufficient structure to fully define the cell wall ( e.g. , methods described herein ) . strains with variable shape as opposed to typical cocci, rod , [0032 ] Methods are described herein which enable the and / or spiral characterization ). The skilled artisan will rec isolation and culturing of L - form bacteria from a subject's ognize that the term “ L - form bacteria ” does not, however , sample ( e.g., a biological sample , such as a blood sample ) . refer to bacteria of the genus Mycoplasma. In some embodiments, methods described by the disclosure [0037 ] In some instances, the term “ L - form bacteria ” provide for the diagnosis of a subject as having an L - form therefore includes strains of bacteria that do not yet include bacterial infection . In some embodiments , where an L - form fully recognizable cell wall structures , but which are tran bacterial infection is determined to be present, the methods sitioning toward cell wall sufficient strains . The term described herein provide for the isolation , culturing , and “ L - form bacteria ” also refers to pleomorphic bacteria which identification of the L - form bacteria underlying the L - form are capable of progressing from a reduced -cell - wall or bacterial infection . In some embodiments , L - form bacteria absent -cell -wall - form toward a classic form with a full cell associated with endometriosis are identified , and it can be determined that the subject has or is at risk of developing wall . The term also includes species and /or strains of bac endometriosis . Such a determination may beneficially teria that are not known to exist in nature in a CWS form , enable more effective and less costly medical care . For but which have been found to reside in one or more samples example , differential treatment may be provided to the in L - form . subject based on the presence or absence of L - form bacteria [ 0038 ] The term “ L - form capable bacteria ” is used herein associated with endometriosis . A positive result may allow to describe bacteria that are found within an L -form sample the subject to forego other endometriosis diagnostic proce and which have been cultured from the L - form morphology into a CWS morphology . Such strains often exhibit flexible dures, which are typically much more invasive and /or costly . morphological characteristics and are able to revert back to an L - form morphology under certain environmental condi Definitions tions . [0033 ] Many of the examples and embodiments described [0039 ] Although the exemplary embodiments described herein are described in the context of detecting the presence herein refer specifically to bacteria , one of skill in the art will of, or likelihood of developing , endometriosis. However , it understand that certain principles disclosed herein may be will be understood that the same principles may also be utilized for culturing, screening, and /or detecting fungi ( e.g., applied to determine the likelihood of fibroid tumors of the yeast ) , protozoans , and other pathogenic microorganisms ovaries , and in particular, submucosal fibroid tumors of the capable of residing intracellularly within host cells and /or ovaries. Thus , where the description refers to “ endometrio capable of being hidden from immune system responses sis -associated L - form bacteria ,” it will be understood that within biological fluids or tissues. US 2020/0237786 A1 Jul. 30 , 2020 4

[0040 ] As used herein , the term “ sample ” refers to a [0044 ] In some embodiments , the presence or absence of biological sample obtained from a subject ( e.g., a subject L -form bacteria in a biological sample is detected relative to having or suspected of having endometriosis ) such as a a control sample . A control sample is generally a biological tissue sample ( e.g. , endometrial tissue , cervical tissue , vagi sample obtained from a subject that is not characterized by nal tissue, uterine tissue, etc.) , whole blood sample , serum one ormore signs, symptoms, or risk factors for endometrio sample , plasma sample , urine sample , and the like. Such sis . In some embodiments , a control sample is obtained from samples are typically obtained from mammalian sources . As a subject that is known not to harbor L - form bacteria ( e.g., used herein , “ sample ” may also refer to mixtures containing a subject that is not infected with L - form bacteria ). In some the tissue / clinical sample . For example , a sample may be embodiments , a control sample is an internal control sample . added to or mixed with a growth medium to promote the An internal control sample generally refers to a biological growth of bacteria within the sample . When such a mixture sample obtained from a subject having or suspected of is further processed (e.g. , transferred , analyzed , monitored , having endometriosis , where the sample is cultured accord stored , etc.) , the mixture may be referred to simply as the ing to standard microbiological culture techniques ( and not " sample. " L -form bacterial culture methods described by the disclo sure ). Diagnosis and Treatment of Endometriosis /Ovarian Fibroid [0045 ] Examples of standard microbiological culture tech Tumors niques include direct ( e.g., visual) examination of the bio [0041 ] In some embodiments , a method for detecting an logical sample to identify the presence of bacteria , non endometriosis -associated L - form bacteria within a subject in selective and selective culture in liquid broth and /or agar order to diagnose the subject as having endometriosis and /or plates, etc. , and are described for example in Washington J an ovarian fibroid tumor includes : ( 1 ) subjecting a sample A. Principles of Diagnosis . In : Baron S , editor. Medical obtained from the subject to conditions that promote the Microbiology . 4th edition . Galveston ( Tex . ): University of culture of L -form bacteria present within the sample; ( 2 ) Texas Medical Branch at Galveston ; 1996. Chapter 10. In detecting the presence or absence of endometriosis -associ some embodiments , the absence of bacteria in an internal ated L - form bacteria as a result of the culturing ; and ( 3 ) biological sample cultured by a standard microbiological based on detecting the presence of endometriosis - associated culture technique and the presence of one or more L - form L - form bacteria , diagnosing the subject as having endo bacteria in a biological sample obtained from the same metriosis or likely having ( e.g., having a higher risk of subject is indicative of the subject being infected with the developing relative to the normal female population ) endo L - form bacteria . In some embodiments , a subject infected metriosis and /or an ovarian fibroid tumor. with the L - form bacteria is diagnosed as having endometrio [ 0042 ] A subject is typically a female mammal. In some sis or having a higher risk of developing ( relative to the embodiments , a subject is a female human . The age of a normal female population ) endometriosis . subject can vary. For example , in some embodiments , a [0046 ] The disclosure is based , in part , on the detection of subject is between 5 years and 85 years old . In some certain genera or species of bacteria ( e.g. , L - form bacteria ) embodiments , a subject is between 8 years and 80 years old . in biological samples obtained from a subject having or In some embodiments , a subject is between 10 years and 60 suspected ofhaving endometriosis . In some embodiments , a years ( e.g. , any age between 10 and 60 years old , inclusive) . subject is determined as having endometriosis based upon In some embodiments , a subject has not undergone meno detection of one or more L - form bacteria selected from pause . In some embodiments , a subject is menstruating . Microbacterium , Bacillus, Micrococcus, and Staphylococ [ 0043 ] Aspects of the disclosure relate to detecting one or CUS . In some embodiments , one or more L - form bacteria is more L - form bacteria in a subject that has or is suspected of 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , or 10 species of L - form bacteria . In having endometriosis and / or ovarian fibroid tumor. As used some embodiments , one or more L -form bacteria is more herein , “ having or suspected ofhaving endometriosis ” refers than 10 species of L - form bacteria . Each species of the one to a subject characterized by one or more signs, symptoms, or more L - form bacteria can vary. For example , 1 species of and / or risk factors for endometriosis . Signs and symptoms Microbacterium , 4 species of Bacillus, and 4 species of of endometriosis include but are not limited to displaced Staphylococcus may be detected in a biological sample . In endometrial tissue , endometriomas, dysmenorrhea (painful another example a single species ofMicrobacterium may be periods) , pelvic pain , cramping, pain with intercourse , pain detected in a biological sample . In some embodiments , at with bowelmovements or urination ,menorrhagia ( excessive least one of the L -form bacteria is of a species set forth in bleeding ), menometrorrhagia (bleeding between periods) , Table 2 . infertility , fatigue , diarrhea , constipation , bloating , and nau [0047 ] Examples of Micrococcus bacteria include Micro sea . Examples of risk factors for endometriosis include but coccus aloeverae, Micrococcus antarcticus, Micrococcus are not limited to retrograde menstruation , surgical scar cohnii , Micrococcus endophyticus, Micrococcus flavus, implantation ( e.g. , as a result of hysterectomy or Caesarian Micrococcus lactis , Micrococcus luteus, Micrococcus lylae , section ) , early age of onset for menstruation , delayed onset Micrococcus mucilaginosis , Micrococcus roseus, Micrococ of menopause , short menstrual cycles, increased levels or cus terreus, Micrococcus mortus, and Micrococcus yun exposure to estrogen , low body mass index , alcohol con nanensis . In some embodiments the Micrococcus is Micro sumption , having one or more relatives ( e.g. , sister ,mother , coccus luteus. aunt, etc. ) having endometriosis , having a medical condition [0048 ] Examples of Bacillus bacteria include B. acid that prevents normal passage of menstrual flow out of the iceler, B. acidicola , B. acidiproducens, B. acidocaldarius, B. body, and uterine abnormalities . Additional risk factors acidoterrestris , B. aeolius, B. aerius, B. aerophilus, B. associated with endometriosis are described , for example by agaradhaerens, B. agri, B. aidingensis, B. akibai, B. alca Hemmings et al . (2004 ) Fertility and Sterility 81 (6 ) : 1513 lophilus, B. algicola , B. alginolyticus, B. alkalidiazotrophi 1521 . cus, B. alkalinitrilicus, B. alkalisediminis , B. alkalitelluris , US 2020/0237786 A1 Jul. 30 , 2020 5

B. altitudinis , B. alveayuensis, B. alvei, B. amyloliquefa spizizenii, B. s . subsp . subtilis , B. taeanensis , B. tequilensis , ciens, B. a. subsp . amyloliquefaciens, B. a. subsp . planta B. thermantarcticus, B. thermoaerophilus, B. thermoamylo rum , B. aminovorans[ 2 ], B. amylolyticus, B. andreesenii, B. vorans, B. thermocatenulatus, B. thermocloacae, B. thermo aneurinilyticus, B. anthracis , B. aquimaris , B. arenosi, B. copriae, B. thermodenitrificans, B. thermoglucosidasius, B. arseniciselenatis , B. arsenicus, B. aurantiacus, B. arvi , B. thermolactis, B. thermoleovorans, B. thermophilus, B. ther aryabhattai, B. asahii , B. atrophaeus, B. axarquiensis, B. moruber, B. thermosphaericus, B. thiaminolyticus, B. thio azotofixans, B. azotoformans, B. badius, B. barbaricus, B. parans, B. thuringiensis , B. tianshenii, B. trypoxylicola , B. bataviensis , B. beijingensis, B. benzoevorans, B. beringen tusciae , B. validus, B. vallismortis , B. vedderi, B. velezensis , sis , B. berkeleyi, B. beveridgei, B. bogoriensis, B. boron B. vietnamensis, B. vieti, B. vulcani, B. wakoensis, B. iphilus, B. borstelensis, B. brevis Migula , B. butanolivorans, weihenstephanensis, B. xiamenensis, B. xiaoxiensis , and B. B. canaveralius, B. carboniphilus, B. cecembensis, B. cel zhanjiangensis . lulosilyticus, B. centrosporus, B. cereus, B. chagannorensis, [0049 ] In some embodiments , the Bacillus is Bacillus B. chitinolyticus, B. chondroitinus, B. choshinensis , B. chun safensis , Bacillus simplex , Bacillus licheniformis . Bacillus gangensis , B. cibi, B. circulans, B. clarkii , B. clausii, B. stratosphericus, Bacillus dretensis , Bacillus subtillis , Bacil coagulans, B. coahuilensis, B. cohnii, B. composti, B. curd lus velezensis . Bacillus cereus, Bacillus subterraneus , lanolyticus, B. cycloheptanicus , B. cytotoxicus, B. daliensis , Bacillus persicus, or Bacillus thuringiensis . B. decisifrondis , B. decolorationis , B. deserti, B. dipsosauri , [0050 ] Examples of Microbacterium bacteria include B. drentensis , B. edaphicus, B. ehimensis , B. eiseniae, B. Microbacterium aerolatum , Microbacterium agarici, Micro enclensis, B. endophyticus, B. endoradicis , B. farraginis , B. bacterium amylolyticum , Microbacterium aoyamense, fastidiosus, B. fengqiuensis , B. firmus, B. flexus, B. Microbacterium aquimaris, Microbacterium arabinogalac foraminis , B. fordii, B. formosus, B. fortis , B. fumarioli, B. tanolyticum , Microbacterium arborescens, Microbacterium funiculus, B. fusiformis, B. galactophilus, B. galactosidilyti arthrosphaerae, Microbacterium aurantiacum , Microbacte cus, B. galliciensis , B. gelatini , B. gibsonii, B. ginsengi , B. rium aurum , Microbacterium awajiense , Microbacterium ginsengihumi, B. ginsengisoli, B. glucanolyticus, B. gor azadirachtae , Microbacterium barkeri, Microbacterium donae , B. gottheilii , B. graminis , B. halmapalus, B. haloal binotii , Microbacterium chocolatum , Microbacterium kaliphilus, B. halochares , B. halodenitrificans, B. halo deminutum , Microbacterium dextranolyticum , Microbacte durans , B. halophilus, B. halosaccharovorans , B. rium enclense , Microbacterium endophyticum , Microbacte hemicellulosilyticus, B. hemicentroti, B. herbersteinensis , B. rium esteraromaticum , Microbacterium flavescens, Micro horikoshii , B. horneckiae , B. horti, B. huizhouensis, B. humi, bacterium flavum , Microbacterium fluvii, Microbacterium B. hwajinpoensis, B. idriensis , B. indicus, B. infantis , B. foliorum , Microbacterium ginsengisoli, Microbacterium infernus, B. insolitus, B. invictae , B. iranensis , B. isabeliae , ginsengiterrae, Microbacterium gubbeenense , Microbacte B. isronensis , B. jeotgali, B. kaustophilus, B. kobensis , B. rium halimionae, Microbacterium halophilum , Microbacte kochii, B. kokeshiiformis, B. koreensis , B. korlensis , B. rium halotolerans, Microbacterium hatanonis [ 3 ] ,Microbac kribbensis, B. krulwichiae , B. laevolacticus, B. larvae, B. terium hominis, Microbacterium humi, Microbacterium laterosporus, B. lautus, B. lehensis , B. lentimorbus, B. hydrocarbonoxydans, Microbacterium hydrothermale , lentus , B. licheniformis , B. ligniniphilus, B. litoralis , B. Microbacterium immunditiarum , Microbacterium imperi locisalis , B. luciferensis , B. luteolus, B. luteus, B. macauen ale , Microbacterium indicum , Microbacterium insulae , sis , B. macerans , B. macquariensis , B. macyae, B. malac Microbacterium invictum , Microbacterium jejuense , Micro itensis , B. mannanilyticus, B. marisflavi , B. marismortui, B. bacterium keratanolyticum , Microbacterium ketosire marmarensis, B. massiliensis , B. megaterium , B. mesonae , ducens, Microbacterium kitamiense , Microbacterium kore B. methanolicus, B. methylotrophicus, B. migulanus, B. ense , Microbacterium kribbense, Microbacterium mojavensis , B. mucilaginosus, B. muralis , B. murimartini, B. kyungheense , Microbacterium lacticum , Microbacterium mycoides, B. naganoensis, B. nanhaiensis , B. nanhaiisedi lacus, Microbacterium lindanitolerans, Microbacterium liq minis , B. nealsonii , B. neidei, B. neizhouensis , B. niabensis , uefaciens, Microbacterium luteolum , Microbacterium lutic B. niacini, B. novalis , B. oceanisediminis, B. odysseyi , B. octi, Microbacterium mangrovi, Microbacterium marinila okhensis , B. okuhidensis , B. oleronius, B. oryzaecorticis, B. cus, Microbacterium maritypicum , Microbacterium oshimensis, B. pabuli , B. pakistanensis , B. pallidus, B. marinum , Microbacterium mitrae , Microbacterium murale , pallidus, B. panacisoli, B. panaciterrae, B. pantothenticus, Microbacterium nanhaiense , Microbacterium natoriense, B. parabrevis , B. paraflexus, B. pasteurii, B. patagoniensis, Microbacterium neimengense , Microbacterium oleivorans, B. peoriae , B. persepolensis , B. persicus , B. pervagus, B. Microbacterium oxydans, Microbacterium paludicola , plakortidis , B. pocheonensis , B. polygoni, B. polymyxa, B. Microbacterium panaciterrae , Microbacterium paraoxy popilliae, B. pseudalcalophilus, B. pseudofirmus, B. dans , Microbacterium petrolearium , Microbacterium phyl pseudomycoides, B. psychrodurans, B. psychrophilus, B. losphaerae , Microbacterium populi , Microbacterium pro psychrosaccharolyticus, B. psychrotolerans, B. pulvifaciens , fundi, Microbacterium proteolyticum , Microbacterium B. pumilus, B. purgationiresistens, B. pycnus , B. qingda pseudoresistens, Microbacterium pumilum , Microbacterium onensis , B. qingshengii , B. reuszeri , B. rhizosphaerae , B. pygmaeum , Microbacterium radiodurans, Microbacterium rigui, B. ruris, B. safensis, B. salarius, B. salexigens, B. resistens, Microbacterium rhizomatis , Microbacterium sac saliphilus , B. schlegelii, B. sediminis , B. selenatarsenatis, B. charophilum , Microbacterium saperdae , Microbacterium selenitireducens, B. seohaeanensis, B. shacheensis , B. schleiferi, Microbacterium sediminicola , Microbacterium shackletonii, B. siamensis , B. silvestris , B. simplex, B. sira sediminis , Microbacterium shaanxiense , Microbacterium lis, B. smithii, B. soli , B. solimangrovi , B. solisalsi, B. soli, Microbacterium sorbitolivorans[ 4 ], Microbacterium songklensis , B. sonorensis , B. sphaericus, B. sporothermo suwonense , Microbacterium terrae, Microbacterium terre durans, B. stearothermophilus, B. stratosphericus , B. sub gens, Microbacterium terricola , Microbacterium testaceum , terraneus, B. subtilis , B. s. subsp . inaquosorum , B. s . subsp . Microbacterium thalassium , Microbacterium trichoth US 2020/0237786 A1 Jul. 30 , 2020 6 ecenolyticum , Microbacterium ulmi, Microbacterium [0055 ] Some embodiments may further include providing xylanilyticum , and Microbacterium yannicii. In some differential treatment based on the results of the foregoing embodiments , the Microbacterium is Microbacterium mari steps. In some circumstances, where a patient is identified as typic??. harboring one or more endometriosis -associated L -form [0051 ] Examples of Staphylococcus include S. argenteus, bacteria, the likelihood of endometriosis being at least part S. arlettae, S. agnetis , S. aureus, S. auricularis, S. capitis, S. of the patient's symptoms may be determined to be likely , caprae, S. carnosus, S. caseolyticus, S. chromogenes , S. and further testing for endometriosis ( e.g., biopsy and /or cohnii, S. condimenti, S. delphini, S. devriesei, S. edaphicus, laparoscopy ) may be justified . On the other hand , where no S. epidermidis , S. equorum , S. felis , S. fleurettii, S. galli endometriosis -associated L - form bacteria are detected , a narum , S. haemolyticus, S. hominis, S. hyicus, S. interme different treatment protocol, such as first testing for other dius, S. kloosii , S. leei , S. lentus, S. lugdunensis , S. lutrae , S. potential causes of the patient's symptoms ( e.g., hormone lyticans, S. massiliensis , S. microti , S.muscae , S. nepalensis , level measurements ) may be called for prior to trying a more S. pasteuri , S. petrasii, S. pettenkoferi, S. piscifermentans, S. invasive procedure . pseudintermedius, S. pseudolugdunensis , S. pulvereri, S. [0056 ] Such differential treatment beneficially allows for a rostri, S. saccharolyticus, S. saprophyticus, S. schleiferi , S. more focused treatment and testing strategy , thereby saving schweitzeri, S. sciuri, S. simiae, S. simulans, S. stepanovicii, time and money as well as potentially lessening patient S. succinus, S. vitulinus, S. warneri, and S. xylosus. In some anxiety and discomfort . In at least some circumstances , the embodiments , a Staphylococcus bacterium is Staphylococ detection of endometriosis -associated L - form bacteria cus epidermis , Staphylococcus petrasii, Staphylococcus allows other potential causes of the patient's symptoms to be equorum , Staphylococcus pasteuri, Staphylococcus speibo ruled out or at least determined as less likely . For example , nae , and Staphylococcus warneri . as described above, where endometriosis -associated bacteria [ 0052] In some embodiments , detecting comprises detect are detected , endometriosis may be at least provisionally ing or having detected one or more additional L - form determined as the most likely cause of the patient's symp bacteria ( e.g. , genus of L - form bacteria that are not Micro toms, and further testing and / or treatment may proceed bacterium , Bacillus, Micrococcus, and Staphylococcus) in accordingly . Alternatively , where no endometriosis -associ the biological sample . Examples of one or more additional ated L -form bacteria are detected within the patient, the L - form bacterial genera include Brachybacterium , Paeniba patient may be considered as likely not having such endo cillus, Planococcus, Pseudomonas, Kocuria , Streptomyces , metriosis , and may be subjected to a different line of further Dietzia , and Amnibacterium . In some embodiments , the one testing in an attempt to identify a different cause of endo or more additional L - form bacteria is a species listed in metriosis or to determine some other cause of underlying Table 2 . symptoms. [0053 ] In some embodiments , the presence of L - form [0057 ] In some circumstances , a patient may be tested bacteria in a biological sample is detected and /or species of prior to experiencing any infertility issues or other symp L -form bacteria are identified after performing a method of toms of endometriosis . For example , as a prophylactic L -form bacterial culture described by the disclosure. In some measure , a patientmay be tested to determine the presence embodiments , L - form bacteria that have been cultured or absence of endometriosis - associated L - form bacteria and according to a method described herein are identified by thus the likelihood that the patient has or may in the future phenotypic analysis ( e.g., morphology ) , for example after develop endometriosis . visual analysis under a microscope. In some embodiments , [0058 ] As described herein , the presence of certain L - form L - form bacteria is identified using a genetic method , for bacteria is correlated to , and may be causally linked to , example nucleic acid sequencing such as 16S rRNA endometriosis and /or fibroid tumors of the ovaries. Using sequencing . In some embodiments , an L - form bacteria is the methods described herein , it has been found that the identified using biochemical assays , for example Gram presence in a patient's sample of certain L - form bacteria , for staining or by chemotaxonomic methods such as analysis of example L - form bacteria of the genus Microbacterium , quinone system or fatty acid profiles , or by restriction highly correlates with the patient having endometriosis enzyme digestion analysis . and /or fibroid tumors of the ovaries. In some embodiments , [0054 ] In some embodiments , the detecting comprises a the species of Microbacterium is Microbacterium maritypi non - culture -based method ( e.g. , a method that does not cum . As described further in the Examples , of females tested require culturing of bacterial cells from the biological which were found to harbor an L - form of Microbacterium sample prior to analysis ) for detecting L - form bacteria (e.g. , maritypicum , more than 80 % had previously indicated that L - form bacteria associated with endometriosis ). In some they suffered from endometriosis or fibroid tumors of the embodiments , one or more L -form bacteria are detected in ovaries . a sample using species - specific markers ( e.g., identification [0059 ] In some embodiments , a patient may harbor an of one or more genes , proteins , metabolites, signaling mol L - form Microbacterium strain along with at least one other ecules, etc. that are unique to a given type , genus, and /or antagonist or symbiont L -form bacterial strain . In some species of bacteria ). In some embodiments , one or more embodiments , a method for diagnosing the patient as having species - specific marker is detected using a hybridization endometriosis and /or an ovarian fibroid tumor includes : ( 1 ) assay, for example nucleic acid sequencing ( e.g., DNA or subjecting a sample obtained from the patient to conditions RNA sequencing ) , such as direct 16S rRNA gene sequenc that promote the culture of L - form bacteria present within ing . In some embodiments , the presence and /or identity of the sample ; (2 ) detecting the presence of Microbacterium L - form bacteria in a biological sample are determined using L - form bacteria , such as Microbacterium maritypicum , as a an analytical method , for example by matrix assisted laser result of the culturing ; (3 ) detecting the presence of at least desorption ionization time of flight (MALDI - TOF) mass one other L - form bacteria strain that is an antagonist or spectrometry . symbiont with the Microbacterium L - form bacteria ; and ( 4 ) US 2020/0237786 A1 Jul. 30 , 2020 7 based on detecting the presence of the Microbacterium carbapenems include but are not limited to Ertapenem , L - form bacteria and the at least one other L - form bacteria Doripenem , Imipenem (or Imipenem /Cilastatin ), and Mero strain , diagnosing the patient as having or likely having penem . endometriosis and/ or an ovarian fibroid tumor. In some [0067 ] In some embodiments , an antibiotic agent is a embodiments, the at least one other L - form bacteria is a cephalosporin . Examples of cephalosporins include but are Bacillus or Staphylococcus strain . not limited to Cefadroxil, Cefazolin , Cephradine , Cephapi [ 0060 ] In some embodiments , the Microbacterium and the rin , Cephalothin , Cefalexin , Cefaclor, Cefoxitin , Cefotetan , at least one other L - form bacteria interact with one another Cefamandole , Cefmetazole , Cefonicid , Loracarbef, Cefpro within the host patient to produce one or more biologically zil, Cefuroxime, Cefixime, Cefdinir , Cefditoren , Cefopera active agents that are correlated with or causally linked to zone , Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten , the patient's symptoms. Thus, in some embodiments , a Ceftizoxime , Moxalactam , Ceftriaxone, Cefepime, Ceftaro method for diagnosing a patient as having endometriosis line fosamil , and Ceftobiprole . and / or an ovarian fibroid tumor includes testing a sample [0068 ] In some embodiments , an antibiotic agent is a obtained from the patient for the sufficient presence of one glycopeptide. Examples of glycopeptides include but are not or more biologically active agents produced by the mixed limited to Teicoplanin , Vancomycin , Telavancin , Dalbavan L - form culture . cin , and Oritavancin . [0061 ] The disclosure relates , in some aspects , to methods [0069 ] In some embodiments , an antibiotic agent is a for treating endometriosis or ovarian fibroid tumor in a lincosamide. Examples of lincosamides include but are not subject in need thereof. In some embodiments, a subject in limited to Clindamycin and Lincomycin . need thereof is a subject who 1 ) exhibits one or more signs, [0070 ] In some embodiments , an antibiotic agent is a symptoms, or risk factors for endometriosis , and /or 2 ) has lipopeptide, for example Daptomycin . been determined to harbor an L - form bacterial infection [0071 ] In some embodiments , an antibiotic agent is a according to culture methods described by the disclosure . In macrolide . Examples of macrolides include but are not some embodiments , a method of treating endometriosis limited to Azithromycin , Clarithromycin , Erythromycin , comprises administering a subject one or more antibiotic Roxithromycin , Telithromycin , and Spiramycin . agents . [0072 ] In some embodiments an antibiotic agent is a [0062 ] As used herein an “ antibiotic agent” refers to a monobactam , for example Azitreonam . small molecule , protein , peptide, polypeptide, or nucleic [0073 ] In some embodiments , an antibiotic agent is a acid that inhibits growth of or destroys ( e.g., kills or results nutrofuran . Examples of nitrofurans include but are not in death of) a microorganism ( e.g., bacteria , fungi , protozo limited to Furacolidone and Nutrofurantoin . ans, yeast, etc.) . In some embodiments, an antibiotic agent [ 0074 ] In some embodiments , an antibiotic agent is an ( e.g. , an antibacterial agent) is an agent that is effective in oxazolidinone. Examples of oxaxolidinones include but are killing or inhibiting growth of L - form bacteria . not limited to Linezolid , Posizolid , Radezolid , and Tor [0063 ] In some embodiments , an antibiotic agent is a ezolid . broad -spectrum antibiotic (e.g. , antibiotics that are effective [0075 ] In some embodiments , an antibiotic agent is a against both Gram - positive and Gram - negative bacteria ), an penicillin or a penicillin derivative . Examples of penicillin antibiotic that inhibits bacterial cell wall growth ( e.g., car or penicillin derivatives include but are not limited to bapenems , beta - lactam antibiotics , glycopeptides, penicil Amoxicillin , Ampicillin , Azlocillin , Dicloxacillin , Flucloxa lins, etc. ), or an antibiotic that inhibits DNA or RNA cillin , Mezlocillin , Methicillin , Nafcillin , Oxacillin , Penicil synthesis or function ( e.g. , aminoglycosides, macrolides , lin G , Penicillin V , Piperacillin , Penicillin G , Temocillin , and quinolones, tetracyclines, etc.) . Examples of antibiotic (anti Ticarcillin . In some embodiments , an antibiotic agent is a bacterial ) agents include aminoglycosides, ansamycins , car penicillin combination , for example Amoxicillin /clavulan bacephems, carbapenems, cephalosporins, glycopeptides, ate , Ampicillin /sulbactam , Piperacillin /tazobactam , and lincosamides, lipopeptides, macrolides, monobactams, Ticarcillin /clavulanate . nitrofurans, oxazolidinones , penicillins, antibacterial poly [0076 ] In some embodiments , an antibiotic agent is an peptides , quinolones and fluoroquinolones , sulfonamides , antibacterial polypeptide , for example Bacitracin , Colistin , tetracyclines, and other agents ( e.g., Clofazimine, Dapsone , or Polymyxin B. Capreomycin , Cycloserine , Ethambutol, Ethionamide, Iso [0077 ] In some embodiments , an antibiotic agent is a niazid , Pyrazinamide, Rifampicin , Rifabutin , Rifapentine , quinolone or a fluoroquinolone. Examples of quinolones and Streptomycin , Arsphenamine, Chloramphenicol, Fosfomy fluoroquinolones include but are not limited to Ciprofloxa cin , Fusidic acid , Metronidazole , Mupirocin , Platensimycin , cin , Enoxacin , Gatifloxacin , Gemifloxacin , Levofloxacin , Quinupristin /Dalfopristin , Thiamphenicol, Tigecycline , Lomefloxacin , Moxifloxacin , Nadifloxacin , Nalidixic acid , Tinidazole , Trimethoprim , etc. ) . Norfloxacin , Ofloxacin , Trovafloxacin , Grepafloxacin , Spar [ 0064] In some embodiments , an antibiotic agent is an floxacin , and Temafloxacin . aminoglycoside . Examples of aminoglycosides include but [0078 ] In some embodiments , an antibiotic agent is a are not limited to Amikacin , Gentamicin , Kanamycin , Neo sulfonamide . Examples of sulfonamides include but are not mycin , Netilmicin , Tobramycin , Paromomycin , Streptomy limited to Mafenide, Sulfacetamide , Sulfadiazine, Silver cin , and Spectinomycin . sulfadiazine, Sulfadimethoxine , Sulfamethizole , Sulfame [ 0065 ] In some embodiments , an antibiotic agent is an thoxazole , Sulfanilimide , Sulfasalazine , Sulfisoxazole , ansamycin . Examples of ansamycins include but are not Trimethoprim -Sulfamethoxazole (Co -trimoxazole ; TMP limited to Geldanamycin , Herbimycin , and Rifaximin . SMX) , and Sulfonamidochrysoidine . [0066 ] In some embodiments , an antibiotic agent is a [0079 ] In some embodiments , an antibiotic agent is a carbaphem ( e.g., Loracarbef ) or a carbapenem . Examples of tetracycline. Examples of tetracyclines include but are not US 2020/0237786 A1 Jul. 30 , 2020 8

limited to Demeclocycline, Doxycycline , Metacycline , detection assay ) obtained from a subject during treatment . In Minocycline , Oxytetracycline , and Tetracycline . some embodiments , treatment is continued for a while ( e.g., [0080 ] The disclosure is based , in part , on the recognition for a week , a month , 1-6 months, or longer ) after there are that certain L - form bacteria are sensitive to ( e.g., not resis no longer any detectable signs of L - form infection in sample tant to treatment with ) specific antibiotic agents . In some obtained from the subject during (or after cessation of) embodiments , resistance to antibiotic agents is tested by a treatment. diffusion tab ( diffusion disk ) test, for example as described [0085 ] Compositions for different routes of administration in Reller et al . (2009 ) Clinical Infectious Diseases , 49 ( 11 ): are well known in the art (see , e.g., Remington's Pharma 1749-1755 . However , the skilled artisan recognizes that ceutical Sciences by E. W.Martin ) . additionalmethods of antibiotic sensitivity testing may also [0086 ] Dosage will depend on the subject and the route of be used . In some embodiments , an L - form bacteria associ administration . Dosage can be determined by the skilled ated with endometriosis ( e.g., Microbacterium maritypicum ) artisan . In some embodiments , a subject is administered a is sensitive to carbapenem antibiotic agents and /or tetracy dose of an antibiotic agent that is consistent with the dosing cline antibiotic agents , for example Imipenem , Meropenem , guidelines set forth in Porter , R. S., Kaplan , J. L., & Merck Doxycycline , or any combination thereof. & Co. ( 2018 ). The Merck manual of diagnosis and therapy . [0081 ] The route of administration to a subject may vary . Whitehouse Station , N.J : Merck Sharp & Dohme Corp. For In some embodiments , an antibiotic agent is administered to example , in some embodiments , a subject is orally admin a subject intravenously . In some embodiments , an antibiotic istered an antibiotic agent at a dosage between about 1 mg agent is administered to a subject orally . In cases where and about 2500 mg per dose ( e.g., any integer between 1 and multiple ( e.g., 2 , 3 , 4, 5 , 6 , 7 , 8, 9 , or 10 ) antibiotic agents 2500 , inclusive) and a frequency of between 1 time and 5 are administered to a subject , each antibiotic agent may be ( e.g., 1 , 2 , 3 , 4 , or 5 ) times per day. In some embodiments , administered via the same route or different routes . a subject is orally administered an antibiotic agent at a [0082 ] The dose and frequency of administration of an dosage between about 10 mg and about 250 mg per dose and antibiotic agent to a subject may vary . Generally , a subject a frequency of between 1 time and 5 times per day. In some is administered a therapeutically effective amount of an embodiments , a subject is orally administered an antibiotic antibiotic agent. A “ therapeutically effective amount" gen agent at a dosage between about 100 mg and about 1000 mg erally refers to the amount of an antibiotic agent that is per dose and a frequency of between 1 time and 5 times per capable of producing a desired result ( e.g. , reduction of day . In some embodiments , a subject is intravenously (IV ) severity or frequency of one or more signs or symptoms of administered an antibiotic agent at a dosage between about endometriosis , and /or reduction or clearance of an L - form 500 mg and about 5 g per four to six hours . In some bacterial infection ) . embodiments , a subject is intravenously ( IV ) administered [0083 ] In some embodiments , an antibiotic agent is an antibiotic agent at a dosage between about 1 g and about administered to a subject prior to the beginning of the 5 g per six to eight hours , 500 mg once per day ( 24 hours) , subject's menstrual cycle (e.g. , after the end of one period of 1 and 3 grams every eighthours , etc. menstrual bleeding and prior to the beginning of the next [0087 ] The disclosure relates, in some aspects , to admin period of menstrual bleeding ) . In some embodiments , a istration of one or more antibiotic agents to a subject to treat subject is administered an antibiotic agent 1, 2, 3 , 4 , 5 , 6 , 7 , endometriosis . As used herein , the terms " treat” and “ treat 8 , 9 , or 10 days prior to the beginning of the subject's ing” mean to reduce the severity or frequency of at least one menstrual cycle . In some embodiments , a subject is admin sign or symptom of a disease or disorder. For example , istered antibiotic agent during menstruation ( e.g. , while treating endometriosis , in some embodiments , relates to the menstrual bleeding is occurring ) . In some embodiments , a reduction of one or more signs or symptoms of endometrio subject is administered an antibiotic agent both prior to and sis , such as displaced endometrial tissue, endometriomas , during the subject's menstrual cycle . In some embodiments , dysmenorrhea (painful periods ), pelvic pain , cramping , pain an antibiotic agent is not administered to a subject while with intercourse , pain with bowel movements or urination , menstrual bleeding is not occurring ( e.g. , in between men menorrhagia ( excessive bleeding ) , menometrorrhagia struation of the subject) . In some embodiments , an antibiotic (bleeding between periods ) , infertility , fatigue , diarrhea , agent is administered to a subject ( e.g., administered once , constipation , bloating, and nausea . In some embodiments , twice , three times , four times, etc. , per day ) for between 1 treating endometriosis refers to the reduction ( e.g. , reduction week and 6 months, between 3 weeks and 10 months , or of about 50 % , 60 % , 70 % , 80 % , 90 % , 99 % , etc.) or clearance between 6 months and 12 months, for example through 1 , 2 , ( e.g. , total killing or removal from a subject such that there 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , or more menstrual cycles. In is an undetectable level of L - form bacteria relative to the some embodiments , an antibiotic agent is administered to a level of L - form bacteria in a subject prior to treatment ) of subject for between 1, 2 , 3 , 4 , 5 , 6 , 7, 8 , 9 , 10 , or more years. one or more L - form bacteria that are associated with endo [ 0084 ] In some embodiments , the duration of the treat metriosis . ment ( e.g., administration of an antibiotic agent to the subject) is determined by the persistence of the L - form Methods of Screening for L - form Bacteria bacterial infection . In some embodiments , a subject is evalu [ 0088 ] In some embodiments , methods of culturing ated after treatment ( e.g., monthly , every six months, or at an L - form bacteria are described by US Patent Publication suitable frequency ) to determine whether the L - form bacte US2016 /0168614 , the entire contents of which are incorpo rial infection has been cleared . In some embodiments , rated herein by reference . treatment is continued until there are no longer any detect [ 0089 ] FIG . 1 illustrates an exemplary method 100 of able signs of L - form bacterial infection in a sample ( e.g., in screening a sample for the presence of L - form bacteria and a blood or other sample described in this application , for culturing/ producing L - form capable bacteria . In some example using a cell growth assay and /or a molecular embodiments , the method includes a step 110 of collecting US 2020/0237786 A1 Jul. 30 , 2020 9

a sample , a step 120 of contacting the sample to a first tion , the dilution of the sample within the first growth growth medium , a step 130 of incubating the inoculated first medium dilutes the concentration of antibodies and other growth medium under a first set of incubation conditions, immune system components present within the blood and a step 140 of monitoring the inoculated first growth sample , also enabling greater growth of the L - form bacteria . medium for the presence of L - form bacteria . [0095 ] In some embodiments , immune system compo [0090 ] In some embodiments , the step 110 of collecting nents may be removed from the sample or from the inocu the sample is performed using standard sample collection lated first growth medium , or can be inactivated by adding techniques, such as a blood draw , tissue swab, plant tissue an inactivating agent, such as a binding compound or extraction , and the like . In some embodiments , the sample is complement inactivator, by adding one or more blocking collected in the same container in which the first growth antibodies , by washing , centrifuging , and /or filtering the medium is contained . Alternatively , the sample may be sample to separate cells from other immune system mol collected in one or more separate containers prior to storage , ecules , or simply by diluting the sample sufficiently within transport, and subsequent transfer to the container holding the growth medium to render the components ineffective. In the first growth medium . preferred embodiments , however, substances that would [0091 ] Various types and /or combinations of growth hamper polymerase chain reaction (PCR ) or other analysis media may be used as the first growth medium . For example , techniques ( such as ethylenediaminetetraacetate (EDTA ) , the first growth medium may be formulated as complex or that would inhibit conversion / reversion to classic form growth media ( e.g. , blood , yeast extract, bile , peptone , ( such as EDTA ), are omitted . serum , and / or starch containing medias ), defined growth [0096 ] After the sample is contacted with the first growth media , or a selective media ( e.g. , nutrient selective for medium in step 120 , the method proceeds to step 130 by mannitol , cysteine, lactose , sucrose , salicin , xylose , lysine , incubating the inoculated first growth medium under a first or combinations thereof; selective based on carbon source , set of incubation conditions . The collected sample is stored nitrogen source, energy source , and /or essential amino acids , at a temperature about body temperature or at a temperature lipids, vitamins, minerals , trace elements , or other nutrients ; lower than about body temperature . For example , the col and /or selective antibiotic / antimicrobial containing media ) . lected sample may be stored at a temperature, constant or Exemplary growth media that may be used in solid ( e.g., fluctuating , within a range or about 20 ° C. to about 40 ° C., with agarose ) or liquid form include R2A , nutrient, choco or within a range of about 25 ° C. to about 35 ° C., or more late blood , blood ,mannitol salt , Vogel Johnson ,Kligler iron , preferably within a range of about 25 ° C. to about 30 ° C., or Simmons citrate , Columbia , cetrimide, xylose - lysine -deoxy about 27 ° C. In preferred embodiments , the inoculated first cholate , tryptic soy , Tinsdale , Phenylethyl alcohol, Mueller growth medium is stored at a temperature below body Hinton , MacConkey, brain -heart infusion (BHI ) , and lyso temperature . geny broth media . [0097 ] It has been surprisingly found that L - form bacteria [0092 ] In preferred embodiments , the first growth medium within a sample grow at a greater rate at temperatures lower is a liquid growth medium . In one particular example , the than body temperature . For example , in human blood growth media is serum ( e.g., human , bovine ) and /or brain samples , which are typically stored at body temperature ( 37 ° heart infusion (BHI ) broth , and may be contacted with the C.) , it has been found that storage at a lower temperature sample as a liquid in suspension with the sample . In pre increases the growth of L - form bacteria within the sample ferred embodiments the growth media is formulated without and enables L - form bacteria , which would otherwise remain substances that would hamper or restrict the growth of any present at non - detectable levels , to grow to observable bacteria found within the sample . For example , the growth levels . Preferably , incubation also omits rocking or shaking media preferably omits antimicrobial enzymes ( e.g., of the growth medium in order to reduce the amount of lysozyme, protease , etc.) , antimicrobial peptides, and contact between any L - form bacteria and any antibodies or immune system components ( e.g., leukocytes , complement other immune components that may be present within the system proteins, antibodies or other immunoglobulins , etc. ). sample . [0093 ] For example , it has been discovered that L - form [0098 ] The inoculated first growth medium is incubated bacteria are often able to reside within a sample at a for a time sufficient to provide growth of any L - form low -grade level without eliciting a full immune response and bacteria present within the sample ( e.g. , for a time sufficient without converting to classic form . The presence of immune to allow any L - form bacteria present within the sample to system components or other growth hampering substances achieve a detectable population ). In some embodiments , this within such samples can prevent the bacteria from being monitoring period can be about 120 hours or even longer manifest in classic form , even though the bacteria are than 120 hours . In more preferred embodiments , this moni present within the sample in L - form . Under such circum toring period can be less than about 120 hours. For example , stances, the removal or dilution of growth hampering sub in some embodiments , themonitoring period can be within stances and /or the transfer of L - form bacteria to growth a range of about 24 hours to about 96 hours , or within a media without growth hampering substances can promote range of about 36 hours to about 84 hours. In other embodi progression of the bacteria within the sample to classic form , ments , the monitoring period is within a range of about 48 and thereby provide faster culture and screening of L - form hours to about 72 hours . bacteria within the sample . [ 0099 ] In some embodiments , a dual track culturing setup [0094 ] In some embodiments , the first set of incubation is followed by subjecting a first set of sample portions to a conditions promote the aging of the sample tissue cells , short- track monitoring period and a second set of sample allowing L - form bacteria present within the cells to grow . portions to a long - track monitoring period , where the short For example , as the cells die and rupture , more L - form and long -track monitoring periods have durations according bacteria are able to escape their intracellular positions and to the above ranges, with the short -track duration being move into the surrounding extracellular medium . In addi shorter than the long - track duration . Such a dual- track setup US 2020/0237786 A1 Jul. 30 , 2020 10

has shown good results by enabling faster results from the state 270 , the cell wall further breaks down and the cell short- track , when possible , withoutmissing the detection of continues to expand toward its limits . At a seventh state 280 , other, slower species and / or strains resulting from the long the cell ruptures due to degradation and excessive internal track . bacterial growth , releasing L - form bacteria into the sur [0100 ] Step 140 of monitoring the first growth medium rounding growth medium . during the monitoring period for the presence of any L - form [0104 ] Preferably , the inoculated first growth medium is bacteria may be performed by transferring the sample or a incubated until at least some ( e.g., 10 % or more , 25 % or portion of the stored sample to a microscope slide, well more , 50 % or more , 75 % or more , 90 % or more ) of the plate , or other such apparatus allowing the microscopic monitored cells of the sample have progressed to a state visualization of the sample or portion of the sample . In where they have ruptured to release intracellular L - form preferred embodiments , in order to avoid the disruption of bacteria . potentially fragile L - form bacteria within the sample or [0105 ] Some embodiments further include a step 150 of portion of the sample collected for microscopic inspection , transferring at least a portion of the inoculated first growth the visual monitoring is carried out without traditional medium to a second growth medium , and a step 160 of staining ( e.g., Gram staining ) or chemical or heat fixing incubating the second growth medium under a second set of steps. For example , the visualmonitoring may be carried out incubation conditions. In preferred embodiments , the second by direct microscopic observation of the sample or portion growth medium is a solid - phase growth medium (e.g. , thereof by preparing a wet -mount , live slide for observation . contained in a plate or slant ) . For example , solid -phase Although microscopy using live slides is the preferred growth media may include one or more of the growth media manner of monitoring for L - form growth , other suitable described above ( e.g. , complex media , defined media ,mini monitoring techniques include spectrophotometric methods mal or selective media ) incorporated into a solid substrate . (including colorimetry and measurement of optical density ) , Suitable solid substrates include those formed with agarose, staining , and measurements of turbidity , total cellular DNA collagen , laminin , elastin , peptidoglycan , fibronectin , and and / or protein levels , electrical field impedance , biolumi the like . nescence , carbon dioxide, oxygen , ATP production or con [0106 ] The second set of incubation conditions includes a sumption , and the like . temperature within a range of about 20 ° C. to about 40 ° C. [0101 ] Monitoring of the first growth medium may be Preferably , the second growth medium is incubated at carried out throughout the monitoring period . For example , approximately body temperature ( about 30 ° C. to 40 ° C. or monitoring may occur periodically according to a set sched about 37 ° C.) . The second growth medium is incubated at ule throughout themonitoring period , such as at set intervals this temperature for a time period of about 24 hours to 96 ( e.g., daily , every 12 hours , every 10 , 8 , or 6 hours , every 4 , hours, or about 36 hours to 84 hours, or about 48 hours to 3 , or 2 hours , hourly , or even more frequently ) . In some 72 hours , or about 60 hours. In some embodiments , the circumstances , a sample may be monitored throughout a temperature is then adjusted to a range that is below body monitoring period , and may fail to exhibit any indication of temperature (e.g. , about 25 ° C. to 35 ° C., or about 25 ° C. to bacterial presence . At this point, in some embodiments , the 30 ° C., or about 27 ° C.) for a time period of about 4 days to method is completed and a negative result is returned ( e.g. , about 30 days, or about 7 days to about 21 days, or about 14 the method either detected or failed to detect the presence of days . In preferred embodiments, the temperature is adjusted any L - form bacteria in the sample ). to a range that is below body temperature for a time period [0102 ] Prior to transferring to a second growth medium , of about 1-7 days , or about 3-5 days . the inoculated first growth medium is preferably incubated [ 0107 ] In some embodiments , the step 150 includes trans until L - form bacteria within the medium have progressed to fer to multiple types of solid - phase growth media in order to a state of sufficient growth . FIG . 2 illustrates a typical isolate multiple strains that may be present within the progression of a red blood cell harboring L - form bacteria sample . For example , a set of agar plates may be prepared once placed under the first set of incubation conditions. A to receive the sample , with several of the agar plates healthy red blood cell 210 that harbors L - form bacteria will containing different forms ofmedia ( such as any of those begin to progress to a first state 220 , where internal pressure types discussed above with respect to the sample collection is created by developing L - form bacteria within the cell. At device , including selective growth media ), and these may be a second state 230 , L - form bacteria begin to transition from further divided by placing one set under aerobic conditions a non -microscopically observable form ( e.g., under about after inoculation and another set under anaerobic conditions 0.05 um ) to an observable form . At a third state 240 , internal after inoculation (e.g. , by placing in a standard anaerobic structures of the red blood cell begin to break down ( e.g. , chamber maintained with carbon dioxide ) . During or after through the action of lysozymes ), freeing up additional incubation , the method can include the step 170 of moni nutrients for L - form growth and creating greater internal toring the second growth medium for bacterial growth (e.g. , pressure within the cell . In some circumstances it has been using one or more of the monitoring techniques described observed that many cells stay at this state for long periods of herein ). time ( e.g. , severalweeks or months ). L - form bacteria appear [ 0108 ] Although defined medias may be used as growth to be present in such cells , but the L - form bacteria are not media in the methods described herein , it has been found released from the cells at detectable levels . When these types that L - form bacteria are able to be efficiently cultured and of cells are present, embodiments utilizing a comminuting detected using various complex medias such as BHI medias step may be particularly advantageous. or those including serum ( as the first and / or second growth [0103 ] In other circumstances , cells continue toward more medias) . Beneficially , the methods described herein have progressed states . At a fourth state 250, outward protrusions enabled the screening of L - form bacteria without the need of the cell become visible through weak spots in the wall of for generally more expensive defined medium formulations. the degrading red blood cell. At a fifth state 260 and a sixth Without being bound to any particular theory , it is thought US 2020/0237786 A1 Jul. 30 , 2020 11

that one or more process steps, such as the particular faster detection and screening of samples having L - form incubation conditions (e.g. , time, temperature ) and /or trans bacteria . In some embodiments , a biological sample ( e.g. , fer steps ( e.g. , transferring bacteria in a manner that enables cells of a biological sample ) are subjected to one or more bacteria within a sample to maintain a hydrated state ) techniques to lyse cells containing L - form bacteria ( e.g., enables L - form bacteria to be cultured without the need for release intracellular L - form bacteria into culture media ) , for custom -made or defined medias. example by subjecting the sample ( or cells ) to one or more [0109 ] Referring back to FIG . 1 , some embodiments fur freeze -thaw cycles or other cell lysis techniques known in ther include a step 180 of isolating bacteria grown on the the art. Although the exemplary method may be used to second growth medium . As growth occurs on the second prepare any of the forms of samples defined above, it may growth medium , some strains of L - form bacteria may tran be particularly useful in preparing samples known to con sition to classic form morphologies and may grow classic tain , or known to be likely to contain , biofilms, root nodules, form colonies on the second growth medium . Such bacteria and /or other aggregates potentially harboring L - form may be transferred to separate media ( e.g., one or more growth . complex , selective, or defined medias described herein ) until [0113 ] FIG . 3 illustrates another exemplary method 300 of a single strain is found on the media , and /or may be sampled screening for L - form bacteria that includes comminution of and further analyzed according to well- known microbiologi the sample . The embodiment shown in FIG . 3 has steps and cal characterization techniques , including microscopic elements similar to the embodiment shown in FIG . 1 , and examination , staining ( e.g. , Gram , Malachite green / Safra like numbers represent like elements . As illustrated , the nin , and acid - fast stains ), and selective growth testing. Other method includes a step 310 of collecting a sample , and a step analytical techniques such as chromatography, gel separa 320 of contacting the sample to a first growth medium . In tion, immunoassays, flow - through assays ( e.g. , plasmon some embodiments , the first growth medium is contained resonance detection ), fluorescent probe binding and mea within a comminution container. The comminution con surement, automated cell/ plate counting, microwell reading, tainer is typically formed as an elongate tube with a rounded DNA hybridization and amplification methods ( e.g., poly bottom portion , or with a tapering (e.g. , conical frustum ) merase chain reaction , strand displacement amplification ), shaped bottom portion . 16S rDNA sequencing, other molecular biological charac terization techniques , and combinations thereofmay also be [0114 ] The comminution container includes a comminut used to analyze bacteria cultured or isolated using the ing media configured to contact the sample and disaggregate methods described herein . biofilms, cell clumps, and other aggregates within the [0110 ] Beneficially , many of the bacteria cultured to a sample . The comminuting media is preferably formed from CWS form using one of the culturing embodiments crushed or shattered glass. Other embodiments may include described herein maintain a flexible morphology capable of comminuting media formed from beads, shards , particles, reverting back to L -form when exposed to appropriate fragments , filaments , or other structures configured to con conditions. tact the sample and disassociate particles within the sample , [0111 ] Any of the foregoing embodiments may also and may be formed out of metal, plastic , ceramic , or other include the addition of a transmembrane protein inhibiting materials or combinations of materials . agent to promote and /or augment the conversion of L - form [0115 ] The exemplary method includes a step 322 of bacteria to CWS form . It is presently theorized that at least comminuting the sample . In some embodiments, the sample some L - form bacteria are capable of resisting a host immune and first growth medium are vortexed ( e.g., by placing the response by modulating or secreting one or more of the comminuting container in a vortex apparatus ) to displace the host's transmembrane proteins in order to inhibit or dampen comminuting media within the liquid and to enable contact the host's immune response . In one example , an L - form between the comminuting media and the aggregated por bacteria may modulate or secrete the CD47 protein in order tions of the sample . In other embodiments , the sample may to inhibit macrophage response as a result of the CD47 be comminuted using magnetic stirring ( e.g., one or more protein engaging with SIRP - a . It is presently believed that magnetic stir bars included in the comminuting media ), or inhibiting one or more of such transmembrane proteins in a by shaking, vibrating, or otherwise displacing the commi collected sample ( e.g., blood sample ) will trigger or aug nuting media . ment the conversion of L - form bacteria to CWS form . Some [0116 ] In some embodiments , after comminuting, the embodiments may therefore include the removal of one or method includes a step 330 of incubating the inoculated first more transmembrane proteins and /or the addition of an growth medium under a first set of incubation conditions and inhibiting agent ( e.g., a targeted antibody) to inhibit one or a step 340 ofmonitoring the inoculated first growth medium more transmembrane proteins in order to trigger or augment for the presence of L - form bacteria . Alternatively , after the conversion of L - form bacteria to CWS form . comminuting , the method can proceed to a step 350 of transferring a portion of the first growth medium to a second Sample Comminution growth medium (preferably a solid growth medium ) without [0112 ] In some circumstances, it may be desirable to prior incubation of the sample . Such embodiments can subject a sample to blending, vortexing, sonication , or other beneficially reduce the culture time required before bacteria disruptive processes or combinations thereof in order to can be isolated , analyzed , and /or harvested . For example , the disassociate biofilms and /or aggregates, to rupture cells , or progression of infected red blood cells shown in FIG . 2 can to otherwise disperse any bacteria and increase exposure to be effectively bypassed or made to progress more rapidly . surrounding growth media ( e.g., prior to culturing of L - form [0117 ] In some embodiments , the method then proceeds bacteria or further screening ) . It has been surprisingly found through a step 360 of incubating the second growth medium that proper use of a comminution step in a screening process under a second set of incubation conditions, a step 370 of can increase yields, reduce culture times , and allow for monitoring the second growth medium for the presence of US 2020/0237786 A1 Jul. 30 , 2020 12

bacteria , and optionally a step 380 of isolating bacteria lant side up . It has been discovered that, at this point in the grown on the second growth medium , as described above. progression of L - form cultures , the L - form bacteria have typically progressed enough and /or the insert has sufficiently Inoculant Transfer interfaced with the solid medium , such that the benefits of [0118 ] FIG . 4 illustrates an exemplary method 400 for repositioning the solid medium to allow evaporation of transferring an inoculant from a first, liquid growth medium water that has built up in the inverted position outweigh the to a second , solid growth medium and incubating the solid detrimental effects , if any, of repositioning . growth medium ( e.g. , as part of the steps 150 and 160 in the [0124 ] In some embodiments, the method further includes embodiment of FIG . 1 or the steps 350 and 360 in the a step 470 of incubating the solid medium for a second embodiment of FIG . 3 ). As shown , the method includes a solid - phase incubation period . The second solid - phase incu step 410 of withdrawing an inoculant from the liquid bation time period is preferably performed in an atmosphere medium , and a step 420 of contacting the inoculant to a having similar relative humidity levels of the first solid surface of a solid medium . phase incubation time period , and for a time period ranging [0119 ] After contacting the inoculant to the solid medium , from about 12 hours to about 84 hours , or about 24 hours to the method includes a step 430 wherein the inoculant is about 72 hours , or about 36 hours to about60 hours, or about immediately ( e.g., within seconds or within about 1 or 2 48 hours . In some embodiments , one or more cultures are minutes) covered by an insert in order to maintain a hydrated further incubated at a temperature in a range that is below state of the inoculant. It has been found that positioning the body temperature (e.g. , about 25 ° C. to about 35 ° C., or insert over the inoculant beneficially enables L - form bacte about 25 ° C. to about 30 ° C., or about 27 ° C.) for a time ria within the inoculant to interface with the solid substrate period of about 4 to about 30 days, or about 7 to 21 about to begin colonization of the solid medium . It is theorized that days, or about 14 days. In preferred embodiments , the one L - form bacteria are often in a hydraulically fragile state at or more cultures are further incubated at a temperature this point in culturing ( e.g., due to reduced or absent cell below body temperature for a period of about 1 to 7 about wall structures ) , and that excessive drying and /or too rapid days, or about 3-5 days. concentrating of solutes within the inoculant containing the [0125 ] In some embodiments , a dual track culturing setup L - form bacteria can inhibit further culturing of the L - form is followed by subjecting a first set of sample portions to a bacteria . short -track monitoring period and a second set of sample [0120 ] In some embodiments , the insert is a glass panel, portions to a long - track monitoring period , where the short glass slide , or other material configured to sit upon the solid and long - track monitoring periods have durations according media and preferably, to maintain position relative to the to the above ranges , with the short -track duration being solid media ( e.g. , through adhesive forces between the inner shorter than the long- track duration . Such a dual- track setup surface of the insert contacting the inoculant and the inocu has shown good results by enabling faster results from the lant) . Other embodiments may include inserts made from short - track ( e.g. , about 1 to about 7 days or about 3 to about rigid or film plastics, ceramics, or other materials . Prefer 5 days ), when possible , without missing the detection of ably , the insert is positioned to eliminate air pockets within other , slower species and / or strains resulting from the long the inoculant between the surface of the solid media and the track ( e.g. , about 7 to about 21 days, or about 14 days ) . inner surface of the insert. In some embodiments , an addi tional amount of inoculant may be contacted to other por EXAMPLES tions of the surface of the solid media not covered by the insert, if any. Example 1 [0121 ] In some embodiments , the method further includes [0126 ] Over 500 separate blood samples were collected a step 440 of positioning the solid medium for incubation ( about 120 of these were from individuals who gave more with the inoculant side facing down . For example , where an than one sample ). More than 30 separate synovial fluid agarose plate is used to contain the solid media , the plate is samples and 1 lymphatic fluid sample were also collected . positioned “ upside down” so that the surface to which the For each sample , about 0.5 ml or less of the sample ( about inoculant and insert were applied faces down . 2 drops ) was added to a tube containing 10-15 mlof bovine [0122 ] In some embodiments , the method further includes serum and a tube containing 10-15 ml of BHI broth . The a step 450 of incubating the solid medium for a first inoculated tubes were incubated at 27 ° C. Development of solid -phase incubation time period of about 4 hours to 24 L - form culture was monitored by preparing wet mount live hours , or about 6 hours to 18 hours, or about 12 hours. The slides daily . Samples were monitored for a period of up to incubation may be carried out under the temperature con 30 days. Samples that showed indications of L - form bacte ditions described in relation to step 160 of FIG . 1. Prefer rial growth were typically incubated for at least 48 hours , ably , the incubation is also carried out in an atmosphere and typically began to show signs of progressive growth having a relative humidity that is sufficient to prevent overly within 48-72 hours . L - form bacteria were not observed to rapid drying of the inoculant. progress to a CWS form while within the broth . [ 0123 ] As explained above , it has been discovered that [0127 ] For samples in which L - form bacterial growth was greater culturing efficiency is made possible by maintaining detected , the broth was used to inoculate a variety of agarose a hydrated state of the inoculants and growth media as the plates (mannitol salt, BHI, tryptic soy , tryptic soy w / 5 % disclosed methods are performed . For example , during the sheep's blood , chocolate blood , Vogel Johnson , Simmons first solid -phase incubation time period , the relative humid citrate , Columbia , brewer's yeast, nutrient, MacConkey ity may be maintained within a range of about 40 % to about agar, starch agar, and Kligler Iron agar) . The inoculant was 100 % , or about 50 % to about 90 % , or about 60 % to about immediately covered with a sterile cover slide to prevent 80 % . In some embodiments , the method further includes a dehydration of L - form bacteria . Extra inoculant was step 460 of repositioning the solid medium with the inocu streaked onto remaining portions of the agarose surface. A US 2020/0237786 A1 Jul. 30 , 2020 13

set of plates was then incubated at 37 ° C. in an aerobic standard direct inoculation technique. The results show that incubation unit , and a set of plates was incubated at 37 ° C. use of the L - form growth protocol can significantly improve in an anaerobic chamber. Sterile water was supplied in order the ability to screen for and then culture and produce L - form to maintain a humid environment within the incubation capable bacteria . areas . The plates were placed agarose - side down for 12 hours and then were flipped to agarose -side up and incu Example 3 bated for an additional 48 hours . Plates were then removed and sealed in a plastic bag in order to retain moisture and [0131 ] Of the subjects tested using the culturing process were further incubated at 27 ° C. for 5 days. At 5 days , plates described in Example 1 , 10 subjects were found to harbor were inspected for growth . Each colony was transferred the Microbacterium maritypicum strain DSM 12512. Of the ( isolated ) to a set of nutrient agar and blood agar ( trypticase 10 subjects with this Microbacterium strain , 8 were female , soy agar with 5 % sheep blood ) plates . and 7 of the 8 had indicated in a health questionnaire that [ 0128 ] Isolated colonies were characterized using a they suffered from endometriosis or fibroid tumors of the BioLog GEN III MicroPlate 96 well plate as well as 16S ovaries . rDNA sequencing . The L - form growth protocols have resulted in the culture and isolation of over 254 unique Example 4 strains of bacteria originally residing in respective samples [0132 ] The culture obtained from one of the 7 female as L - form bacteria . subjects self -identifying as having endometriosis was fur ther examined . As shown in FIG . 5 , the culture obtained Example 2 from the subject's sample produced a visible purple hue [0129 ] A comparative study was conducted to compare a thought to be indicative of production of one or more standard culturing process to the process of Example 1. Each biologically active agents such as an enzyme. Genetic analy sample was divided into two portions. The first portion was sis showed the culture to be a mixed pair ofMicrobacterium used to directly inoculate two nutrient agars , which were maritypicum and Staphylococcus epidermidis , shown in then incubated and monitored for growth . The second por FIG . 6. Surprisingly , upon isolation , neither strain produced tion was used as inoculant in the L - form growth protocol of the purple hue . When reunited as a co -culture , the purple hue Example 1. Results of the comparative study are shown in returned . Table 1 ( samples which showed no growth in either protocol [0133 ] Each of the Microbacterium maritypicum cultures obtained from each of the 7 female subjects who reported are omitted ). suffering from endometriosis or ovarian fibroid tumors were found to produce a purple or orange pigment when co TABLE 1 cultured with any other L - form capable bacteria isolated Bacteria from the subject (which group consisted of Staphylococcus cultured Bacteria cultured Sample via direct via process of and Bacillus species ). Type Example 1 [ 0134 ] The culture from the one female subject who was inoculation found to harbor L - form Microbacterium maritypicum , but Blood No growth Acintobacter genomospecies 15tu who did not indicate suffering from endometriosis or ovarian Bacillus pumilus/ safensis fibroid tumors , was also tested in co - culture . Surprisingly , Bordetella parapertussis none of the mixed cultures from this subject produced a Simplicispira metamorpha pigment ; however , when the subject's Microbacterium Micrococcus luteus A Bacillus salentarsenatis / jeotigaii maritypicum was cultured with a Staphylococcus or Bacillus Moraxella canis strains from any of the 7 other subjects , the mixed culture Unknown Rod did exhibit production of a pigment. Blood Bacillus pumilus/ Bacillus pumilus/ safensis safensis Bacillus pumilus/ safensis Staph . capitis ss Bacillus pumilus / safensis Example 5 urealyticus Bacillus thuringiensis / cereus Bacilus Vallismortis /subtilis [0135 ] Biological samples (blood samples) were obtained Blood No growth Bacillus plakortidis from 16 subjects . Each subject exhibited one or more signs Brachybacterium sacelii (26C ) or symptoms of endometriosis and had been previously Unknown Bacteria diagnosed with endometriosis by conventional examination Blood No growth Bacillus pumilus/ safensis Blood No growth Bacillus pumilus/ safensis (e.g. , physical exam , laparoscopy, etc.) . Blood samples were Blood No growth Bacillus lichenformis also obtained from 12 healthy subjects , having no prior Bacillus lichenformis signs, symptoms or previous diagnosis of endometriosis . Staphylococcus intermedius Blood No growth Bacillus pumilus/ safensis [0136 ] Each sample was divided into two portions. The Blood No growth Bacillus pumilus/ safensis first portion ( internal control) was used to directly inoculate Micrococcus luteus B two nutrient agars , which were then incubated and moni Corynebacterium terpenotabidum tored for growth ( standard direct inoculation ) . The second Blood No growth Bacillus pumilus/ safensis portion was used as inoculant in the L - form growth protocol Blood No growth Unknown rod described in Example 1. No bacterial growth was observed in cultures produced from the healthy volunteer samples . [0130 ] As shown, growth and culture of L - form bacteria to Cultures were subjected to 16S ribosomal sequencing in identifiable classic form was achieved using the process of order to identify the bacterial strains present. Results of the L -form growth protocol of Example 1 , even for many comparative study for subjects having signs or symptoms of samples which gave no results and no growth under a endometriosis are shown in Table 2 . US 2020/0237786 A1 Jul. 30 , 2020 14

TABLE 2 Subject 1609 1842 1888 1905 1985 3025 3104 3164 1619 3016 1545 1642 1644 1538 1636 1661 Total Bacteria Bacillus X X X X X 5 safensis Micrococ X X X X X 5 cus luteus Brachyb X acterium hainane nse Bacillus X 1 simplex Paenibac X illus lautus Bacillus X X X 3 lichenifor mis Micrococ X X 2 cus aloeverae Staphylo X 1 coccus petrasii Bacillus X 1 stratosp hericus Bacillus X drentensis Planococ X cus antarcticus Pseudom X 1 onas parafulva Staphylo X x x X X 5 coccus epidermi dis Bacillus X X X X X X 6 subtilis Bacillus X 1 velezensis Kocuria X 1 himachal ensis Staphylo X 1 coccus equorum Staphylo X coccus pasteuri Streptom X 1 yces speibonae Dietzia X 1 maris Microbac X terium barkeri Planococ X cus kocurii Staphylo X 1 coccus warnei Microbac X X X X X X X X 8 terium maritypic um Bacillus X X 2 cereus Bacillus X subterra neus US 2020/0237786 A1 Jul. 30 , 2020 15

TABLE 2 - continued Subject 1609 1842 1888 1905 1985 3025 3104 3164 1619 3016 1545 1642 1644 1538 1636 1661 Total Microbac X 1 terium flavum Amnibac X terium kyonggie nse Bacillus X 1 Persicus Bacillus X 1 thuringie nsis

[0137 ] It was observed that 8 of the 16 subjects tested TABLE 3 - continued (50 % ) were infected with L - form Microbacterium maritypi cum and presented with signs and symptomsof endometrio Most Effective Most Effective sis . Additionally , it was observed that 7 of the 8 subjects Subject IV Antibiotic Oral Antibiotic Resistance ? ( ~ 87 % ) that were infected with L - form M. maritypicum 1636 Imipenem , Meropenem Doxycycline Metronidazole , were co - infected with at least one L - form Bacillus and /or Clindamycin , Oxacillin L - form Staphylococcus bacterial species . No bacterial 1661 Imipenem Doxycycline Metronidazole , growth was observed in the internal control samples from Clindamycin each of the endometriosis subjects . Example 6 Example 7 [0138 ] Bacterial strains identified in Example 5 were [0139 ] Based upon the results of the diffusion disk sensi tested for antibiotic sensitivity by a diffusion disk test. Table tivity testing described in Example 6 , subjects identified as 3 describes the most effective intravenous ( IV ) and oral being infected with L - form M. maritypicum are adminis antibiotic agents tested against each strain . Also described in tered the antibiotic agent that is effective against M. mari Table 3 are antibiotic agents that bacterial strains cultured typicum are not resistant) . Preferably, none of the other from each subject were observed to display resistance. In the L -form bacterial strains identified in a given subject (e.g. , majority of samples, resistance to Metronidazole was Bacillus , Staphylococcus , etc. ) are resistant to the selected observed for M. maritypicum . Resistance to Oxacillin was antibiotic . As a result of being administered the antibiotic also observed in the M. maritypicum samples . agent, subjects are expected to have a reduction ( or clear ance ) of L - form bacteria and improvement in one or more TABLE 3 signs or symptoms of endometriosis . In some embodiments , Most Effective Most Effective subjects that are administered an antibiotic agent have Subject IV Antil tic Oral Antibiotic Resistance ? increased fertility relative to their fertility prior to being 1609 Meropenem Doxycycline Metronidazole , administered the antibiotic agent. Oxaillin , Cefuroxime 1842 Meropenem Doxycycline Metronidazole , [0140 ] Although the foregoing has been described in some Oxacillin detail by way of illustrations and examples for purposes of 1888 Amoxiclav clarity and understanding, it will be understood by those of 1905 Imipenem Amoxiclav , skill in the art that numerous and various modifications can Penicillin 1985 Rifapin Metronidazole be made without departing from the spirit of the present 3025 Imipenem , Meropenem Amoxiclav Oxacillin , Clinamycin disclosure. Therefore , it should be clearly understood that 3104 Imipenem , Meropenem Doxycycline the forms disclosed herein are illustrative only and are not 3164 imipenem , Meropenem Doxycycline Metronidazole intended to limit the scope of the present disclosure , but 1619 Imipenem , Meropenem Doxycycline Metronidazole , Oxacillin rather to also cover all modification and alternatives coming 3016 Amoxiclav Metronidazole , with the true scope and spirit of the invention . Oxacilin , Cefuroxime, Sulfa - Trimeth , What is claimed is : Penicillin , Clindamycin 1545 Doxycycline Metronidazile , 1. A method for detecting an L - form bacterial infection in Oxacillin , Sulfa Trimeth , Clindamycin a subject in order to diagnose the subject as having endo 1642 Doxycycline Metronidazole , metriosis and /or an ovarian fibroid tumor, the method com Oxacillin , Cefuroxime, prising : Sulfa - Trimeth , Amoxiclav subjecting a sample obtained from the subject to condi 1644 Cefuroxime Metronidazole , tions that promote the culturing of L -form bacteria Oxacillin , Clindamycin present within the sample ; 1538 Imipenem , Meropenem Amoxiclav Metronidazole Clindamycin detecting the presence or absence of bacteria of the genus Microbacterium as a result of the culturing ; and US 2020/0237786 A1 Jul. 30 , 2020 16

based on detecting the presence or absence of Microbac 12. The method of any one of claims 1 through 11, terium bacteria , diagnosing the subject as likely having wherein at least one of the first and second growth media is or not having endometriosis and/ or an ovarian fibroid a complex media . tumor . 13. The method of any one of claims 1 through 12, further 2. The method of claim 1 , further comprising : comprising comminuting the sample prior to transferring the based on detecting the presence of Microbacterium bac sample to the second growth medium in order to increase culture growth and /or decrease culture time . teria , continuing one or more additional tests for diag 14. The method of claim 13 , wherein comminution is nosing endometriosis and / or an ovarian fibroid tumor; performed in a comminuting container containing a com or minuting media , the comminuting media being configured to based on failing to detect the presence of Microbacterium contact portions of the sample to increase exposure of bacteria , foregoing one or more additional tests for L - form bacteria within the sample to surrounding growth diagnosing endometriosis and /or an ovarian fibroid media . tumor, or continuing further testing that is not specific 15. A method for treating endometriosis in a subject, the to diagnosing endometriosis and /or an ovarian fibroid method comprising obtaining or having obtained a biologi tumor. cal sample from the subject; detecting or having detected in 3. The method of claim 1 or claim 2 , wherein the culturing the biological sample one or more L - form bacteria , each comprises: L -form bacteria having a genus selected from Microbacte contacting the sample to a first growth medium ; rium , Bacillus, Micrococcus, and Staphylococcus; and incubating the first growth medium under a first set of administering to the subject a therapeutically effective incubation conditions ; amount of an antibiotic agent based upon the presence of the transferring at least a portion of the first growth medium , one or more bacteria in the biological sample . as an inoculant, to a second growth medium under 16. The method of claim 15 , wherein the subject has or is conditions that maintain a hydrated state of the inocu suspected of having endometriosis , optionally wherein the lant; and subject is characterized by one or more signs, symptoms, or incubating the second growth medium under a second set risk factors associated with endometriosis . of incubation conditions . 17. The method of claim 15 or 16 , wherein the subject is 4. The method of any one of claims 1 through 3, wherein a human . the Microbacterium bacteria is Microbacterium maritypi 18. The method of any one of claims 15 to 17 , wherein the ???. biological sample is blood . 5. The method of any one of claims 1 through 4 , further 19. The method of any one of claims 15 to 18 , wherein the comprising detecting the presence of at least one other detecting comprises the method of any one of claims 1 to 14 . L - form bacteria that is an antagonist or symbiont with the 20. The method of any one of claims 15 to 19 , wherein at Microbacterium bacteria , and based on detecting the pres least one of the L - form bacteria is of a species set forth in ence of the Microbacterium bacteria and the at least one Table 2 . other L - form bacteria , diagnosing the patient as having or 21. The method of any one of claims 15 to 20 , wherein the likely having endometriosis and /or an ovarian fibroid tumor. Micrococcus is Micrococcus luteus. 22. The method of any one of claims 15 to 20 , wherein the 6. The method of claim 6 , wherein the at least one other Bacillus is Bacillus safensis , Bacillus simplex , Bacillus L - form bacteria is a Staphylococcus or Bacillus species . licheniformis , Bacillus stratosphericus, Bacillus dretensis , 7. The method of any one of claims 1 through 6 , wherein Bacillus subtillis , Bacillus velezensis , Bacillus cereus, Bacil themethod has an accuracy for positive results that is higher lus subterraneus, Bacillus persicus, or Bacillus thuringien than about 50 % , higher than about 60 % , higher than about sis . 70 % , higher than about 80 % , or higher than about 85 % . 23. The method of any one of claims 15 to 20 , wherein the 8. The method of any one of claims 1 through 7 , wherein Microbacterium is Microbacterium maritypicum . the first growth medium is a liquid , and wherein the second 24. The method of any one of claims 15 to 23 , wherein the growth medium is a solid . detecting comprises detecting or having detected one or 9. The method of claim 8 , wherein the inoculant is added more additional L - form bacteria in the biological sample . to a surface of the second growth medium , the method 25. The method of claim 24 , wherein the one or more further comprising placing an insert over the inoculant to additional L - form bacteria are of a genus selected from seal at least a portion of the inoculantbetween the surface of Staphylococcus, Brachybacterium , Paenibacillus, Plano the second growth medium and the insert . coccus, Pseudomonas, Kocuria , Streptomyces, Dietzia , and 10. The method of claim 9 , wherein the second set of Amnibacterium . incubation conditions includes a first solid - phase incubation 26. The method of claim 25 , wherein the Staphylococcus time period and a second solid -phase incubation timeperiod , is Staphylococcus epidermis, Staphylococcus petrasii , the second growth medium being positioned inoculant side Staphylococcus equorum , Staphylococcus pasteuri, Staphy down for the first solid - phase incubation time period and lococcus speibonae , and Staphylococcus warneri. inoculant side up for the second solid -phase incubation time 27. The method of any one of claims 15 to 26 , wherein the period . antibiotic agent is a broad - spectrum antibiotic , optionally 11. The method of any one of claims 1 through 10 , wherein the broad -spectrum antibiotic is a beta - lactam anti wherein the first and second growth media are independently biotic . selected from the group consisting of: mannitol salt, Kligler 28. The method of claim 27 , wherein the broad -spectrum iron , Vogel Johnson , Columbia blood , brain heart infusion antibiotic is a carbapenem , optionally wherein the car (BHI ) , nutrient, bovine serum , and human serum . bapenem is Imipenem or Meropenem . US 2020/0237786 A1 Jul. 30 , 2020 17

29. The method of claim 27 , wherein the broad - spectrum antibiotic is a tetracycline , optionally wherein the tetracy cline is Doxycycline . 30. The method of claim 27 , wherein the broad -spectrum antibiotic is Amoxiclav . 31. The method of claim any one of claims 15 to 30 , wherein the antibiotic agent is administered intravenously . 32. The method of any one of claims 15 to 30 , wherein the antibiotic agent is administered orally . 33. The method of any one of claims 15 to 32 , wherein the antibiotic agent is administered to the subject during men struation of the subject.