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Conceptual Design of a Universal Donor Screening Approach for Vaginal Microbiota Transplant

The success of fecal microbiota transplant (FMT) in treating recurrent Clostridioides difficile infection has led to growing excitement about the potential of using transplanted human material as a therapy for a wide range of diseases and conditions related to microbial dysbiosis. We anticipate that the next frontier of microbiota transplantation will be vaginal microbiota transplant (VMT). The composition of the vaginal microbiota has broad impact on sexual and reproductive health. The vaginal microbiota in the “optimal” state are one of the simplest communities, dominated by one of only a few species of Lactobacillus. Diversity in the microbiota and the concomitant depletion of lactobacilli, a condition referred to as bacterial vaginosis (BV), is associated with a wide range of deleterious effects, including increased risk of acquiring sexually transmitted infections and increased likelihood of having a preterm birth. However, we have very few treatment options available, and none of them curative or restorative, for “resetting” the vaginal microbiota to a more protective state. In order to test the hypothesis that VMT may be a more effective treatment option, we must first determine how to screen donors to find those with minimal risk of pathogen transmission and “optimal” vaginal microbiota for transplant. Here, we describe a universal donor screening approach that was implemented in a small pilot study of 20 women. We further characterized key physicochemical properties of donor cervicovaginal secretions (CVS) and the corresponding composition of the vaginal microbiota to delineate criteria for inclusion/exclusion. We anticipate that the framework described here will help accelerate clinical studies of VMT.

The success of fecal microbiota transplant (FMT) in treating recurrent Clostridioides difficile infection has led to growing excitement about the potential of using transplanted human material as a therapy for a wide range of diseases and conditions related to microbial dysbiosis. We anticipate that the next frontier of microbiota transplantation will be vaginal microbiota transplant (VMT). The composition of the vaginal microbiota has broad impact on sexual and reproductive health. The vaginal microbiota in the "optimal" state are one of the simplest communities, dominated by one of only a few species of Lactobacillus. Diversity in the microbiota and the concomitant depletion of lactobacilli, a condition referred to as bacterial vaginosis (BV), is associated with a wide range of deleterious effects, including increased risk of acquiring sexually transmitted infections and increased likelihood of having a preterm birth. However, we have very few treatment options available, and none of them curative or restorative, for "resetting" the vaginal microbiota to a more protective state. In order to test the hypothesis that VMT may be a more effective treatment option, we must first determine how to screen donors to find those with minimal risk of pathogen transmission and "optimal" vaginal microbiota for transplant. Here, we describe a universal donor screening approach that was implemented in a small pilot study of 20 women. We further characterized key physicochemical properties of donor cervicovaginal secretions (CVS) and the corresponding composition of the vaginal microbiota to delineate criteria for inclusion/exclusion. We anticipate that the framework described here will help accelerate clinical studies of VMT.

INTRODUCTION
Evidence continues to accumulate that demonstrates the critical role that bacteria play in human health and disease. It is often cited that the commensal bacteria colonizing our epithelial surfaces, glands, and fluids outnumber the cells that make up our bodies (Abbott, 2016;Sender et al., 2016). It has even been suggested that the gut microbiota should be considered an additional organ (Baquero and Nombela, 2012;Liu, 2016). The gut microbiota is perhaps the most extensively characterized, and has been shown to influence a wide range of diseases and disorders affecting the gut and beyond (Clemente et al., 2012;Valdes et al., 2018). Indeed, the premise of microbiota transplantation originated in the gut with the development of fecal microbiota transplantation (FMT) as a strategy for treating recurrent Clostridioides difficile (C. difficile) infection. The tremendous clinical success of FMT has led to explorations in using FMT for treating inflammatory bowel disease, obesity, liver disease, depression, food allergies, antibiotic resistance, malnutrition, multiple sclerosis, and more (Borody et al., 2013). Further, FMT has motivated the study of other forms of microbiota transfer, including skin microbiota transplant (Myles et al., 2018;Perin et al., 2018) and vaginal microbiota transfer from mother to babies born by Cesarean section (Dominguez-Bello et al., 2016).
Despite being one of the simpler commensal bacteria communities, cervicovaginal microbiota play a key role in sexual and reproductive tract health. The earliest attempts to characterize the cervicovaginal microbiota were conducted in the mid 1800s, a time when puerperal sepsis killed at least 15% of women giving birth in Europe and America (Hallett, 2005). In 1879, Louis Pasteur reported observation of streptococci in the blood of women with puerperal fever (Dunn, 2005). He believed this may have resulted from mechanical trauma that allowed bacteria in the vagina to enter the bloodstream. Later in 1892, Doderlein reported that women who had Grampositive, lactic acid-producing bacilli in their vagina at the time of childbirth were less likely to develop puerperal sepsis after delivery (Doderlein, 1892;Thomas, 1928). Although there has been increasing awareness of the broad spectrum of "normal" (Smith and Ravel, 2017;Anahtar et al., 2018), it is generally considered that the "optimal" vaginal microbiota communities are dominated by one of only a handful of species of Lactobacillus (Linhares et al., 2011;Petrova et al., 2015). Diversity in the vaginal microbiota and lack of dominance by Lactobacillus species can be described clinically as bacterial vaginosis (BV), a condition that has been linked to increased risk of sexually transmitted infection acquisition and transmission (Cherpes et al., 2003;Wiesenfeld et al., 2003;Allsworth et al., 2008;Atashili et al., 2008;Cohen et al., 2012), urinary tract infections (Sumati and Saritha, 2009;Stapleton, 2016), and fertility and pregnancy outcomes (Hyman et al., 2014;Petricevic et al., 2014;García-Velasco et al., 2017;Stout et al., 2017). Further, rates of BV relapse after standard antibiotic treatment can be as high as 70% within 3 months (Larsson and Forsum, 2005). Beyond BV, vaginal microbiota have also been implicated in recurrent yeast infections (Zhou et al., 2009;Liu et al., 2013), colonization with group B streptococcus (Rosen et al., 2017;van de Wijgert, 2017), and potentially reproductive tract cancers (Xu et al., 2014;Kyrgiou et al., 2017). Many have drawn attention to the limitations of currently available treatments for BV and the need for innovation in approaches for modifying the vaginal microbiota (Bradshaw and Sobel, 2016;Martin and Marrazzo, 2016).
Vaginal microbiota transplant (VMT) has the potential to revolutionize the way we view and treat conditions affecting the female reproductive tract. Unfortunately, study of vaginal microbiota in preclinical animal models is severely limited by the fact that dominance of the vaginal microbiota and acidification by Lactobacillus species is a uniquely human phenomenon (Miller et al., 2016;Witkin and Linhares, 2017). Even our primate cousins have low vaginal colonization by Lactobacillus species, and it has been suggested that the normal rhesus macaque vaginal microbiota is a good model for human BV (Spear et al., 2010;Mirmonsef et al., 2012;Yildirim et al., 2014). Unlike FMT, there is no previous history of anecdotal clinical implementation of VMT. However, there is significant epidemiological evidence of vaginal microbiota transfer between women who have sex with women (WSW) (Marrazzo et al., 2002(Marrazzo et al., , 2009Vodstrcil et al., 2015). Thus, a logical next step is to determine whether cervicovaginal secretions (CVS) can be used to transplant vaginal microbiota from a donor to a recipient in a clinical setting. Undoubtedly, ensuring safety and tolerability are top priorities. Here, we describe the development of a universal donor screening protocol intended for maximal risk reduction to mitigate potential transmission of infectious pathogens, as well as collection, characterization, and testing procedures for donor CVS.

Ethics Statement
The sample collection and testing procedures described here were approved by the Johns Hopkins University Institutional Review Boards as a part of study IRB00131437. Informed consent was obtained from all human subjects prior to participation.

Screening Questionnaire
We have developed a questionnaire for pre-screening potential VMT donors. The questionnaire includes all questions listed in the FDA Guidance for Industry for Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) (Section IV, Donor screening, §1271.75, section E). In addition, we included screening questions that are consistent with what has been observed to impact vaginal microbiota and stability of vaginal microbiota communities, including sexual history, sexual behavior, and vaginal product usage. We also included questions about medical history, and travel history (e.g., potential exposure to Zika or Ebola) that could have an impact on risk of incident sexually transmitted infections. For the pilot donor screening study described herein, we used an abbreviated questionnaire with the primary goal of correlating testing outcomes with self-reported sexual behavior, vaginal symptoms, vaginal product usage, and self-reported history of reproductive tract and sexually transmitted infections.
Participant demographics and questionnaire data can be found in Table 1.

Test List
The list of clinical tests and laboratory characterizations with their submeasures and readouts with normal ranges, if applicable, can be found in Supplementary Tables 1 and 2 Mycoplasma genitalium, and Human papilloma viruses (HPV), as well as quantitative polymerase chain reaction (qPCR) and 16S rDNA sequencing to determine composition of vaginal microbiota communities. As 16S rDNA sequencing runs typically require batching of samples into a 96 well plate format, we sought to develop a qPCR approach that was predictive of the relative abundance of Lactobacillus species and Gardnerella vaginalis and could be readily performed on individual samples on the same day for rapid screening and eligibility determination.

Sample Collection
Potential study participants were recruited from a list of participants from the investigator's prior studies that (i) had agreed to be contacted for potential participation in future studies, and (ii) had provided Lactobacillus-dominated CVS samples as part of these prior studies. To be included in the study, all participants identified as female and were pre-menopausal between the ages of 18-45 yrs. The potential participants were informed that at the time of sample collection, they must not be currently menstruating or within 3 days of their last menstrual period, they must be currently healthy and free of vaginal symptoms, and must not have used vaginal products or had vaginal intercourse in the prior 3 days. Participants were advised to drink extra water in the time leading up to their appointment to ensure proper hydration for blood collection. Participants were consented using an interactive question and answer approach that explained the purpose of the study, the associated risks, and the testing information that was to be obtained. The participants signed a consent form for HIV testing in the state of Maryland. The participants then filled out the abbreviated questionnaire and asked questions for clarification as needed. The participants were then given a bag containing 5 unwrapped vaginal swabs [2 × BD Eswabs (Becton Dickinson), 1 × Mini-tip flocked swabs with viral transport media (Becton Dickinson), 1 × Digene HC2 DNA collection brushes (Qiagen), 1 × Aptima vaginal swab (Hologic)], a Softdisc menstrual fluid collection device (The Flex Company), a sterile wrapped urine specimen cup, and a 50 mL conical tube (Corning Falcon). The participants were shown diagrams to instruct them on how to use each swab and how to insert the Softdisc to collect CVS using a previously described method (Boskey et al., 2003). It was emphasized that the participants take their time collecting each specimen carefully, to ensure that none of the materials touched unintended surfaces, and to return for replacement swabs if any were dropped or contacted a surface other than the vaginal wall. The participant then went to a self-locked single stall restroom for sample self-collection. Upon their return to the clinical area, the participant was prepared for blood collection. The total volume of blood collected across various tubes to perform all tests was ∼50 mL. Specimens were then grouped for immediate overnight shipping to Memorial Blood Centers, transport to the JHMI Medical Laboratories, and transport on ice to the respective laboratory facilities for testing and characterization. Three participants had menstrual blood in their CVS, either trace amounts still present despite reporting 3 days post the end of their menstrual period, or because of an unanticipated early start to their period. They returned within 2-20 days to provide another CVS sample and vaginal Eswab for 16S rDNA sequencing, which were the samples used for the data reported herein.

Panther System
Aptima (Hologic) swabs were transported on ice and stored at 4 • C for <30 days until testing by the Aptima Combo 2 for chlamydia and gonorrhea, by Aptima TV for trichomonas, and by Aptima MG for Mycoplasma genitalium.

Human Papilloma Virus Assay
The Digene HC2 vaginal brushes were transported on ice and stored at 4 • C for <60 days until testing. The Roche Linear Array HPV Genotyping Test is a qualitative test that detects 37 human papillomavirus genotypes including 17 high risk types, 15 lowrisk types, and 5 unknown-risk/probable-high-risk (pHR) types (Muñoz et al., 2003;de Villiers et al., 2004). The tests were performed according to the manufacturer's protocol (Woo et al., 2007), with broad-spectrum amplification and reverse line blot hybridization for genotype discrimination (Gravitt et al., 1998;Low et al., 2015).

CVS Characterization
Specimens were transported from the clinical location to the laboratory in a cooler on ice. Immediately upon return to the laboratory, the 50 mL conical tube containing the Softdisc was centrifuged at 1,000 RCF for 2 min to collect the CVS. The CVS was transferred to a 1.5 mL Eppendorf tube using a 50 µL Wiretrol (Drummond Scientific). The approximate sample volume was noted (average 0.25 ± 0.14 mL, range 0.1-0.5 mL).
We were able to characterize 3 out of 4 Amsel's criteria for BV diagnosis, including: (i) pH >4.5, (ii) basic amine "fishy" odor upon mixing with 10% KOH (positive whiff test), and (iii) presence of clue cells in the wet mount. A sample had to meet all 3 criteria to be categorized as BV based on Amsel's criteria. CVS sample pH was measured using a Mettler Toledo EL20 pH meter with a micro-combination pH electrode MI-411 (Microelectrodes, Inc.). Slides were prepared for wet mount by rolling a swab coated in CVS on a standard microscope slide followed by the addition of 10 µL of normal saline and covered with a glass coverslip. The wet mount slide was observed for the presence of clue cells using differential interference contrast (DIC) microscopy. The whiff test was performed by dipping a cotton swab in the CVS, pipetting 100 µl of 10% KOH onto the swab, and using a gloved hand to waft air over to determine whether a fishy odor was produced. Another swab covered in CVS was rolled onto a second standard microscope slide and left to air dry for gram staining and Nugent scoring for diagnosis of BV (Nugent et al., 1991). For lactic acid measurements, ∼10 µL of CVS was transferred to a preweighed Eppendorf tube to obtain the sample mass (6-20 mg). The CVS was diluted with 490 µL of normal saline and frozen at −20 • C until performing the assay. The diluted samples were thawed and centrifuged at 1000 RCF for 5 min to pellet mucus solids. The supernatant was processed per manufacturer's instruction in a 96 well plate format using a D/L-lactic acid kit (R-Biopharm).

Multiple Particle Tracking to Assess HIV-1 Virion Mobility
Fluorescently labeled HIV-1 pseudoviruses were prepared as previously described (Nunn et al., 2015;Hoang et al., submitted). HIV virions (0.3 µL) were pipetted into 20 µL of undiluted CVS in a custom-made glass slide with a circular sample well, gently mixed, and immediately sealed with a glass coverslip. Twenty second videos of viral motion were recorded at room temperature using a Zeiss Axio Observer inverted epifluorescence microscope equipped with a 100x/1.46 NA oil-immersion objective and an EM-CCD camera (Evolve 512; Photometrics). The image resolution was 25 nm/pixel and sequential images were captured at a frame rate of 15 Hz. A minimum of 5 videos were collected per CVS sample. Virion trajectories were analyzed using automated MATLAB-based particle tracking software with a minimum of 16 frames (∼1 s) of consecutive tracking as previously published (Suh et al., 2005;Lai et al., 2007;Schuster et al., 2015).

DNA Extraction
BD Eswab fluid (150 µL) or 10 mg of CVS were resuspended in 180 µL of lysis buffer (1% Triton X-100, 20 mM Tris-HCl pH 8.0, 2 mM EDTA) with 20 mg/mL of lysozyme and incubated for 1 h at 37 • C. Following lysozyme treatment, DNA was extracted using the DNeasy R Blood and Tissue kit (QIAGEN R , Hilden, Germany), including the addition of 20 µL of proteinase K and incubation for 40 min at 56 • C for pretreatment of gram-positive bacteria. Following DNA extraction, DNA concentrations were measured by NanoDrop.

DNA Amplification/Library Preparation
Library preparation was performed by the JHMI Deep Sequencing and Microarray Core at the Johns Hopkins Medical Institute according to the Illumina 16S metagenomic library preparation protocol (Illumina, San Diego, CA) with primers that amplified the V4 region of the 16S gene. Paired-end sequencing of the pooled library was performed with the MiSeq system (Illumina, San Diego, CA), generating 2 × 250 reads. Adaptor and barcode trimming was also performed by the JHMI Deep Sequencing and Microarray Core at the Johns Hopkins Medical Institute.
Sequence Processing QIIME 1.9.1 was used to join paired reads, demultiplex samples, identify and filter out chimeras using USEARCH 6.1 (Edgar, 2010), perform open-reference OTU picking and make taxonomic assignments (Caporaso et al., 2010). Joining required a minimum of 15 base calls of overlap and <20% dissimilarity between matching sequences. Quality control during demultiplexing required 60% of base calls in a read to have a quality score above 17 with no more than 9 consecutive base calls below 17. The reference database used for OTU picking and taxonomic assignments is a custom reference database that combined an existing vaginal microbiome 16S database (Srinivasan et al., 2012) with the V4 regions of additional species represented in the VaHMP V1-V3 database (Fettweis et al., 2012).

Sequencing Data Analysis
Reads were rarefied in R (R 2017, R version 3.4.3) and kmeans clustering was performed to assign samples to CSTs. To compare vaginal swab and CVS samples, Inverse Simpson Indices were calculated in R using the vegan (2.5-4) diversity command and a Bray-Curtis dissimilarity matrix, and NMDS analysis was performed using the vegan metaMDS command and a Bray-Curtis dissimilarity matrix. Stress of the NMDS analysis was 0.1725. PERMANOVA statistics were calculated using the vegan adonis command.

Quantitative PCR and Standard Curve Generation
Quantitative PCR qPCR of vaginal samples and cultured bacteria was performed on Applied Biosystems, QuantStudio 3. Bacterial species-specific primers (Integrated DNA Technologies) for the 16S rRNA gene were used at a concentration of 100 nM (Supplementary Table 3) (Jespers et al., 2012). To standardize the qPCR, a fixed volume (2 µL) of DNA was used for cultured standards, CVS, and swab samples. The program started with an initial incubation at 95 • C for 20 s, followed by 40 cycles with denaturing at 95 • C for 1 s followed by anneal/extend at 60 • C for 20 s. Data is shown for qPCR of CVS; the correlation coefficients for Ct values between CVS and swab was 0.97 for L. crispatus, 0.96 for L. iners, 0.96 for L. jensenii, 0.96 for L. gasseri, and 0.85 for G. vaginalis.

Species-Specific qPCR Standard Curve Generation
Bacteria were cultured overnight in liquid media (see above) then diluted 1:10 into fresh media and incubated for an additional 3-12 h to minimize the amount of dead bacteria. The subcultures were serially diluted 10-fold 5 times in triplicate. Serial dilutions were then plated as described above and used for DNA extraction. Following qPCR, the cycle thresholds (Ct) of the dilutions were plotted against the log of the CFU concentration for each species. Linear fitting provided an equation that was used to predict the CFU concentration in CVS based on the Ct for each species of bacteria.

Calculation of Lactobacilli Fraction
To calculate the fraction of a particular species of Lactobacillus using 16S sequencing data, the number of reads assigned to that species was divided by the total number of reads assigned to all Lactobacilli. To calculate the predicted fraction of a particular species of Lactobacillus using qPCR data, the predicted concentration (CFU/mL) for a particular species was divided by the total predicted CFU/mL for all Lactobacilli species.

Statistical Analysis
PERMANOVA statistics were calculated on the Bray-Curtis dissimilarity command using the adonis command of the vegan package in R. Inverse Simpson indices were compared using the student's t-test. Correlation analysis and significance for qPCR standard curves and comparisons between specific Lactobacillus species representation from 16S rDNA sequencing and qPCR was performed in GraphPad Prism 8.1.0.

Participant Demographics
Participant demographics are shown in Table 1. The median age of the participants was 26.5 yrs with a range of ages from 23 to 35 yrs. Two of the 20 (10%) of participants were Hispanic or Latino. The majority of participants were White (12/10, 60%) ( Table 1).

Notable Findings From Self-Reported Questionnaires
The participants answered questions about their sexual history, history of sexually transmitted infections, vaginal symptoms, and vaginal product use. A summary of key questionnaire data is provided in Table 1. Birth control usage was high, with participants reporting None (4/20, 20%), Condoms (2/20, 10%), Oral contraceptives (5/20, 25%), and copper or progestin intrauterine device (IUD) (9/20, 45%). Two participants (10%) reported BV in the past, one of which appeared to have active BV by Amsel's criteria (also noted below) and one who had the appearance of mixed bacteria on wet mount and gram staining. Prior human papilloma virus (HPV) infection was reported by one participant (5%). Prior yeast infection was reported by 12/20 participants (60%). One participant reported prior UTI. Prior chlamydia infection or treatment for potential exposure was reported by 4/20 participants (25%). Prior herpes infection was reported by 1 participant who was also on viral suppressive therapy (also noted below). Self-reporting of vaginal symptoms was rare and did not correlate well with other test results that were criteria for exclusion. For example, vaginal discharge (2/20, 10%) was reported by two participants with Lactobacillusdominated microbiota, and not by the one participant that apparently had active BV. Vaginal product use was also rarely reported and did not correlate with other negative findings. Interestingly, the 1 participant that reported use of boric acid did not report prior incidence of BV, which is the situation where boric acid is most likely to be used. There was a wide range of reported total numbers of sexual partners (0-29). The number of new sexual partners during the past month was low (0-1), and the percentage of participants reporting that their current male partner was circumcised was high (15/20, 75%). All participants in the study reported no tobacco use and no prior pregnancies resulting in birth of a baby. Many of the participants also had some extent of medical records that were accessible to supplement the self-reported questionnaire answers. It was noted that one participant was on testosterone therapy, presumably as part of gender affirming therapy.

Blood Tests
As expected, a significant proportion (50%, 10/20) of the participants were CMV positive. Thus, while stool donors are typically not tested for CMV (Woodworth et al., 2017), we intend to ensure that CVS from CMV positive donors is used only in CMV positive recipients. Additionally, we found that 5/20 (25%) were positive for HSV-1 IgG, and two of these participants noted having oral cold sores in the past on follow-up. Out of an abundance of caution, we suggest that this be an exclusion criterion for potential VMT donors. Two participants out of 20 (10%) were HSV-2 IgG positive, where one was unaware of any past symptoms of infection and tested negative on the swabbased herpes NAT, and the other was on viral suppressive therapy. As expected, all participants (20/20, 100%) had rubella IgG and varicella zoster virus (VZV) IgG present due to past vaccination or exposure. Further, all participants tested were negative for rubella and VZV IgM, indicating there was no active infection. Likewise, 16/20 (80%) of participants were reactive for hepatitis A (HAV) IgG, but all participants tested were negative for HAV IgM. All participants (20/20) tested negative for current hepatitis B (HBV) and C (HCV) infections. One participant's bloodwork was indicative of microcytic anemia, which was consistent with a family history of thalassemia reported to the study team doctor on follow-up. While thassalemia does not have known impact on immunity or vaginal microbiota, for early investigations of VMT, a participant with thassalemia may be excluded under investigator discretion.

Swab Tests
We observed that there was not necessarily concordance between self-reporting of past yeast infection (12/20, 60%) and the presence of culturable yeast from a vaginal swab (6/20, 30%). Three of the participants that had culturable yeast did not report prior yeast infection, and 9 of the participants that reported past yeast infection did not have yeast reported after fungal culture. Regardless, all six participants with cultivable yeast (typically Candida albicans) did not have any symptoms of a yeast infection on follow-up. Bacteria species not considered to part of the normal urogenital flora were detected in 4/20 (20%) of participants. Two of these participants had culturable Staphylococcus bacteria but reported no symptoms of active infection on follow-up. One of the participants had light growth of numerous bacteria species including Escherichia coli and Bacteroides species, but reported that she dropped the swab on the floor when the study team doctor contacted for follow-up. One of the participants had moderate growth of Corynebacterium species on culture, which are typically innocuous. However, this participant was also the only participant (1/20, 5%) to test positive for Mycoplasma genitalium, and was instructed to see her physician on follow-up. All participants (20/20) tested negative for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis. One 1/20 participants (5%) tested negative for all strains of HPV, though another 6/20 (30%) had only "weak" or "very weak" positives that may warrant additional confirmatory testing. All participants (20/20, 100%) were negative for HPV16 and 18, the strains responsible for most HPV-related cancers. Given the intermittent nature of viral shedding, we suggest that vaginal swabs be collected with every donor CVS sample to test for HPV and herpes viruses.

Urine Tests
All participants tested negative for pregnancy, Chlamydia trachomatis, and Neisseria gonorrhoeae.

CVS
One participant did not report any history of BV, but the CVS sample characteristics were indicative of BV by fulfilling the 3 Amsel's criteria tested (positive whiff test, pH 4.62, visible clue cells/biofilm in wet mount). The presence of a polymicrobial bacteria community was confirmed by 16S rDNA sequencing (see below) and Nugent score (8-10). One participant had no notable exclusion criteria based on the questionnaire and screening tests, but the CVS itself appeared ovulatory (egg-white appearance, spinnbarkeit).

Microbiota Communities Were Consistent Between Swabs and CVS
There have been differing reports as to whether the sampling location and sampling method affects the composition of the vaginal microbiota (Kim et al., 2009;Virtanen et al., 2017). The Softdisc method described here collects material from the entirety of the cervicovaginal canal, which is then pooled together during centrifugation. Thus, we used 16S rDNA sequencing to characterize the vaginal microbiota communities for paired CVS and vaginal swab samples to determine whether the results would be similar. As shown in Figure 1, we found that 19/20 (95%) of the participants had CVS dominated by Lactobacillus spp., which was consistent with testing negative for BV based on the three Amsel's criteria characterized. CVS samples clustered into four distinct community state types (CSTs), three of which were predominated by Lactobacillus spp. (L. crispatus, L. iners, or a L. crispatus/L. iners mix). When comparing sequencing of DNA extracted from CVS samples and vaginal swabs, paired CVS samples and vaginal swabs were not discernible from each other using NMDS analysis (Figure 2A) and applying PERMANOVA did not reveal a significant difference between the distribution of the CVS samples or vaginal swabs (p = 0.986). The paired samples from each participant clustered into concordant CSTs and did not show a significant difference in diversity, as measured by the inverse Simpson Index (p = 0.7138) (Figure 2B).

Standardized qPCR Correlates With the Relative Abundance of Specific Bacterial Species
Because 16S rDNA gene sequencing is a high-throughput approach, samples are typically batched together in large numbers. For the purpose of screening CVS samples to rapidly determine individual donor and sample eligibility, we sought to determine whether qPCR could be used to provide rapid compositional information that was reflective of sequencing. Using species-specific primers for L. crispatus, L. jensenii, L. iners, L. gasseri, and G. vaginalis, we performed 5 qPCR reactions on DNA extracted from each CVS sample. By comparing the qPCR results with the relative abundance obtained from the 16S sequencing, we determined that in the case of G. vaginalis, a Ct cutoff of 20 readily distinguished three samples that had >10% relative abundance (red bars in the last three columns in Figure 1) from samples with low relative abundance of G. vaginalis ( Figure 3A). Furthermore, Ct < 20 reliably predicted dominance by Lactobacillus species (Figure 3A,  Supplementary Figure 1A), though not necessarily the relative abundance of each species in mixed samples. Therefore, to allow rapid predictive CST classification before 16S rDNA sequencing, we generated qPCR standard curves from laboratory strains of L. crispatus, L. jensenii, L. iners, and L. gasseri to relate Ct values with the concentration of colony forming units (CFU) per volume across four logs in concentration in serial dilutions. Strong linearity was observed in all four species over four logs in bacteria concentration (insets in Figure 3B, Supplementary Figure 1B). The comparison of the predicted fractional representation based on qPCR and the fractional representation based on the 16S rDNA sequencing is shown for the dominant species (L. crispatus, L. iners) in Figure 3B and for the minor species (L. jensenii, L. gasseri) in Supplementary Figure 1B. In all four cases, we observed strong correlations between the predicted relative abundance based on qPCR and the relative abundance based on 16S rDNA sequencing.

CVS Physicochemical Properties Correlate With the Presence of Lactobacillus
The protective role of the lactic acid and low pH of the vagina appears to be important for maintaining vaginal health (Olmsted et al., 2005;Aldunate et al., 2013;Hearps et al., 2017;Tachedjian et al., 2017), suggesting that the physicochemical characteristics of the CVS may be more important than the particular species  present. As shown in Figure 4A, dominance by L. crispatus is generally associated with lower pH. In comparison, samples dominated by L. iners or a mixture of L. crispatus and L. iners had a slightly higher average pH, though still below the clinical cutoff for BV (>4.5, gray dotted line). Indeed, only the one sample that was polymicrobial had a pH above 4.5 ( Figure 4A). We also found the relative concentrations of the D-and L-isomers of lactic acid (D-LA, L-LA) were reflective of the bacteria present in the CVS sample. Namely, there were relatively higher levels of D-LA compared to L-LA in CVS samples dominated by L. crispatus, where the opposite was true in samples dominated by L. iners ( Figure 4B). For the samples that were a mixture of L. iners and L. crispatus, the concentrations for D-LA and L-LA were more similar, with the exception of one sample that had higher D-LA concentrations (Figure 4B), which may reflect the metabolic contributions of both species. The total concentration of lactic acid (D + L) generally increased with decreasing pH, meaning that L. crispatus-dominated samples typically had higher total LA concentrations, though there also were L. iners-dominated samples and L. crispatus/L. iners mix samples within the cluster (Figure 4C). We further found that the ability of the CVS to adhesively trap fluorescently labeled HIV-1 virions correlated with the Lactobacillus spp. present. L. crispatus-dominated CVS consistently trapped virions ( Supplementary Figure 2A), supporting the mucosal barrier function to pathogens. However, virion mobility was more variable in L. iners and L. iners/L. crispatus mix CVS samples, where some samples trapped virions and other samples allowed for permissive diffusion of virions ( Supplementary Figure 2A). Generally, virion mobility increased with increasing CVS pH, with the transition being relatively sharp around pH 4.2 (Supplementary Figure 2B).
Thus, as pH is relatively easy to measure in real time and is an indicator of dominance by Lactobacillus spp. and the amount of lactic acid produced, we suggest a pH cutoff of ≤4.2 for use in VMT. Similarly, we found that a cutoff pH of ≤4.2 also correlated well with scoring negative for BV by Nugent (≤3) (Figure 4D). A few samples that were Lactobacillus-dominated by sequencing and pH < 4.5 had high Nugent scores in the intermediate (4-6) or BV (7-8) range. Two of these samples were dominated by L. iners but had significant amounts of Gardnerella vaginalis (Nugent scores 6 and 8). Based on these data, we suggest that Nugent score ≤2 is sufficient to identify Lactobacillus-dominated samples with high lactic acid content and low pH suitable for VMT.

Potential Eligibility Findings
The full lists of clinical and laboratory tests performed can be found in Supplementary Tables 1, 2, respectively. FDA testing requirements indicate that a potential donor of leukocyte-rich cells or tissues that is not sexually intimate with the recipient must be tested for HIV (types 1 and 2), HBV, HCV, Treponema pallidum; HTLV (types 1 and 2), CMV, Chlamydia trachomatis, and Neisseria gonorrhea. We propose that for VMT, a potential donor candidate should also be tested for HAV, HSV-1/2, VZV, EBV, rubella virus, Toxoplasma gondii, HPV, Trichomonas vaginalis, and Mycoplasma genitalium, cultivable yeast/fungi, and cultivable bacteria. The test list employed here included redundant serological testing and vaginal swab-based testing of herpes viruses (HSV-1/2, VZV). All active infections should be excluded. Evidence of past exposure to CMV, VZV, and rubella (including vaccination) should be matched between donors and recipients. We suggest that the evidence of past exposure to HSV-1 or HSV-2 should be an exclusion criterion. The presence of cultivable yeast in the absence of symptoms of an active yeast infection and/or evidence of yeast growth in the wet mount preparation need not be an exclusion criterion unless the participant indicates having had a history of yeast infections (>1 in the past). We suggest similar guidelines that only UTI occurring on more than 1 occasion be grounds for exclusion. The HCT/P guidelines stipulate chlamydia infection is only an exclusion if it has occurred in the prior 12 months, though we suggest that infections occurring >12 months ago should be considered along with other history and could potentially be grounds for exclusion at investigator discretion upon review. The presence of cultivable bacteria that are not considered "normal" urogenital flora (terminology used by the pathology laboratory) would result in exclusion. We suggest that self-reported product use need not be an exclusion criteria alone, but that donor participants should be instructed to avoid insertion of any vaginal products (including tampons) during the entire CVS collection period. Our results suggest that there is not a clear rationale for setting a maximum limit for number of sexual partners for inclusion as a CVS donor. However, we suggest that donor participants should be required to avoid participating in vaginal, rectal, or oral intercourse, as well as use of sex toys, digital penetration, etc. during the entire CVS collection period. As the effects of exogenous hormones on the vaginal microbiota are not well-studied, we determined that this would be an exclusion criteria for participating as a VMT donor. Further, while the procedures for participation as a donor do not likely pose any risk to pregnant women, we propose that the potential impacts of hormones and other factors on the vaginal microbiome during pregnancy be grounds for exclusion. Ovulatory CVS or CVS collected at the time of menses would not be ideal for transplant due to low bacteria density and elevated pH, but would not exclude a donor from providing additional CVS samples in the future. For the purposes of screening a potential donor's vaginal microbiota composition, our results suggest that qPCR can be used to perform rapid characterization of the relative abundances of the four common Lactobacillus species that is predictive of the relative abundance by 16S rDNA sequencing. We prefer the use of CVS isolated with the menstrual cup collection approach to a vaginal swab for this purpose, because with isolated CVS, sample mass can be standardized. In contrast, the amount of CVS collected with a vaginal swab is not known or easily standardized, which would affect the threshold Ct value between samples. Of course, use of this qPCR approach would require individual labs to generate standard curves using reference bacteria and standardization using individual instrumentation and reagents. Using our qPCR instrument and a standard mass of 10 mg CVS, we found that Ct < 20 (>10% relative abundance by 16S sequencing) for G. vaginalis should be an exclusion criterion. Similarly, dominance by Lactobacillus species was predicted by Ct < 20. Together with Nugent scoring, pH measurement, and lactic acid measurements, dominance by Lactobacillus species can be quickly confirmed in individual CVS samples. Further, all samples should still be characterized by confirmatory 16S rDNA sequencing, which is standard in the field. Also, we would suggest that a standard plating method and counting of CFUs should be used to determine the overall Lactobacillus "dose." While the HIV mobility assay is informative and correlative with other CVS physicochemical properties, the equipment and materials required are specialized and need not be considered a standard screening approach.
Based on the test results and exclusion criteria discussed above, we found that 7/20 (35%) of our participants may be eligible for CVS donation as part of a future VMT study. However, we anticipate that the actual success rate for participation as a CVS donor in a clinical trial will be much lower than 35% for several reasons. This small pilot study used an abbreviated screening questionnaire, as described in the Materials and Methods. The participants were selected from a pool of participants from the study team's prior clinical studies, increasing the likelihood that they would have Lactobacillusdominated vaginal microbiota and fulfill the donor criteria. Of note, the majority of the participants were White or Asian, which is consistent with the observation that White and Asian women in the U.S. are less likely to have BV (Allsworth and Peipert, 2007;Fettweis et al., 2014;Peebles et al., 2019). However, efforts should be made to recruit a racially/ethnically diverse donor pool, as the potential impact of race/ethnicity on the success of VMT is unknown. Additionally, the more stringent cut-off values suggested based on the participants in this study (Nugent ≤ 2,pH ≤ 4.2) may need to be adjusted for a larger and more racially/ethically and/or geographically diverse donor pool. Further, the "universal" donor approach would typically consist of an initial full blood, swab, and urine screening like that described here, followed by a 30-60 day period of sequential CVS sample collections (excluding menses and ovulation if not using hormonal contraceptives). Out of an abundance of caution, we propose that donors be willing to abstain from vaginal intercourse and vaginal product use during the full duration of CVS collection, which was not an eligibility criterion for this small pilot study. Although not performed here, we also suggest that every donor CVS sample be tested for the presence of semen with a test like the ABAcard P30 test for the forensic identification of semen. We further propose that due to the intermittent nature of viral shedding (Wald et al., 1995;Phipps et al., 2011;Gravitt and Winer, 2017), vaginal swabs be collected to test for herpes viruses and HPV at the time of every CVS sample. After collection of the last CVS sample, the donor should go through the full blood, swab, and urine testing procedure again after a quarantine period of ∼30 days. It is likely that this series of additional testing will result in additional screening failures.

DISCUSSION
Although the first modern literature report of fecal microbiota transplant (FMT) was documented in 1958 (Eiseman et al., 1958), it was not until 2013 that the first randomized clinical trial for treating recurrent C. difficile infection with FMT was reported (van Nood et al., 2013). Although the study did not involve blinding, it was considered to unequivocally represent the potential of FMT as a safe and effective treatment (Sampath et al., 2013). The prior year, the FMT process was greatly streamlined by demonstrating that equivalent efficacy could be achieved with (i) stool from standard volunteer donors rather than donors identified by the patient, and (ii) stool frozen prior to use (Hamilton et al., 2012). These findings paved the way for various stool banks around the country that screen potential donors and collect, process, and freeze stool for distribution to clinical providers. OpenBiome, a non-profit that reportedly shipped over 43,000 treatments since starting its service in 2013, was an early innovator in developing thorough screening and quarantining procedures for frozen, ready-to-use stool preparations to increase patient access and safety 1 . The development of the FMT field is an obvious source of inspiration for initiating study of other forms of microbiota transplantation, such as VMT. Thus, our goal here was to conceptualize a universal VMT donor screening process that is uniquely suited to what we understand about vaginal microbiota and sexually transmitted infections. For example, unique considerations for VMT donor participants include the number of sexual partners, practice of certain sexual behaviors and frequency, and use of vaginal products. Further, any history of sexually transmitted infections should be grounds for exclusion. The stringency of the universal donor screening process inherently limits the availability of eligible donor participants, which is why OpenBiome reports only 3% of their participants screened are deemed eligible for stool donation (Dubois et al., 2015) 1 . It is possible that if VMT were to be successful, a similar model for donor screening with CVS banking and distribution could be implemented. Further, the development of a rationally designed biotherapeutic product using clinical outcome data from VMT studies would be a logical next step, both from a safety perspective as well as the much larger potential patient population. The trajectory of the FMT field is similarly informative in this regard, and will aid in accelerating the development of other microbiota-based therapeutic strategies.
Much of what we understand about the dynamics of vaginal microbiota was learned in studies that employed vaginal swabs, self-collected or physician collected, as the means for isolating bacteria (Aagaard et al., 2012;Gajer et al., 2012;DiGiulio et al., 2015). Other studies have reported the physicochemical properties of undiluted CVS or cervicovaginal lavage fluids, which gives us added indirect measures of bacteria composition and some aspects of metabolic function (Lai et al., 2009a(Lai et al., ,b, 2010O'Hanlon et al., 2011;Chappell et al., 2014;Nunn et al., 2015). However, we and others have begun characterizing both the CVS and the composition of the vaginal microbiota to begin to correlate community structure and function (Nunn et al., 2015;Hoang et al., submitted). Here, we further demonstrate that the community structure is strikingly similar whether using a vaginal swab or an aliquot of the CVS as the biological matrix for DNA extraction, which supports the use of vaginal swabs as a reliable and valid representation of the native mucosal environment. Similar to what was described for Lactobacillus bacteria in culture, we found that the relative concentrations of D-and L-lactic acid were reflective of the Lactobacillus spp. present in the CVS (Witkin et al., 2013). We also found that L. crispatus-dominated CVS tended to have higher total lactic acid content, lower pH, and more effective immobilization of fluorescently-labeled HIV virions, which is consistent with prior observations made by our group and others (Nunn et al., 2015;O'Hanlon et al., 2019). Our data shown here also suggested that using a CVS pH cut-off of ≤4.2 and a Nugent score cut-off of ≤2 would be most suitable for VMT, which is slightly more stringent than the criteria defined for categorizing samples as negative for BV (Amsel et al., 1983;Nugent et al., 1991).
The procedures and data shown herein are not intended to be wholly inclusive of every consideration for screening potential donors for VMT. Further, to operate under an investigational new drug application (IND), there are additional considerations for Chemistry, Manufacturing, and Controls (CMC). In the case of stool transplants, which were known to be efficacious for treating recurrent C. difficile prior to regulation by the FDA, we have yet to identify relevant measures of potency. In contrast to a small molecule drug or biologic, we do not know the key "active" component in stool, and thus cannot quantify the concentration or activity of that component. Further, the clinical success of using processed stool with 10% glycerol added prior to freezing (a standard approach for freezing bacteria stocks) was reported without characterization of the stool before and after freezing (Hamilton et al., 2012;Satokari et al., 2015). Many studies since have looked at the effect of processing and storage on the stability of particular bacteria or the overall bacteria community Fouhy et al., 2015), though there is still not a general consensus on what is important for a successful clinical outcome. In the case of VMT, donor CVS is largely considered a monoculture of one Lactobacillus species, and thus, lends itself to performing characterization of the colony forming units (CFU) per unit volume before and after freezing. We can also determine the effect of cryoprotectants and other media on the Lactobacillus viability, which is an area of active study in our group. Of note, however, although we can directly characterize the "potency" (concentration in CFU/mg) of the Lactobacillus, it is also possible that the lactic acid, mucins, or other components of the host environment could play a role in the potential success of the transplantation process. While vaginal microbiota can be considered relatively monomicrobial, there is also evidence here and described by others that more than one species of Lactobacillus, such as L. crispatus and L. iners, can cohabitate (Ravel et al., 2011;Gajer et al., 2012). Further, although other species may be in the minority, it has yet to be determined whether the overall community structure with major and minor players is important. We anticipate that the trajectory of VMT will likely follow that of FMT, wherein there are many parallel efforts in academia and industry to determine what minimum cultivable components can be manufactured to produce uniform, standardized products with similar therapeutic efficacy as stool (Kelly et al., 2015;Hoffmann et al., 2017;Ott et al., 2017).
In addition to intensive and thorough screening of potential donor participants for VMT, it is also important to consider testing of potential recipient participants. An inherent risk of the VMT procedure is transmission of a sexually transmitted pathogen. For various reasons associated with participant safety and for determining the overall safety of the VMT procedure, the baseline infection status of the potential recipient should be known. Thus, potential recipients should be screened similarly to potential donor participants, but the inclusion and exclusion criteria would differ. In addition to the obvious consideration that BV would be an exclusion for donors but not for recipients, it is also feasible that a controlled herpes infection or HPV would not necessarily be an exclusion criterion for potential recipient participants. Because of the transient and intermittent nature of viral shedding, potential recipient participants would ideally be screened at multiple time points prior to undergoing the VMT procedure to maximize detection of asymptomatic viral shedding of HSV-1, HSV-2, and HPV. Further, samples for testing should be taken immediately prior to the VMT procedure, because the results will help interpretation of any notable findings that may occur during recipient follow-up, including whether they may be attributable to the donor CVS. As the field of VMT is in its infancy, ensuring safety and tolerability of the procedure is of paramount importance in the first clinical studies. Various standardized adverse event grading systems are available, such as the Division of AIDS (DAIDS) Female Genital Grading Table for Use in Microbicide Studies for local vaginal/reproductive tract adverse events. Further, follow-up testing of recipient participants can also determine whether a potential shift in vaginal microbiota composition impacts future risk of acquiring sexually transmitted infections. It is also conceivable that as VMT may be explored for other indications, additional follow-up considerations may be timing of childbirth and birth outcomes, or recurrence of conditions such as urinary tract infection or yeast infection.
Another important aspect to consider is the VMT procedure itself. As we describe, the donor CVS was collected using a non-absorbent polymer-based cup, allowing for collection of undiluted material by centrifugation. However, similar to FMT, dilution with fluid such as sterile saline would ease loading into a device for application and administration to the recipient. Thus, we anticipate that similar to FMT, dosing would be determined by the ratio of sample mass to diluent fluid volume. This is in contrast to the dosing approach for probiotic products, where the concentration of CFU/dose can be standardized during the manufacturing procedure-with CVS donation, the mucus itself may be as important as the bacterial colony count. However, in the case of CVS, we observe a wide range of CFU/mg between participants, which is also reflective of the functional differences of the dominant bacteria. As such, when working with CVS from individual donors, it is not straightforward to standardize the dose based on the concentration of CFU. This is perhaps particularly true for Lactobacillus species found in CVS, because when they are the predominant species, they are present in lower numbers of CFU than the total number of CFU when the community is polymicrobial (Hill, 1993). Indeed, the ability of lactobacilli to colonize and compete out other bacterial species in CVS does not appear to be directly dependent on their concentration.
In the discussion of how microbiota transplantation must be regulated differently than standard drugs and biologics, it is worth noting that VMT and vaginal microbiota transfer from mother to babies born by Cesarean section, or "vaginal seeding, " uses the same "drug." However, the nature of the screening procedures and the handling of the CVS are quite different, and thus, VMT has many more parallels to FMT than vaginal seeding. For example, the notion of universal donors cannot apply to vaginal seeding, as the donor must be the mother. Thus, screening failure rules out the possibility of conducting the procedure at all. Further, because of the relevant time constraints, the vaginal seeding procedure also rules out the possibility of performing thorough testing of the CVS at the time of collection (1 h before Cesarean section) and quarantining in the frozen state until test results are known. The CVS collection procedure for vaginal seeding is also quite different and requires no subsequent processing; absorbent gauze or swabs are used to "soak up" the CVS for direct transfer to the baby's skin after birth. Thus, the definition of the "dose" is also different, in that a single dose would just be the amount of CVS that is collected in the gauze while it is left in the mother's vagina. Finally, the use of the term "transfer" rather than "transplant" reflects the fact that, by definition, transplanted material is placed in the same area of the body from which it was obtained. In the case of vaginal seeding, the CVS is swabbed onto the baby's mouth, nose, and skin in order to mimic the process of passing through the vaginal canal during birth. In contrast, VMT is a true transplant of CVS from the vagina of the donor to the vagina of the recipient.

CONCLUSION
This project represents a multidisciplinary effort to establish a protocol for comprehensive screening and characterization of donor CVS for the purpose of VMT, including a parallel rigorous screening process for potential recipients of VMT. The limitations of this pilot study include the small sample size and selection of participants from past study participant pools known to the study team to have optimal microbiota, which would not be generalizable to larger populations. It is possible that the requirement for sexual abstinence throughout the CVS sample collection period, or the requirement for providing multiple sequential self-collected swabs and CVS samples intensively over a month-long period, may constrain enrollment in future studies. The stringency of our exclusion criteria for donors, motivated by the ethical and clinical imperative to avoid potential transmission of pathogens, is both necessary and challenging from the perspective of recruitment. However, once a donor has been identified, using this protocol, as a safe donor, she could ideally donate CVS on multiple appropriately screened occasions; the idea of a "super-donor" with no identified past or current infections and with favorable Lactobacillus-dominated microbiota is one that should be explored and is of potential high impact to the project and the field. It is certain that as the field of microbiota transplant expands, unique regulatory questions and dynamic scientific, ethical, and clinical considerations will continue to arise (Hoffmann et al., 2017). These should be met with equally dynamic innovative approaches that incorporate optimized human safety considerations while recognizing the potential value of microbiota transplantation in addressing BV and its varied adverse impacts on human health.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. As of submission, the raw sequencing data is not publicly available, but public links or the data itself will be supplied on request.

AUTHOR CONTRIBUTIONS
LE, EW, CG, PG, EF, CH, RC, and JC contributed conception and design of the study. LE wrote the first draft of the manuscript and KD, SB, and EW wrote sections of the manuscript. KD, SB, FZ, HZ, TH, LE, CG, and PG conducted experiments, oversaw staff testing of clinical specimens, and/or collected clinical specimens. EW, EF, and CH oversaw the clinical study and coordinated clinical activities. KD, SB, FZ, HZ, TH, PG, EW, AA, and LE performed data analysis and/or data interpretation. All authors contributed to the manuscript revision, read, and approved the submitted version.

FUNDING
We received regulatory guidance from the Institute for Clinical and Translational Research (ICTR) funded by a grant from the NIH through NCATS (UL1TR001079). We also acknowledge funding from the Burroughs Wellcome Fund Preterm Birth Initiative (grant 1015020). KD was supported by a Hartwell Foundation Postdoctoral Fellowship, SB was supported by the Doctoral Diversity Program (DDP), HZ was supported by a National Science Foundation Graduate Research Fellowship, and TH was supported by a PhRMA Foundation Pre Doctoral Fellowship.


extfieldsofstudy: Medicine Chemistry

text:
Transformation Cycle of Magnetosomes in Human Stem Cells: From Degradation to Biosynthesis of Magnetic Nanoparticles Anew.

The nanoparticles produced by magnetotactic bacteria, called magnetosomes, are made of a magnetite core with high levels of crystallinity surrounded by a lipid bilayer. This organized structure has been developed during the course of evolution of these organisms to adapt to their specific habitat, and is assumed to resist degradation, able to withstand the demanding biological environment. Herein, we investigated magnetosomes structural fate upon internalization in human stem cells using magnetic and photothermal measurements, electron microscopy, and X-ray absorption spectroscopy. All measurements first converge to the demonstration that intracellular magnetosomes can experience an important biodegradation, with up to 70 % of their initial content degraded, and associated with the progressive storage of the released iron in the ferritin protein. It correlates with an extensive magnetite to ferrihydrite phase transition. Remarkably, the ionic species delivered by this degradation could be used by the cells to biosynthesize magnetic nanoparticles anew. In this case, cells magnetism first decreased with magnetosomes being dissolved, but then cells re-magnetized entirely, evidencing the neo-synthesis of biogenic magnetic nanoparticles. Bacteria-made biogenic magnetosomes can thus be totally remodeled by human stem cells, into human cells-made magnetic nanoparticles.

controversial, as a few studies also reported their progressive degradation over time, in protozoans, or in mice, 19,33,34 but not yet in human cells.
In this study, we monitored the magnetosomes intracellular integrity in human stem cells over the course of a month. The magnetism of the cells was primarily used as quantitative fingerprint of the magnetosomes magnetic signature and photothermia was used as additional assessment of magnetosome integrity. Transmission electron microscopy (TEM) and high resolution TEM (HR-TEM) allowed to localize and monitor in situ the crystal structures at the nanoscale. Time-resolved X-ray absorption spectroscopy (XAS) studies in the near edge (XANES) and extended (EXAFS) regimes at the Fe K-edge provided the iron oxidation state during the long-term intracellular processing of the magnetosomes in the cells. 35 Magnetosomes were internalized in human mesenchymal stem cells and the labeled cells were then maintained under two distinct culture conditions. First, the cells were assembled in a 3D cell-spheroid kept in medium supplemented with insulin, transferrin and selenous acid (ITS) to ensure the spheroids viability over months. Such culture method was previously used as a model to monitor synthetic nanoparticles degradation on the long-term and quantitatively. 2,36 In this case, a progressive and extensive degradation of the magnetosomes occurred, associated with the change of iron oxidation state over 21 days detected by XAS and confirmed by HR-TEM.
Monolayer (2D) stem cells culture was next performed in absence of transferrin and selenium.
In this condition, small (8-nm) synthetic nanoparticles were found to be transformed into de novo magnetic nanoparticles biosynthesized from degradation products. 37 Under this culture conditions, magnetosomes were found to be degraded during the first days upon their internalization in stem cells, evidenced by the decrease of their magnetization (and at the same time their magnetite-mediated photothermal efficiency 24 ), but a few days later, cells remagnetized almost totally (and re-generated heat equally): magnetic nanoparticles were being neo-synthesized. TEM and HR-TEM confirmed the presence of these newly biosynthesized nanoparticles, bearing a magnetite structure. Magnetosomes can thus be precursors of a biogenic synthesis of magnetic nanoparticles in human cells.
Magnetosomes are thus fully integrated in human stem cells where they are first converted from magnetite to ferrihydrite, the reverse cycle to the one observed during magnetosomes synthesis. 17 Magnetosomes can then be biologically reshaped into neo-synthesized magnetic nanoparticles such as obtaining double bio-made magnetic nanoparticles: first synthesized in the bacteria then degraded in human stem cells prior to being biosynthesized anew.

Magnetosomes are internalized in human stem cells and the cellular magnetization is the fingerprint of the intracellular dose
Low doses of magnetosomes (50-nm size range, Figure 1A) from 15 to 120 μM of Fe were incubated with human mesenchymal stem cells for a short-time-period (30 min). Two hours upon internalization, cells observed by TEM displayed magnetosomes located only within the cytoplasm, confined in endosomes ( Figures 1B and 1C, Figure S1). No magnetosome were observed outside the cells. Size analyses extrapolated from the TEM images ( Figure 1D) indicated similar diameters for the magnetosomes observed before and after cell internalization, thus indicating that the process itself does not alter their structural integrity and that internalization is size-independent. Spheroids were then formed based on 200 000 magnetosomes-containing cells each. In few hours the spheroids became cohesive, and their magnetization was measured at the single spheroid level revealing a ferromagnetic signal characteristic of magnetite nanocrystals (see Figure S2 for the hysteresis in the magnetization cycles). A linear dose-dependent intake was observed by saturation magnetization values ( Figure 1E, in µemu, at room temperature) that increased with the incubation dose. These values can be directly converted into the mass (pgFe) of magnetosomes per cell (using the magnetosomes mass magnetization of 86 emu g −1 of iron) as shown in Figure 1F, evidencing an efficient internalization.

Time-resolved magnetization reveals first that magnetosomes can be degraded in human stem cells
For the degradation model, the stem cells-based spheroids were cultured for 21 days in specific medium leading to tissue maturation ( Figure 2A) and extracellular matrix production ( Figure 2B). Magnetization at 300 K of the spheroids was measured at day 0 (right after the internalization, as showed in Figure 1E) and at days 1, 3, 9, and 21 ( Figures 2C and 2D, Figures   S3 and S4). Average measurements of the saturation magnetization (more than 5 independent spheroids) are presented in Figure 2E for all initial doses. These values are a direct indicator of the magnetic integrity of the magnetosomes, therefore the decrease in magnetism reveals the degradation of their structure, as previously reported with other magnetic nanoparticles. 2 Here, a progressive magnetization decrease from day 0 to day 21 was observed for all concentrations. Importantly, the intracellular degradation was found to be very significant, reaching almost up to 70% of the initial content. This relative quantity of degraded magnetosomes appears to be the inverse function of the initial intracellular content ( Figure   S5): for 1.3, 2.4, 5.3 and 10 pg of iron per cell, decreased degradation rates were at 69, 64, 57 and 46 %, respectively. It indicates that the cells can only degrade a certain amount of magnetosomes relative to the initial dose. In addition, as most important to validate these results, we checked that the magnetic signal decrease was not due to magnetosome excretion out of the cells by performing ICP measurements over time. The total amount of iron in the spheroids was found to be constant until the last measurements after 21 days ( Figure 2F). The decrease in magnetization is therefore only due to the dissolution of the magnetite core into non-magnetic iron compounds, and not to magnetosomes leakage from the cells, or from the spheroids. As additional analytical method, the magnetite-produced photothermal heating of the magnetosomes provided another indicator of their structural integrity (magnetite content), also confirming a progressive degradation of the magnetosomes ( Figure 2G and 2H).

Electron microscopy observation confirms the degradation of magnetosomes and evidences the loading of released iron into ferritin
TEM images were taken at day 21 for structural observation of the magnetosomes and their degradation products ( Figure 3). First, the amount of intact magnetosomes observed at day 21 was much lower than at day 0, for the same incubation concentration. Remaining magnetosomes appeared generally intact (high resolution TEM image shown in Figure S6), with average size of 42 ± 10 nm (distribution presented in Figure S7), slightly reduced compared to day 0 (46 ± 9 nm), but not significantly. More importantly, we noticed the presence of new, smaller structures, with spherical shape and mean diameter of 5.3 ± 1.1 nm (distribution shown in Figure 3E), that were present in high number in both the cytoplasm and the endosomes at day 21 ( Figure 3B for cytoplasm and Figure 3C and 3D for endosomes, more examples in Figure S8). EDS analysis was performed on a zone containing these small nanoobjects in endosomes and cytoplasm, revealing a significant presence of iron ( Figure S9). Both size and iron content are typical of the ferritin protein complex loaded with iron, here originating from the magnetosomes degradation and absent at day 0 ( Figure S1). This ferritin protein has already been identified as taking in charge and storing free iron released from the degradation of magnetic nanoparticles. 2 Interestingly, its shell also provides a ferroxidase activity (H-subunit) allowing the oxidation of free Fe 2+ ions to Fe 3+ ions. Finally, analyzing gene expression by RT-PCR, we observed that this ferritin H subunit was significantly upregulated following magnetosomes degradation, in a dose-dependent manner ( Figure 3F). Not only this confirms the implication of the ferritin in magnetosome degradation, but also indicates that the resulting Fe 2+ ions were likely oxidized to produce Fe 3+ -based ferrihydrite in the ferritin.
Importantly, nanocrystalline ferrihydrite is antiferromagnetic below 330 K, 38 justifying the absence of its contribution to the magnetic signal of magnetite, thus the total magnetization decreases because of the transformation of the ferromagnetic magnetite signal into antiferromagnetic ferrihydrite.

Time-resolved structural X-ray absorption and HR-TEM studies demonstrate the iron state
transition, in the degradation model, from magnetite to ferrihydrite X-ray absorption spectra at the near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) measurements were performed at the Fe K-edge (7112 eV) 39 to quantitatively investigate the iron phases over the maturation of the spheroid tissue. Because of the spheroid configuration, it was possible to handle it under synchrotron radiation, and perform the measurement at the spheroid scale. XANES allows probing the coordination and oxidation state of absorbing Fe ions while EXAFS gives information about their local environment, including the interatomic distances and the coordination numbers of surrounding shells. These two complimentary techniques can thus reveal the iron short-range geometry and identify the iron phases. 35 They were recently applied to study biological systems. 17,18,40 Evolution of the XANES spectra during tissue maturation (days 0, 3, 9, and 21) was assessed and compared to the magnetite and ferrihydrite reference spectra (Figures 4A and 4B and Figure S10). 17,41 The XANES spectra showed a clear shift of the absorption edge over the 21 days of tissue maturation, direct evidence of iron oxidation. This shift is consistent with a change of iron oxide structure from magnetite (composed of Fe 2+ and Fe 3+ ) to ferrihydrite (composed of Fe 3+ only).
The XANES pre-edges features are reported in Figure 4B: the intensity variation indicates a gradual change on the symmetry around the Fe atoms over tissue maturation corroborating the change from magnetite (the most intense pre-edge peak) to ferrihydrite (the least intense pre-edge peak) due to their different percentage of Fe cations in tetrahedral sites. 42 The Fourier transform (FT) for the EXAFS signals was also performed ( Figure 4C), and the two distinct averaged distances corresponding to Fe−O and Fe−Fe shells were analyzed. No trend is noticed in the first coordination shell (Fe-O) over the degradation time. By contrast, as the degradation time increased, the evolution of magnetosomes towards ferrihydrite was evidenced by the interatomic distance of the second coordination shell (Fe-Fe) that decreased and reduced its neighbors approaching to the values of ferrihydrite (see Figure 4D and Table   S1), from a semi-quantitative analysis. It confirms the conversion from magnetite to ferrihydrite over time.

Magnetosome biodegradation can be followed by endosomal biosynthesis of magnetic nanoparticles anew
We next cultured the stem cells in monolayers (2D) over a period of 3 weeks, with limited cell division imposed by the selected medium. Importantly, this culture medium does not contain transferrin and selenium (contrary to the spheroid condition), which were shown to maintain the degradation process at the ferrihydrite-loaded ferritin stage, upon the typical iron metabolism pathway. In this culture condition, the ionic species delivered by degradation could also be used to induce a magnetic biogenic synthesis, as just demonstrated for small synthetic magnetic nanoparticles. 37 After magnetosomes internalization, stem cells were seeded in well-plates and kept in culture for 21 days. The magnetism of these cells, measured after 1, 3, 9, and 21 days of culture, demonstrated a different pattern from the previous degradation in spheroids: magnetization first decreased between day 0 and 3, but then  Figure   5E, with average values presented in Figure 5F, and compared to the ones reached upon degradation only. It correlates perfectly with the biosynthesis pattern observed via magnetometry and definitely contrasts with the biodegradation situation. Moreover, it suggests that the biosynthesized nanoparticles are made of magnetite, because the photothermal potential of magnetic nanoparticles comes from the magnetite structure (Fe 2+ to Fe 3+ valence band transition), and is much lower in the case of maghemite composition. 43 Electron microscopy observations further evidenced the endosomal biosynthesis of magnetic nanoparticles anew. Figure 6 shows TEM images taken after 9 ( Figure 6A-C) and 21 days ( Figure 6D-F) of culture (see Figure S12 for additional images). It demonstrates the progressive formation of magnetic nanoparticles, confined in the endosomes. In particular, these nanoparticles appeared to be associated with specific subcellular compartments "fingerprint"like body rich of membranes tightly packed. These neo-synthesized nanoparticles are 7-8 nm in diameter (distribution shown in Figure 6G), are well defined, and are far from the original magnetosome structures. Finally, Figure 6H shows HR-TEM images of the newly biosynthesized nanoparticles, and Figure 6I presents the HR-TEM electron diffraction profile of a zone containing only the biosynthesized nanoparticles. These particles displayed the distinctive rings of magnetite/maghemite composition, which further evidenced the magnetic biosynthesis of nanoparticles in human stem cells, using the ionic iron species delivered by magnetosome degradation. One remaining question concerns the fate of the biological membrane surrounding the magnetosomes prior to their internalization in stem cells, which can be clearly detected on TEM images when in solution ( Figure S13). Thanks to uranyl acetate membrane staining, these magnetosome surrounding membranes can still be detected inside the cells the first days after their incorporation (Figures S14 and S15 at days 0 and 1, respectively). Their presence is less obvious at further days (Figures S16 and S17 at days 3 and 9, respectively), and almost none can be observed on remaining magnetosomes at day 21 ( Figure S18). At day 21, the biosynthesized magnetic nanoparticles do not seem to present any biological shell ( Figure S18), yet more difficult to detect due to their smaller size. However, it remains possible that these nanoparticles are integrated within the ferritin protein core.
Fluorescence tracking of the Venus tag included in the magnetosome membranes was also performed over time ( Figure S19) evidencing a delocalization from sharp endosomal confinement at day 0 to diffuse endosomal and cytoplasmic localization at day 21.

CONCLUSIONS
With the ever increasing number of theranostics approaches using magnetosomes, it is crucial to understand their long-term fate after internalization in human cells. Here, we show that magnetosomes processing by human stem cells results in two biotransformation phenomena. In one case, magnetosomes are progressively degraded while iron state evolves from magnetite to ferrihydrite. Magnetic measurements and transmission electron microscopy indicate the appearance of 4-6 nm sized structures; these structures are shown to be ferrihydrite stored in the ferritin, which oxidize the free Fe 2+ ions coming from magnetosomes degradation. In the second case, magnetosomes were first degraded but then dynamically transformed into magnetic nanoparticles biosynthesized in situ, within the endosomal compartments of the cells, evidenced by magnetic, photothermal, and HR-TEM analyses. The intracellular transition of the iron state from magnetite to ferrihydrite then cycles back to magnetite and demonstrates a complete transformation cycle of magnetosomes within human cells. Not only it reveals that magnetosomes can be degraded and fully integrate the iron metabolism of stem cells, but also that these cells are capable of biosynthesizing magnetic nanoparticles. This dissolution / biosynthesis mechanism resembles to the one observed with other nanomaterial compositions, such as hydroxyapatite nanocrystals that are fully dissolved and re-precipitate as needle-like crystallites within one day. 44 The natural presence of magnetite nanoparticles has been detected in a variety of human cells including in the brain, heart, spleen, liver, ethmoid bone, and tumors, [45][46][47][48][49] and has sometimes been designated as an indicator of neurodegenerative diseases. [50][51][52] However, the reason for their presence remains unexplained. A recent study showed humans are capable of sensing the geomagnetic field of the Earth and correlates this feature with the presence of magnetite in the brain. 53 It recalls the capacity of magnetotactic bacteria to align along the magnetic field of the Earth, a property that has largely been attributed to the magnetosomes. Here, the biosynthesis of magnetite nanoparticles in human cells, based on magnetosomes degradation products, may be one crucial step toward understanding a possible magnetoreception aptitude in humans.

Magnetite magnetosomes were biosynthesized under strict genetic control by
Magnetospirillum magneticum strain AMB-1 (ATCC700264). By genetic manipulation the Venus fluorescent reporter and the RGD peptide were inserted in the lipid membrane by genetic manipulation to enable the tracking of the membrane by fluorescence microscopy, and to enhance the cell internalization, respectively. 21 Magnetosome production and purification were performed as already described. 24 Briefly, the genetically modified AMB-1 bacterial strain was grown in a 6 L bioreactor and cells from late exponential-phase culture were harvested by centrifugation (7500 G, 10 min, 4°C). The pellet (≈19 g per 6 L) was resuspended in 100 mL of purification buffer (20 mM HEPES, 1 mM EDTA, 0.9% NaCl, 8% glycerol pH 7.5 in presence of a cocktail of proteases) then re-pelleted (7500 G, 10 min, 4°C).
The cells were then disrupted 3 times with a French press (1000 PSI, 4°C). The tubes were left for 30 min at 4°C in contact of a magnet (MACSi-MAG tm separator, Miltenyi Biotec), resulting in magnetosomes collection. The unretained fraction was removed and the magnetosome fraction was resuspended in 45 mL of buffer. This magnetic purification step was performed 5 times with buffer 1 (without anti protease) then 5 times with buffer 2 (20 mM HEPES, 0.9% NaCl, 8% glycerol, pH 7.5). After the washing steps, the magnetosomes were resuspended in buffer 3 (20 mM HEPES, 8% glycerol, pH 7.5), at a concentration of 3 g L −1 of iron and 100-μL aliquots were flash-frozen in liquid nitrogen then stored at −80°C. As previously reported, this protocol ends up with magnetite nanoparticles. 54

Cell culture, magnetosomes internalization and long-term cell monitoring
Human mesenchymal stem cells (hMSC) were purchased from Lonza and were cultured in hMSC-basal medium at 37°C, 5% pCO2 and 95% relative humidity. At 90% confluence, cells were incubated with magnetosomes diluted in serum-free RPMI, previously sonicated for

Quantification of the internalized magnetic iron by VSM
Magnetic iron in the hMSCs right after magnetosomes incubation and in samples fixed at different maturation times was systematically measured by magnetometry using a PPMS device equipped with a vibrating sample magnetometer (VSM) option (Quantum Design). The analysis was performed at 300 K, between 0 and 25 000 Oe and provides the saturation magnetization of the sample (in emu), giving directly the amount of magnetic iron (in grams) based on the magnetosomes saturation magnetization (86 ± 3 emu g −1 ). To evaluate further the hysteresis loop of the ferromagnetic magnetosomes upon field shift, a cyclic magnetometry analysis was also performed from 1500 Oe to −1500 Oe and back to 1500 Oe.

Quantification of the internalized total iron by ICP
Total iron content was analyzed by elemental analysis of the samples using ICP-AES (iCAP 6500, Thermo). The cells at different stage of maturation (days 1, 3, 9 and 21) were digested in concentrated HNO3 and recovered with a 2% HNO3 water solution.

Transmission electron microscopy of magnetosomes and by-products of their degradation
The samples fixed at day 0 and day 21 were contrasted with Oolong Tea Extract (OTE) 0.5% in 0.1 M Na cacodylate buffer, post-fixed with 1% osmium tetroxide containing 1.5% potassium cyanoferrate, gradually dehydrated in ethanol (30% to 100%) and gradually embedded in epoxy resins. Ultrathin slices (70 nm) were collected onto 200 mesh copper grids and counterstained with lead citrate prior to being observed with a Hitachi HT 7700 TEM operated at 80 kV and images were acquired with a charge-coupled device camera (AMT). The sizes of the nanoparticles at Day 0 (compared with the one of the magnetosomes in solution) and of the magnetosomes and iron deposits in the ferritins at Day 21 were measured by the Image J software (at least 400 diameters measured per condition). The same procedure without the lead counterstain have been applied for the preparation of the high-resolution TEM samples, which have been observed with a JEOL 2100F operating at 200 kV, equipped with a Schottky emission gun, an ultra-high-resolution pole piece, and a Gatan US 4000 CCD camera.

Ferritin Heavy chain expression by Real-Time PCR
Total RNA was extracted from spheroids formed from cells incubated with magnetosomes at

Laser-induced heating measurements
Photothermal analysis of samples at different maturation stages was carried out using a 808nm CW laser diode coupled to an optic fiber (Laser Components S.A.S). Fixed samples were suspended in 5 µL PBS in a 0.5 mL tube and were illuminated with a power density of 0.3 W cm −2 for 2 min. The temperature elevation (ΔT) was recorded using an infrared thermal imaging camera (FLIR SC7000). A decrease in photothermal efficiency is an indirect proof of the magnetosomes degradation and of their biotransformation from a more (magnetite) to a less (ferrihydrite) plasmonic material. 36

XANES/EXAFS analysis (Synchrotron radiation)
XAS measurements were performed on the biodegradation samples that consist in dense cellpellets easily transferable, by contrast with the independent cells of the biosynthesis model.  Figure S10). From 4 to 5 spheroids were used to obtain the sample spectra at different maturation time. Several scans were taken in order to obtain a good signal-to noise ratio. Data analysis was carried out using Athena program 4 identifying the beginning of the absorption edge, E0, and the pre-edge and post-edge backgrounds. The spectra were subjected to a background subtraction and normalized by the edge jump. In the XANES regime, the edge position of samples was analyzed since it is an indication of the average oxidation state of Fe, with edge increasing to higher energies when the oxidation state is incremented. On the other hand, the XANES pre-edge peak increases in intensity as the degree of non-centrosymmetry of the absorbing atom environment increases, e.g. in presence of a tetrahedral site. Magnetite is a spinel iron oxide where Fe cations occupy twice octahedral sites than tetrahedral sites, while in ferrihydrite the tetrahedral Fe occupies a lower percentage. The software Arthemis 55 was used to process the EXAFS signal as a function of the modulus of the photoelectron wavevector, k, (k) in the range of 2.8 and 9.5 Å −1 . The amplitude reduction factor S 0 2 , the interatomic distance R and the Debye-Waller (DW) factor σ 2 for the two shell distances are used as free parameters for each fitting.

Statistical analysis
All values are reported as means and standard error of the mean. Significant differences were determined using a Tukey's test in one-way analysis of variance (ANOVA).

ASSOCIATED CONTENT Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: It contains additional TEM images ( Figures S1, S8, S12, S13, S14, S15, S16, S17 and S18), additional HR-TEM and EDS analysis ( Figure S6 and Figure S9, respectively), typical magnetization curves ( Figures S2, S3 and S4), the analysis of the degradation rate as a function of the dose (Figure S5), the size distribution of magnetosomes in cells ( Figure S7), the XANES absorption reference spectra ( Figure S10), the analysis of cellular magnetism over time for endothelial cells ( Figure S11), confocal microscopy visualization of the Venus probe embedded in the magnetosome membrane ( Figure S19) and parameters extracted from the EXAFS experiment (Table S1).