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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Cell. Infect. Microbiol.</journal-id>
<journal-title>Frontiers in Cellular and Infection Microbiology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Cell. Infect. Microbiol.</abbrev-journal-title>
<issn pub-type="epub">2235-2988</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fcimb.2024.1403782</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Cellular and Infection Microbiology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Anti-chlamydial activity of vaginal fluids: new evidence from an <italic>in vitro</italic> model</article-title>
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<contrib-group>
<contrib contrib-type="author" equal-contrib="yes">
<name>
<surname>Morselli</surname><given-names>Sara</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="author-notes" rid="fn002"><sup>&#x2020;</sup></xref>
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<contrib contrib-type="author" equal-contrib="yes">
<name>
<surname>Ceccarani</surname><given-names>Camilla</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<xref ref-type="author-notes" rid="fn002"><sup>&#x2020;</sup></xref>
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<contrib contrib-type="author">
<name>
<surname>Djusse</surname><given-names>Marielle Ezekielle</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
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<contrib contrib-type="author">
<name>
<surname>Laghi</surname><given-names>Luca</given-names>
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<xref ref-type="aff" rid="aff4"><sup>4</sup></xref>
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<contrib contrib-type="author">
<name>
<surname>Camboni</surname><given-names>Tania</given-names>
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<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author">
<name>
<surname>Consolandi</surname><given-names>Clarissa</given-names>
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<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
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<contrib contrib-type="author" corresp="yes">
<name>
<surname>Foschi</surname><given-names>Claudio</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff5"><sup>5</sup></xref>
<xref ref-type="author-notes" rid="fn001"><sup>*</sup></xref>
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<contrib contrib-type="author" equal-contrib="yes">
<name>
<surname>Severgnini</surname><given-names>Marco</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<xref ref-type="author-notes" rid="fn003"><sup>&#x2021;</sup></xref>
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<contrib contrib-type="author" equal-contrib="yes">
<name>
<surname>Marangoni</surname><given-names>Antonella</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="author-notes" rid="fn003"><sup>&#x2021;</sup></xref>
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<aff id="aff1"><sup>1</sup><institution>Section of Microbiology, Department of Medical and Surgical Sciences, Alma Mater Studiorum - University of Bologna</institution>, <addr-line>Bologna</addr-line>, <country>Italy</country></aff>
<aff id="aff2"><sup>2</sup><institution>Institute of Biomedical Technologies, National Research Council</institution>, <addr-line>Segrate</addr-line>, <country>Italy</country></aff>
<aff id="aff3"><sup>3</sup><institution>National Biodiversity Future Center S.c.a.r.l.</institution>, <addr-line>Palermo</addr-line>, <country>Italy</country></aff>
<aff id="aff4"><sup>4</sup><institution>Department of Agricultural and Food Sciences, University of Bologna</institution>, <addr-line>Cesena</addr-line>, <country>Italy</country></aff>
<aff id="aff5"><sup>5</sup><institution>Microbiology Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna</institution>, <addr-line>Bologna</addr-line>, <country>Italy</country></aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Keiji Nagano, Health Sciences University of Hokkaido, Japan</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Junji Matsuo, Health Sciences University of Hokkaido, Japan</p>
<p>Kensuke Shima, University of L&#xfc;beck, Germany</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Claudio Foschi, <email xlink:href="mailto:claudio.foschi2@unibo.it">claudio.foschi2@unibo.it</email>
</p>
</fn>
<fn fn-type="equal" id="fn002">
<p>&#x2020;These authors have contributed equally to this work and share first authorship</p>
</fn>
<fn fn-type="equal" id="fn003">
<p>&#x2021;These authors have contributed equally to this work and share last authorship</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>07</day>
<month>06</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>14</volume>
<elocation-id>1403782</elocation-id>
<history>
<date date-type="received">
<day>19</day>
<month>03</month>
<year>2024</year>
</date>
<date date-type="accepted">
<day>01</day>
<month>05</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2024 Morselli, Ceccarani, Djusse, Laghi, Camboni, Consolandi, Foschi, Severgnini and Marangoni</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Morselli, Ceccarani, Djusse, Laghi, Camboni, Consolandi, Foschi, Severgnini and Marangoni</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<sec>
<title>Introduction</title>
<p>We assessed the <italic>in vitro</italic> anti-chlamydial activity of fresh vaginal secretions, deciphering the microbial and metabolic components able to counteract <italic>Chlamydia trachomatis</italic> viability.</p>
</sec> <sec>
<title>Methods</title>
<p>Forty vaginal samples were collected from a group of reproductive-aged women and their anti-chlamydial activity was evaluated by inhibition experiments. Each sample underwent 16S rRNA metabarcoding sequencing to determine the bacterial composition, as well as <sup>1</sup>H-NMR spectroscopy to detect and quantify the presence of vaginal metabolites.</p>
</sec>
<sec>
<title>Results</title>
<p>Samples characterized by a high anti-chlamydial activity were enriched in <italic>Lactobacillus</italic>, especially <italic>Lactobacillus crispatus</italic> and <italic>Lactobacillus iners</italic>, while not-active samples exhibited a significant reduction of lactobacilli, along with higher relative abundances of <italic>Streptococcus</italic> and <italic>Olegusella</italic>. <italic>Lactobacillus gasseri </italic>showed an opposite behavior compared to <italic>L. crispatus</italic>, being more prevalent in not-active vaginal samples. Higher concentrations of several amino acids (i.e., isoleucine, leucine, and aspartate; positively correlated to the abundance of <italic>L. crispatus</italic> and <italic>L. jensenii</italic>) lactate, and 4-aminobutyrate were the most significant metabolic fingerprints of highly active samples. Acetate and formate concentrations, on the other hand, were related to the abundances of a group of anaerobic opportunistic bacteria (including <italic>Prevotella, Dialister, Olegusella, Peptostreptococcus, Peptoniphilus, Finegoldia</italic> and <italic>Anaerococcus</italic>). Finally, glucose, correlated to <italic>Streptococcus, Lachnospira</italic> and <italic>Alloscardovia</italic> genera, emerged as a key molecule of the vaginal environment: indeed, the anti-chlamydial effect of vaginal fluids decreased as glucose concentrations increased.</p>
</sec>
<sec>
<title>Discussion</title>
<p>These findings could pave the way for novel strategies in the prevention and treatment of chlamydial urogenital infections, such as lactobacilli probiotic formulations or lactobacilli-derived postbiotics.</p>
</sec>
</abstract>
<kwd-group>
<kwd>Chlamydia trachomatis</kwd>
<kwd>lactobacilli</kwd>
<kwd>vaginal microbiome</kwd>
<kwd>metabolome</kwd>
<kwd>women&#x2019;s health</kwd>
</kwd-group>
<counts>
<fig-count count="3"/>
<table-count count="1"/>
<equation-count count="0"/>
<ref-count count="50"/>
<page-count count="10"/>
<word-count count="5585"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-in-acceptance</meta-name>
<meta-value>Extra-intestinal Microbiome</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<label>1</label>
<title>Introduction</title>
<p><italic>Chlamydia trachomatis</italic> (CT), an obligate intracellular pathogen, represents the most common bacterial sexually transmitted infection (STI) worldwide, with a significant clinic, economic and public health impact (<xref ref-type="bibr" rid="B11">ECDC, 2024</xref>; <xref ref-type="bibr" rid="B47">World Health Organization (WHO), 2015</xref>). In women, urogenital CT infections (i.e., cervicitis, urethritis) are often characterized by the absence of symptoms, potentially resulting in a range of serious sequelae and complications including pelvic inflammatory disease, ectopic pregnancy, and infertility (<xref ref-type="bibr" rid="B18">Haggerty et&#xa0;al., 2010</xref>; <xref ref-type="bibr" rid="B28">Menon et&#xa0;al., 2015</xref>).</p>
<p>In recent years, the correlation between CT presence and the microbial/metabolic composition of the vaginal environment has garnered particular attention, elucidating mechanisms involved in the protection from chlamydial infection (<xref ref-type="bibr" rid="B33">Parolin et&#xa0;al., 2018a</xref>; <xref ref-type="bibr" rid="B5">Ceccarani et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B43">Tamarelle et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B37">Raimondi et&#xa0;al., 2021</xref>).</p>
<p>Indeed, it has been shown that a vaginal microbiome dominated by certain species of <italic>Lactobacillus</italic> is crucial for preventing CT infections (<xref ref-type="bibr" rid="B29">Nardini et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B10">Di Pietro et&#xa0;al., 2022</xref>). The protective role of lactobacilli against CT is exerted through different mechanisms that act on both extracellular and intracellular steps of the chlamydial cycle. These include the production of various antibacterial compounds (e.g., lactic acid, hydrogen peroxide, bacteriocins, and biosurfactants), competitive exclusion for epithelial adhesion, and immunomodulation (<xref ref-type="bibr" rid="B48">Younes et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B34">Parolin et&#xa0;al., 2018b</xref>; <xref ref-type="bibr" rid="B49">Zalambani et&#xa0;al., 2023</xref>).</p>
<p>The protective role of lactobacilli is strengthened by the demonstration that a higher risk of CT transmission and acquisition is reported in cases of bacterial vaginosis (BV), a clinical condition characterized by the depletion of <italic>Lactobacillus</italic> and an overgrowth of anaerobic genera such as <italic>Gardnerella, Prevotella, Megasphaera</italic>, and <italic>Atopobium</italic>, has been reported (<xref ref-type="bibr" rid="B46">Wiesenfeld et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B1">Abbai et&#xa0;al., 2016</xref>). Changes in bacterial communities during BV conditions are accompanied by significant alterations in vaginal metabolite composition, characterized by a decrease in lactate concentration and higher levels of biogenic amines and short-chain organic acids (<xref ref-type="bibr" rid="B5">Ceccarani et&#xa0;al., 2019</xref>).</p>
<p>To date, only a few studies have focused on the <italic>in vitro</italic> interaction between vaginal fluids/secretions and CT, and many aspects of the mechanisms involved in the activity against chlamydial infectivity remain to be fully elucidated (<xref ref-type="bibr" rid="B26">Mastromarino et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B29">Nardini et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B49">Zalambani et&#xa0;al., 2023</xref>). Therefore, the aim of the present work was to assess the <italic>in vitro</italic> anti-chlamydial activity of fresh vaginal secretions collected from reproductive-aged women, deciphering the microbial composition (microbiome analysis by 16S rRNA metabarcoding sequencing) and the metabolic components (metabolome analysis by <sup>1</sup>H-NMR) of the vaginal fluids associated with protection from CT infection. Understanding the microbial/metabolic fingerprints of vaginal fluids related to their activity against CT could pave the way for the development of novel antimicrobial-free strategies for the prevention and treatment of chlamydial urogenital infections.</p>
</sec>
<sec id="s2" sec-type="materials|methods">
<label>2</label>
<title>Materials and methods</title>
<sec id="s2_1">
<label>2.1</label>
<title>Study cohort and sample collection</title>
<p>Between September and November 2022, 10 women were enclosed in the study. The women were volunteers and were selected from those who had expressed interest in participating in this study. All of them were attending degree courses at the University of Bologna, Italy. At the enrollment, exclusion criteria were: (i) antibiotic use in the month prior to sampling; (ii) menstruating at the time of sampling; (iii) HIV infection; (iv) presence of chronic conditions (e.g., diabetes, autoimmune diseases, malignancies); (v) pregnancy; (vi) age&lt;18 years.</p>
<p>Demographic, clinical, and behavioral data were recorded from each participant. Each woman underwent four self-collected vaginal samplings within 40 days, at four different time points (starting from the first day of menstrual cycle, volunteers were asked to submit their samples at the following time points: 5-7 days, 12-16 days, 21-25 days, 32-40 days). As summarized in <xref ref-type="fig" rid="f1"><bold>Figure&#xa0;1</bold></xref>, two vaginal swabs were collected at each time point. At first, the secretions were collected with a sterile cotton bud, and it was immediately re-suspended in 1.8 mL of sterile saline, while, as a second step, secretions were collected with a flocked swab (E-swab Copan, Brescia, Italy). Both swabs were processed within 10&#xa0;min of collection. The swab suspended in saline was vortexed, and 300 &#xb5;L of the suspension were sampled to test the anti-chlamydia activity (&#x2018;whole fraction&#x2019;). Subsequently, the remaining suspension was centrifuged at 10,000 &#xd7; g for 15&#xa0;min: 300 &#xb5;L of the supernatant were used to test the anti-chlamydia activity (&#x2018;cell-free supernatant fraction&#x2019;), while 700 &#xb5;L were preserved at -80&#xb0;C until the metabolomic analysis by means of <sup>1</sup>H-NMR spectroscopy (see specific paragraph below). Finally, the residual supernatant portion was discarded, and the pellet was resuspended in 1.5 mL of sterile saline: 300 &#xb5;L of this pellet solution were used to test anti-chlamydia activity (&#x2018;pellet fraction&#x2019;), while the remaining 1200 &#xb5;L were used to extract nucleic acids for microbiome analysis (see specific paragraph below).</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Workflow of the experiments performed in this study. For each woman, the vaginal sampling was performed 4 times during a period of 40 days.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1403782-g001.tif"/>
</fig>
<p>The E-swab, after being vortexed, was used for both microscopic evaluation and microbiological tests, using, at first, the Nugent score for a preliminary assessment of the vaginal flora composition (<xref ref-type="bibr" rid="B31">Nugent et&#xa0;al., 1991</xref>). According to this score, women were grouped as follows: &#x201c;H&#x201d; group (normal lactobacilli-dominated microbiota, score 0-3), &#x201c;I&#x201d; group (intermediate microbiota/flora, score 4-6), &#x201c;BV&#x201d; group (bacterial vaginosis, score 7-10) (<xref ref-type="bibr" rid="B50">Zozaya-Hinchliffe et&#xa0;al., 2010</xref>). Moreover, Seeplex STI Master Panels 1 and 3 (Seegene, Seoul, KR) were performed following the manufacturer&#x2019;s instructions, to investigate the potential presence of the most common urogenital STIs agents (i.e., <italic>Chlamydia trachomatis</italic>, <italic>Neisseria gonorrhoeae</italic>, <italic>Trichomonas vaginalis</italic> and <italic>Mycoplasma genitalium</italic>), and <italic>Candida</italic> spp. A positive result would have led to exclusion from the study.</p>
<p>A written informed consent was obtained from all participants and the Bioethics Committee of the University of Bologna (protocol number 0122421) approved the protocol of the study.</p>
</sec>
<sec id="s2_2">
<label>2.2</label>
<title>Cell line and <italic>Chlamydia trachomatis</italic> strain</title>
<p>HeLa cells (ATCC<sup>&#xae;</sup> CCL-2) were used as an <italic>in vitro</italic> model for experiments. Cells were grown in individual tubes containing sterile coverslips (Thermo Fisher Scientific, Waltham, MA, USA) in 5% CO<sub>2</sub> at 37&#xb0;C. Cells were cultivated in DMEM medium (EuroClone, Pero, Italy), supplemented with 10% fetal bovine serum and 1% L-glutamine, without antibiotics.</p>
<p><italic>Chlamydia trachomatis</italic> strain GO/86, serovar D, was used for the present study (<xref ref-type="bibr" rid="B23">Marangoni et&#xa0;al., 2015a</xref>). This strain was clinically isolated in 1986 from a urethral swab sent to the Microbiology Laboratory of IRCSS Azienda Ospedaliero-Universitaria of Bologna, Italy, for routine diagnostic procedures and it belongs to the laboratory collection. The purification of elementary bodies (EBs) and the evaluation of the infectivity titer (expressed as inclusion forming units IFU/mL) have been described in detail elsewhere (<xref ref-type="bibr" rid="B14">Foschi et&#xa0;al., 2019</xref>).</p>
</sec>
<sec id="s2_3">
<label>2.3</label>
<title>Evaluation of the anti-chlamydial activity of vaginal fluids</title>
<p>To study the ability of the whole vaginal fluids and their fractions (i.e., bacterial pellets and supernatants) to directly counteract CT viability, &#x2018;inhibition experiments&#x2019; were performed in triplicate for each woman at each time point, as follows. HeLa cells were seeded in individual tubes and allowed to reach a total cell number of approximately 5 &#xd7; 10<sup>5</sup>. Afterwards, for each single experiment, 5 &#xd7; 10<sup>4</sup> CT EBs were re-suspended for 30&#xa0;min at 37&#xb0;C with 5% CO<sub>2</sub> in four separate solutions containing (i) 100 &#xb5;L of the whole vaginal fluid, (ii) 100 &#xb5;L of the vaginal cell-free supernatant, (iii) 100 &#xb5;L of the vaginal pellet (re-suspended in sterile saline) and (iv) 100 &#xb5;L of sterile saline (used as controls). At the end of the incubation, the samples were inoculated into HeLa cells, grown in DMEM medium in individual tubes containing sterile coverslips, and centrifuged at 530 &#xd7; g for 1h. At the end of the centrifugation, culture medium was replaced with fresh DMEM, and HeLa cells were incubated at 37&#xb0;C with 5% CO<sub>2</sub> for 48h.</p>
<p>CT infection was estimated by counting the number of IFUs by direct immunofluorescence, using a fluorescein-conjugated anti-chlamydial LPS monoclonal antibody (Meridian, Cincinnati, OH, USA). Slides were observed under an epi-fluorescence microscope (Eclipse E600, Nikon, Tokyo, Japan). The number of IFUs was counted in 30 randomly chosen 200&#xd7; microscopic fields.</p>
<p>Results were expressed as the percentage (average percentage &#xb1; standard deviation) of CT infectivity, comparing the number of IFUs from the individual experiments to the control tubes (number of IFU/field of the controls ranged between 60 and 85). Tested fractions were arbitrarily divided into three groups based on their anti-chlamydial activity: &#x2018;High activity&#x2019; (infectivity reduction of 61-100% compared to control), &#x2018;Intermediate activity&#x2019; (infectivity reduction of 41-60% compared to control), and &#x2018;No activity&#x2019; (infectivity reduction of 0-40% compared to control).</p>
</sec>
<sec id="s2_4">
<label>2.4</label>
<title>Microbiome analysis</title>
<p>DNA extraction from vaginal swabs was carried out by means of the Versant molecular system (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) (<xref ref-type="bibr" rid="B24">Marangoni et&#xa0;al., 2015b</xref>). Afterwards, the V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified, according to the 16S metagenomic sequencing library preparation protocol (Illumina, San Diego, CA). Final indexed libraries were prepared in a equimolar (4 nmol/L) pool, then denaturation and dilution to 6 pmol/L were performed, before loading onto the MiSeq flow cell (Illumina) (<xref ref-type="bibr" rid="B39">Severgnini et&#xa0;al., 2021</xref>). Sequencing was performed by a paired 2 &#xd7; 300 bp run.</p>
<p>Amplicon sequence variants (ASVs) were identified from 16S paired-end sequencing using the Divisive Amplicon Denoising Algorithm (DADA2, version 1.18.0; <xref ref-type="bibr" rid="B3">Callahan et&#xa0;al., 2016</xref>) pipeline, including filtering and trimming of the reads. Reads per sample were trimmed to 6,800 reads in order to compensate for the sequencing unevenness of the samples and to provide a consistent minimum amount for the downstream analysis, carried out through the &#x201c;phyloseq&#x201d; package (version 1.34.0) (<xref ref-type="bibr" rid="B27">McMurdie and Holmes, 2013</xref>). Alpha-diversity evaluation was performed according to several microbial diversity metrics (i.e., Chao1, Shannon Index, Observed Species); the Faith&#x2019;s phylogenetic tree diversity metric (&#x201c;PD whole tree&#x201d;) was elaborated through the &#x201c;btools&#x201d; package (<ext-link ext-link-type="uri" xlink:href="https://github.com/twbattaglia/btools">https://github.com/twbattaglia/btools</ext-link>). Beta-diversity analysis was conducted using the unweighted Unifrac distance and the principal coordinates analysis (PCoA).</p>
<p>Taxonomy was assigned to the ASVs using the 8-mer-based classifier from the 11.5 release of the RDP database and using the GTDB 16S rRNA database (release r207) (<xref ref-type="bibr" rid="B32">Parks et&#xa0;al., 2022</xref>).</p>
<p><italic>Lactobacillus</italic> species-level characterization was performed as in <xref ref-type="bibr" rid="B40">Severgnini et&#xa0;al. (2022)</xref>, by BLAST-aligning all reads belonging to the Lactobacillaceae family to a custom reference database made up collecting all available reference sequences in NIH-NCBI database (<ext-link ext-link-type="uri" xlink:href="ftp://ftp.ncbi.nlm.nih.gov/genomes/GENOME_REPORTS/prokaryotes.txt">ftp://ftp.ncbi.nlm.nih.gov/genomes/GENOME_REPORTS/prokaryotes.txt</ext-link>) of 17 species commonly found in the vaginal environment. The dataset included 2,403 sequences belonging to 17 species and 6 genera (i.e., <italic>Lacticaseibacillus</italic>, <italic>Lactiplantibacillus</italic>, <italic>Lactobacillus</italic>, <italic>Levilactobacillus</italic>, <italic>Ligilactobacillus</italic>, and <italic>Limosilactobacillus</italic>). Potential matches were filtered in order to retrieve an unequivocal classification for each read.</p>
</sec>
<sec id="s2_5">
<label>2.5</label>
<title>Metabolome analysis</title>
<p>Metabolomic analysis was performed by means of a <sup>1</sup>H-NMR spectroscopy: 100 &#x3bc;L of a D<sub>2</sub>O solution of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) 10 mM set to pH 7.0 were added to 700 &#xb5;L of the cell-free supernatants of the vaginal swabs. <sup>1</sup>H-NMR spectra were recorded at 298&#xa0;K with an AVANCE III spectrometer (Bruker, Milan, Italy), operating at a frequency of 600.13 MHz, equipped with Topspin software (Ver. 3.5) (<xref ref-type="bibr" rid="B15">Foschi et&#xa0;al., 2018</xref>). The signals originating from large molecules were suppressed by a CPMG filter of 400 spin-echo periods, generated by 180&#xb0; pulses of 24 &#x3bc;s separated by 400 &#x3bc;s (<xref ref-type="bibr" rid="B45">Ventrella et&#xa0;al., 2016</xref>). To each spectrum, line broadening (0.3&#xa0;Hz) and phase adjustment were applied by Topspin software, while any further spectra processing, molecules quantification and data mining step were performed in R computational language (version 4.0.5) by means of in house-developed scripts. The spectra were aligned towards the TSP signal, set at &#x2212;0.017 ppm in agreement with Chenomx software data bank (version 8.3, Chenomx Inc., Edmonton, Alberta, Canada). The spectra were then baseline-adjusted by means of peak detection according to the &#x201c;rolling ball&#x201d; principle implemented in the &#x201c;baseline&#x201d; R package (<xref ref-type="bibr" rid="B19">Kneen and Annegarn, 1996</xref>; <xref ref-type="bibr" rid="B21">Liland et&#xa0;al., 2010</xref>). The signals were assigned by comparing their chemical shift and multiplicity with Chenomx software data bank. Molecules were quantified in the first sample acquired by employing the added TSP as an internal standard. To compensate for differences in sample amount, any other sample was then normalized to such sample by means of probabilistic quotient normalization (<xref ref-type="bibr" rid="B8">Dieterle et&#xa0;al., 2006</xref>). Integration of the signals was performed for each molecule by means of rectangular integration.</p>
</sec>
<sec id="s2_6">
<label>2.6</label>
<title>Statistical analysis</title>
<p>The Fisher-Freeman-Halton test, an extension to Fisher&#x2019;s exact test, was employed to determine the association between the classification of the samples based on the anti-chlamydial activity and that on the Nugent score (<xref ref-type="bibr" rid="B17">Freeman and Halton, 1951</xref>).</p>
<p>For microbiome analysis, statistical evaluation of the alpha-diversity indices was performed by a two-sample Mann-Whitney U-test, whereas beta-diversity differences were assessed by a permutation test with pseudo F-ratios (&#x201c;adonis&#x201d; test) (<ext-link ext-link-type="uri" xlink:href="http://CRAN.Rproject.org/package=vegan">http://CRAN.Rproject.org/package=vegan</ext-link>). Comparisons of microbial relative abundance and metabolite quantities were performed using the Dunn&#x2019;s test for multiple comparisons (Kruskal-Wallis test), with the Bonferroni or Benjamini-Hochberg corrections for multiple testing. An adjusted p-value &lt;0.05 was considered as statistically significant.</p>
<p>Metabolite concentrations were correlated to bacterial composition by calculating Spearman&#x2019;s correlation coefficient between metabolites and bacterial genera present &#x2265;1% in at least 1 sample. We performed a Spearman&#x2019;s rank-based correlation between genus relative abundances and metabolite quantities, selecting only those with p-value&lt;0.05 (i.e., correlation significantly different from 0). To better visualize patterns of positively correlated bacteria and metabolites, a heatmap was drawn, clustering correlation coefficients for metabolites and bacteria (using Pearson&#x2019;s correlation as clustering metric and average linkage).</p>
</sec>
<sec id="s2_7">
<label>2.7</label>
<title>Data availability</title>
<p>Raw sequencing data of 16S rRNA gene are available at NCBI Short-reads Archive (SRA) with BioProject accession number PRJNA1062308 (<ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/sra/PRJNA1062308">https://www.ncbi.nlm.nih.gov/sra/PRJNA1062308</ext-link>). Raw metabolomic data are available as a <xref ref-type="supplementary-material" rid="SM2"><bold>Supplementary Material (Data Sheet S1)</bold></xref>.</p>
</sec>
</sec>
<sec id="s3" sec-type="results">
<label>3</label>
<title>Results</title>
<sec id="s3_1">
<label>3.1</label>
<title>Study population and anti-chlamydial activity of vaginal secretions</title>
<p>Ten reproductive-aged (mean age of 27.5 &#xb1; 2.6 years) normal-weight (mean BMI of 20.0 &#xb1; 1.3) women were included in the study. All the participants denied the use of hormonal contraceptives, and only one reported a smoking habit of approximately five cigarettes/day. Detailed information for each participant is reported in <xref ref-type="supplementary-material" rid="SM2"><bold>Supplementary Table&#xa0;1</bold></xref>.</p>
<p>The 40 vaginal samples (i.e., 4 samples per subject), collected throughout the study period, were categorized based on microscopic Nugent score, as follows: 30 (75%) showed a lactobacilli-dominated composition (&#x2018;H&#x2019; group), 3 (7.5%) were characterized by an intermediate flora (&#x2018;I&#x2019; group), while the remaining 7 (17.5%) harbored a BV-associated microbial composition (&#x2018;BV&#x2019; group). Two participants in the latter group reported burning sensations and white-grey discharges throughout the entire study period.</p>
<p>The anti-chlamydial activity of each vaginal specimen was tested in an <italic>in vitro</italic> model. At first, to mimic what happens in the natural course of chlamydial infection, we assessed the ability of the whole vaginal sample to counteract CT viability. Subsequently, in order to understand the components associated to the activity against CT, for each sample, we also tested (i) the cellular fraction (i.e., bacterial pellet), and (ii) the supernatant (i.e., cell-free component).</p>
<p>By means of preliminary experiments, we excluded a toxic/cytolytic effect of vaginal pellets on Hela cells (data not shown).</p>
<p>Samples were categorized into three groups based on the extent of the reduction in CT viability. Overall, 11/40 vaginal supernatants were highly active against CT, 15/40 showed intermediate activity, whereas 14/40 were not active. Regarding pellet fractions, 19/40 were classified as highly active, 15/40 as intermediate active, and 6 as not active. The detailed results are reported in <xref ref-type="supplementary-material" rid="SM2"><bold>Supplementary Table&#xa0;2</bold></xref>. Classification of the pellets according to their anti-chlamydial activity was not independent of the vaginal status of the women, as determined by the Nugent score. In fact, all 19 pellets with high anti-CT activity and the majority (10 out of 15) with intermediate activity belonged to the &#x2018;H&#x2019; group (lactobacilli-dominated women), whereas not-active pellets were, in 5 out of 6 cases, from BV women. These differences were highly significant (p&lt;0.001, Fisher-Freeman-Halton test); as a matter of fact, the two variables could not be considered independent and disentangled.</p>
</sec>
<sec id="s3_2">
<label>3.2</label>
<title>Microbiome composition: association with the anti-CT activity</title>
<p>The cellular fraction (i.e., pellet) of each vaginal sample was subjected to 16S rRNA metabarcoding sequencing to determine the bacterial composition. High-throughput sequencing generated 1,861,458 read pairs (average: 46,536 &#xb1; 26,151) over the 40 samples. After filtering, denoising, and chimera removal, 858,455 reads (average: 21,461 &#xb1; 9,329; range: 7,967 - 44,830) were obtained, forming a total of 1,130 amplicon sequence variants (ASVs).</p>
<p>The average bacterial composition across all the samples at the phylum level was dominated by Firmicutes (85.7% &#xb1; 18.1%), Actinobacteria (9.3% &#xb1; 13.0%), Bacteroidetes (3.4% &#xb1; 5.2%), and Proteobacteria (1.0% &#xb1; 1.7%), which accounted for &gt;99% of the overall average relative abundance. At the genus level, as expected, <italic>Lactobacillus</italic>, which accounted for up to 78.5% on average, comprised most of the composition, with minor contributions from <italic>Bifidobacterium</italic>, <italic>Prevotella</italic>, and <italic>Olegusella</italic> (average rel. ab. in the range 3.0% - 3.5%), <italic>Streptococcus</italic>, <italic>Finegoldia</italic> and <italic>Anaerococcus</italic> (average rel. ab. in the range of 1.0% - 1.2%) (<xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2A</bold></xref>).</p>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>Microbiome analysis. <bold>(A)</bold> Overall composition of the dataset. Stacked bars show the genus-level relative abundance for each patient, subdivided by the different sampling time-points, labelled by their pellet activity classification (yellow = high; red = intermediate; blue = not active). <bold>(B)</bold> Alpha-diversity analysis of the fluid activity evaluation according to the Observed species, Chao1, Shannon index, PD whole tree metrics. <bold>(C)</bold> Principal coordinates analysis reporting the dataset&#x2019;s beta-diversity for each fluid activity group. <bold>(D)</bold> Genus-level mean relative abundance for high, intermediate, and not active groups. <bold>(E)</bold> Species-level characterization of the <italic>Lactobacillus</italic> genus abundance according to the fluid anti-CT activity subgroups.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1403782-g002.tif"/>
</fig>
<p>The bacterial community from highly and intermediately active pellets was consistently characterized by fewer species than that from not-active pellets (p &#x2264; 0.01 for both high and intermediate vs. not-active for Chao1, Observed species, Shannon, and PD whole tree metrics). Similarly, the bacterial profiles resulted clearly separated in the PCoA plots (p=0.043, intermediate vs. not active, unweighted UniFrac distance; p=0.012, high vs. not active, unweighted UniFrac distance; p=0.005, high vs. not active, weighted UniFrac distance) (<xref ref-type="fig" rid="f2"><bold>Figures&#xa0;2B, C</bold></xref>).</p>
<p>The composition of highly and intermediately active pellets was quite comparable, with a non-significant tendency in the latter group towards a reduction in members of the genus <italic>Lactobacillus</italic> (mean rel. ab.: 89.3% vs. 75.1%, in high and intermediate, respectively) and an increase in the genera <italic>Olegusella</italic> (&lt;0.1% vs. 2.6%, respectively) and <italic>Streptococcus</italic> (0.3% vs. 2.1%). On the other hand, deep changes in the microbial signatures were evident in not-active pellets, which, compared to the highly active ones, showed a significant reduction in <italic>Lactobacillus</italic> (mean rel. ab: 89.3% vs. 52.9%, in the high and not-active groups, respectively, p=0.006), a significant increase in <italic>Streptococcus</italic> (mean rel.ab: 0.3% vs. 2.1%, in the high and not-active groups, respectively, p=0.002) and <italic>Olegusella</italic> (mean rel. ab: &lt;0.1% vs. 14.5% in the high and not-active groups, respectively; p=0.010). The same differences were highlighted in the comparison between the microbial profiles of intermediately- and not-active pellets, together with an increase in <italic>Bifidobacterium</italic> (p=0.030), <italic>Finegoldia</italic> (p=0.049), and <italic>Anaerococcus</italic> (p=0.020) in not-active pellets. Among <italic>Bifidobacterium</italic>, a robust share (corresponding to 1.3%-2.4% of the average abundance) was due to <italic>B. vaginalis</italic>, a recently proposed reclassification of <italic>Gardnerella vaginalis</italic> (unpublished data, presented by Barisic et&#xa0;al, 2019; Using Whole-Genome Sequencing to Revise the Classification of Bifidobacterium and Gardnerella Genera; available at: <ext-link ext-link-type="uri" xlink:href="https://delivery-files.atcc.org/api/public/content/255989-Using-WholeGenome-Sequencing-to-Revise-the-Classification-of-the-Bifidobacterium-and-Gardnerella-Gen">https://delivery-files.atcc.org/api/public/content/255989-Using-WholeGenome-Sequencing-to-Revise-the-Classification-of-the-Bifidobacterium-and-Gardnerella-Gen</ext-link>) as contained in the 16S rRNA database (i.e., GTDB) used for the taxonomic classification of our samples (<xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2D</bold></xref>).</p>
<p>Interestingly, a notable difference in the bacterial species belonging to the <italic>Lactobacillus</italic> genus could be observed, since <italic>L. crispatus</italic> was significantly more abundant in highly- and intermediately active pellets (mean rel. ab. &gt;50.0%) than in not-active ones (mean rel. ab. 2.8%), whereas the opposite was observed for <italic>L. gasseri</italic> (mean rel. ab. &lt;12.0% vs 40.8% in high/intermediate and not-active pellets, respectively). <italic>L. jensenii</italic> (mean rel. ab. 5.0 vs &lt;=0.1% in high and intermediate/not-active pellets, respectively) represented a hallmark of highly active pellets (<xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2E</bold></xref>; <xref ref-type="supplementary-material" rid="SM2"><bold>Supplementary Table&#xa0;3</bold></xref>). As expected, the microbiome profiles of samples stratified on the basis of the Nugent score largely resembled those based on the anti-chlamydial activity. Samples from women with BV were characterized by higher biodiversity (p&lt;0.02 for all the tested alpha-diversity metrics) and a different microbial profile (p=0.018 and p=0.006 for the unweighted and weighted Unifrac distances, respectively) than those from healthy women (H). A difference was somehow evident also for the women classified as having an &#x201c;intermediate&#x201d; vaginal community, which resulted statistically separated from both H and BV women for both the unweighted and the weighted UniFrac distance-based profiles (p=0.033 and p=0.012 for I vs H and I vs BV, respectively). As reported above, the <italic>Lactobacillus</italic> abundance was a hallmark of the H group, as well as <italic>Olegusella</italic> and <italic>Streptococcus</italic> abundances were fingerprints of BV condition (<xref ref-type="supplementary-material" rid="SM2"><bold>Supplementary Figure&#xa0;1</bold></xref>).</p>
</sec>
<sec id="s3_3">
<label>3.3</label>
<title>Association between anti-chlamydial activity and vaginal metabolites</title>
<p>Fifty-seven metabolites were detected and quantified by <sup>1</sup>H-NMR spectroscopy on the vaginal fluid supernatants. These molecules mainly belong to groups of short-chain fatty acids (SCFAs), organic acids, amino acids, and biogenic amines (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Data Sheet S1</bold></xref>). <xref ref-type="table" rid="T1"><bold>Table&#xa0;1</bold></xref> lists the metabolites whose concentrations differed significantly between samples, stratified by anti-chlamydial activity.</p>
<table-wrap id="T1" position="float">
<label>Table&#xa0;1</label>
<caption>
<p>Concentration (mM) of vaginal metabolites determined by <sup>1</sup>H-NMR spectroscopy, stratified by the anti-chlamydial activity.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="top" align="center">Metabolites</th>
<th valign="top" align="center">High<break/>activity (n=11)</th>
<th valign="top" align="center">Intermediate<break/>activity (n=15)</th>
<th valign="top" align="center">No<break/>activity (n=14)</th>
<th valign="top" align="center"><italic>p value</italic>
</th>
<th valign="top" align="center">High activity<break/>vs<break/>Intermediate</th>
<th valign="top" align="center">High activity<break/>vs<break/>No activity</th>
<th valign="top" align="center">Intermediate<break/>vs<break/>No activity</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="center"><bold>4-Aminobutyrate</bold>
</td>
<td valign="top" align="center">0.17 &#xb1; 0.19</td>
<td valign="top" align="center">0.091 &#xb1; 0.13</td>
<td valign="top" align="center">0.0074 &#xb1; 0.01</td>
<td valign="top" align="center">0.0092</td>
<td valign="top" align="left"/>
<td valign="top" align="center">**</td>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"><bold>Aspartate</bold>
</td>
<td valign="top" align="center">0.098 &#xb1; 0.026</td>
<td valign="top" align="center">0.072 &#xb1; 0.023</td>
<td valign="top" align="center">0.055 &#xb1; 0.018</td>
<td valign="top" align="center">0.0001</td>
<td valign="top" align="left"/>
<td valign="top" align="center">****</td>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"/>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="center">0.0147</td>
<td valign="top" align="center">*</td>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"><bold>Glucose</bold>
</td>
<td valign="top" align="center">0.038 &#xb1; 0.042</td>
<td valign="top" align="center">0.071 &#xb1; 0.062</td>
<td valign="top" align="center">0.37 &#xb1; 0.40</td>
<td valign="top" align="center">0.0044</td>
<td valign="top" align="left"/>
<td valign="top" align="center">**</td>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"/>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="center">0.0055</td>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="center">**</td>
</tr>
<tr>
<td valign="top" align="center"><bold>Glutamine</bold>
</td>
<td valign="top" align="center">0.12 &#xb1; 0.055</td>
<td valign="top" align="center">0.15 &#xb1; 0.044</td>
<td valign="top" align="center">0.20 &#xb1; 0.090</td>
<td valign="top" align="center">0.0221</td>
<td valign="top" align="left"/>
<td valign="top" align="center">*</td>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"><bold>Isoleucine</bold>
</td>
<td valign="top" align="center">0.13 &#xb1; 0.030</td>
<td valign="top" align="center">0.11 &#xb1; 0.037</td>
<td valign="top" align="center">0.079 &#xb1; 0.037</td>
<td valign="top" align="center">0.0045</td>
<td valign="top" align="left"/>
<td valign="top" align="center">**</td>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"><bold>Lactate</bold>
</td>
<td valign="top" align="center">8.46 &#xb1; 1.79</td>
<td valign="top" align="center">7.45 &#xb1; 2.22</td>
<td valign="top" align="center">4.57 &#xb1; 1.47</td>
<td valign="top" align="center">0.0001</td>
<td valign="top" align="left"/>
<td valign="top" align="center">****</td>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"/>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="center">0.0005</td>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="center">***</td>
</tr>
<tr>
<td valign="top" align="center"><bold>Leucine</bold>
</td>
<td valign="top" align="center">0.32 &#xb1; 0.060</td>
<td valign="top" align="center">0.26 &#xb1; 0.086</td>
<td valign="top" align="center">0.19 &#xb1; 0.090</td>
<td valign="top" align="center">0.0009</td>
<td valign="top" align="left"/>
<td valign="top" align="center">***</td>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"><bold>Sarcosine</bold>
</td>
<td valign="top" align="center">0.030 &#xb1; 0.014</td>
<td valign="top" align="center">0.022 &#xb1; 0.015</td>
<td valign="top" align="center">0.008 &#xb1; 0.0045</td>
<td valign="top" align="center">0.0002</td>
<td valign="top" align="left"/>
<td valign="top" align="center">***</td>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"/>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="center">0.014</td>
<td valign="top" align="left"/>
<td valign="top" align="left"/>
<td valign="top" align="center">*</td>
</tr>
<tr>
<td valign="top" align="center"><bold>Taurine</bold>
</td>
<td valign="top" align="center">0.39 &#xb1; 0.10</td>
<td valign="top" align="center">0.44 &#xb1; 0.088</td>
<td valign="top" align="center">0.55 &#xb1; 0.16</td>
<td valign="top" align="center">0.008</td>
<td valign="top" align="left"/>
<td valign="top" align="center">**</td>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"><bold>Tyrosine</bold>
</td>
<td valign="top" align="center">0.107 &#xb1; 0.021</td>
<td valign="top" align="center">0.090 &#xb1; 0.027</td>
<td valign="top" align="center">0.071 &#xb1; 0,030</td>
<td valign="top" align="center">0.0073</td>
<td valign="top" align="left"/>
<td valign="top" align="center">**</td>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center"><bold>Valine</bold>
</td>
<td valign="top" align="center">0.15 &#xb1; 0.032</td>
<td valign="top" align="center">0.13 &#xb1; 0.032</td>
<td valign="top" align="center">0.11 &#xb1; 0.038</td>
<td valign="top" align="center">0.038</td>
<td valign="top" align="left"/>
<td valign="top" align="center">*</td>
<td valign="top" align="left"/>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>Results are reported indicating mean &#xb1; standard deviation. Asterisks indicate significant variations (p&lt;0.05, after Benjamini-Hochberg correction) in metabolites concentration between groups. Differences were evaluated by Kruskal-Wallis test followed by Dunn&#x2019;s Multiple Comparison test. *p&lt;0.05 **p&lt;0.01 ***p&lt;0.001 ****p&lt;0.0001.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<p>Except for aspartate, there were no discernible changes in the metabolic profiles of the highly active and intermediately active samples overall. We noticed that a high anti-CT activity was associated with significantly higher levels of 4-aminobutyrate, aspartate, isoleucine, lactate, leucine, sarcosine, tyrosine, and valine, compared to not-active samples. The concentration of glucose tended to increase from the highly active (lowest levels) to the not-active samples (highest levels).</p>
</sec>
<sec id="s3_4">
<label>3.4</label>
<title>Correlation between vaginal microbiome and metabolome</title>
<p>We performed a correlation analysis aimed at relating the vaginal microbial composition to the metabolite concentrations using Spearman&#x2019;s rank correlation to determine monotonically increasing or decreasing relationships (<xref ref-type="fig" rid="f3"><bold>Figure&#xa0;3</bold></xref>).</p>
<fig id="f3" position="float">
<label>Figure&#xa0;3</label>
<caption>
<p>Heatmap showing the Spearman correlation between the main microbial taxa and metabolites. Only taxa present at &gt;1% in at least 10% of the samples are displayed. Statistically significant correlations (p&lt;0.05) are indicated by a star. Both taxa and metabolites are clustered according to Pearson&#x2019;s correlation and average linkage. For representation purposes, taxa abundance is shown in a log<sub>2</sub> scale, whereas lactate abundance scale is split.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1403782-g003.tif"/>
</fig>
<p>A group of bacteria, primarily lactobacilli plus <italic>Ureaplasma</italic> and <italic>Clostridium</italic>, showed a positive correlation with several amino acids (leucine, isoleucine, asparagine, valine, tyrosine, tryptophan, phenylalanine, and aspartate), their degradation products (sarcosine and 5-aminopentanoate), lactate, and 4-aminobutyrate. In particular, the highly abundant metabolite lactate (lactic acid) was significantly correlated to <italic>L. crispatus</italic> (r=0.432), <italic>L. jensenii</italic> (r=0.431), and other unclassified members of <italic>Lactobacillus</italic> genus (r=0.502); moreover, leucine (r between 0.345 and 0.568) and isoleucine (r comprised between 0.333 and 0.470) were positively correlated to <italic>L. crispatus</italic>, <italic>L. iners</italic>, <italic>L. jensenii</italic>, unclassified members of <italic>Lactobacillus</italic>, and <italic>Ureaplasma</italic>.</p>
<p>On the other hand, a further <italic>Lactobacillus</italic> species, <italic>L. gasseri</italic> showed a correlation pattern more similar to that of bacteria from <italic>Streptococcus</italic>, <italic>Lachnospira</italic> and <italic>Alloscardovia</italic> genera, with a high positive correlation to sugars (maltose, r=0.427; fructose, r= 0.608; glucose, r=0.709), glycerol (r=0.459), taurine (r=0.389), glutamine (r=0.435), uridine (r=0.351), creatinine (r=0.399), and o-acetylcarnitine (&#x3c1;=0.465). In general, all the interactions between <italic>L. gasseri</italic> and the metabolites were inversely correlated with those of <italic>L. crispatus</italic> and <italic>L. jensenii</italic>.</p>
<p>Finally, a third group of bacterial taxa (i.e., <italic>Prevotella</italic>, <italic>Dialister</italic>, <italic>Olegusella</italic>, <italic>Peptostreptococcus</italic>, <italic>Peptoniphilus</italic>, <italic>Finegoldia</italic> and <italic>Anaerococcus</italic>) showed a moderate positive correlation to alanine (r between 0.236 and 0.455), acetate (r between 0.138 and 0.353), formate (r between 0.345 and 0.568), and 2,3-butanediol (r between 0.120 and 0.466). A non-significant trend towards a positive correlation was also observed for dimethylamine (DMA), cadaverine, and phthalate.</p>
</sec>
</sec>
<sec id="s4" sec-type="discussion">
<label>4</label>
<title>Discussion</title>
<p>In healthy reproductive-aged women, the vaginal microbiome, generally, shows a predominance of members of the <italic>Lactobacillus</italic> genus and most women display the prevalence of one species among <italic>L. crispatus, L. iners, L. jensenii</italic>, and <italic>L. gasseri</italic> (<xref ref-type="bibr" rid="B38">Ravel et&#xa0;al., 2011</xref>). Vaginal resident lactobacilli act as a line of defense against both exogenous and endogenous pathogens, through different mechanisms, including the use of nutrients, establishment and maintenance of low pH levels, and production of antimicrobial compounds, as bacteriocins, peroxides, and organic acids (<xref ref-type="bibr" rid="B44">Valore et&#xa0;al., 2002</xref>; <xref ref-type="bibr" rid="B33">Parolin et&#xa0;al., 2018a</xref>).</p>
<p>In the case of BV, the abundance of vaginal <italic>Lactobacillus</italic> may decrease significantly, causing an increase in pH levels and the proliferation of different anaerobes, such as <italic>Gardnerella</italic> and <italic>Prevotella</italic> (<xref ref-type="bibr" rid="B7">Coudray and Madhivanan, 2020</xref>). This dysbiotic condition is usually accompanied by a peculiar metabolic fingerprint associated with a higher risk of STI acquisition, including CT infection (<xref ref-type="bibr" rid="B7">Coudray and Madhivanan, 2020</xref>).</p>
<p>Thus, the evaluation of the microbial and metabolic components of the vaginal fluids capable of either protecting or favoring CT infection is particularly intriguing.</p>
<p>To the best of our knowledge, this is the first study assessing the anti-chlamydial activity of fresh vaginal fluids, investigating the vaginal bacterial taxa and molecules associated with a significant reduction/abolishment of CT viability. Firstly, we found that a vaginal microbiome enriched in <italic>Lactobacillus</italic>, especially <italic>L. crispatus</italic> and <italic>L. iners</italic>, was associated with a significant anti-chlamydial activity. This microbial fingerprint was accompanied by high levels of lactate, leucine, and isoleucine; these metabolites are typically found in lactobacilli-dominated vaginal environments (<xref ref-type="bibr" rid="B13">Foschi et&#xa0;al., 2022</xref>).</p>
<p>It is well known that <italic>L. crispatus</italic> represents the hallmark of vaginal health and eubiosis, being indicated as one of the most active species against several urogenital pathogens, including CT (<xref ref-type="bibr" rid="B29">Nardini et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B33">Parolin et&#xa0;al., 2018a</xref>; <xref ref-type="bibr" rid="B6">Chen et&#xa0;al., 2022</xref>). This <italic>Lactobacillus</italic> species is able to produce significant levels of lactate, which is believed to play a central role in host defense against CT, acting on both the extracellular and intracellular phase of chlamydia developmental cycle (<xref ref-type="bibr" rid="B12">Edwards et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B49">Zalambani et&#xa0;al., 2023</xref>).</p>
<p>On the other hand, <italic>L. iners</italic> has been usually considered as a &#x2018;transitional&#x2019; species, characterized by poor activity against urogenital pathogens (<xref ref-type="bibr" rid="B36">Petrova et&#xa0;al., 2015</xref>). Although a <italic>L. iners</italic>-dominated vaginal microbiota may be suboptimal compared with a <italic>L. crispatus</italic>-one for CT infection, it has been recently shown that <italic>L. iners</italic> is the most common species in a large subset of women worldwide, being its presence associated with unprotected sex practices and younger age (<xref ref-type="bibr" rid="B16">France et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B30">Novak et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B4">Carter et&#xa0;al., 2023</xref>). Thus, the relatively young age of our cohort could explain the findings of the present study. Nevertheless, further investigations are needed to better understand the impact of <italic>L. iners</italic> on common cervicovaginal infections.</p>
<p>Interestingly, we observed that <italic>L. gasseri</italic> displayed an opposite behavior compared to <italic>L. crispatus</italic>, being more present in not-active samples and correlated to glucose, taurine, and glutamine, three metabolites more abundant in vaginal secretions with no activity against CT.</p>
<p>Several works underline that, even in presence of a strain-specific activity, <italic>L. gasseri</italic> exerts a reduced and more heterogenous spectrum of activity against CT, compared to <italic>L. crispatus</italic> (<xref ref-type="bibr" rid="B29">Nardini et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B2">Argentini et&#xa0;al., 2022</xref>).</p>
<p>Other interesting data emerged when looking at microbial/metabolic features of vaginal fluids associated with a poor protection from CT infection.</p>
<p>Not-active vaginal samples presented a significant reduction in <italic>Lactobacillus</italic>, along with a significant increase in <italic>Streptococcus</italic> and <italic>Olegusella</italic>. This latter Gram-positive bacterium was recently described as a new member of the Coriobacteriaceae family and was isolated from the vaginal flora of a patient with vaginal dysbiosis (<xref ref-type="bibr" rid="B9">Diop et&#xa0;al., 2016</xref>). <italic>Olegusella</italic>, along with other anaerobes such as <italic>Gardnerella</italic>, <italic>Finegoldia</italic>, and <italic>Anaerococcus</italic>, takes part to the polymicrobial environment typically found during BV, characterized by high levels of SCFAs (e.g., acetate, formate) and biogenic amines (i.e., DMA, cadaverine) (<xref ref-type="bibr" rid="B5">Ceccarani et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B20">Laghi et&#xa0;al., 2021</xref>).</p>
<p>In our experimental model, glucose represented a key metabolite, with vaginal samples with the highest levels of glucose showing the poorest anti-CT activity, whereas highly active samples were characterized by the lowest levels of glucose. This finding could be ascribed to several aspects: (i) the increased glucose consumption by some vaginal <italic>Lactobacillus</italic> strains is associated with a significant reduction in chlamydial elementary bodies infectivity (<xref ref-type="bibr" rid="B29">Nardini et&#xa0;al., 2016</xref>), (ii) sugars, as glucose, can increase CT infectivity and virulence, by modulating the expression/exposure of chlamydial membrane ligands (<xref ref-type="bibr" rid="B25">Marziali et&#xa0;al., 2020</xref>).</p>
<p>We are aware of some limitations in the present study. Firstly, information about the hormonal levels of participants and their sexual practices was not available. Therefore, larger studies with more detailed data are necessary to elucidate the impact of clinical and behavioral factors on the vaginal inhabitants.</p>
<p>Moreover, examining additional components of the vaginal environment, such as the presence of antibacterial peptides, enzymes, pro/anti-inflammatory cytokines, and microbial biosurfactants, will be crucial to better understand the factors associated with the anti-chlamydial effect.</p>
<p>In conclusion, we have highlighted the microbial and metabolic components of fresh vaginal fluids capable of counteracting CT viability in an <italic>in vitro</italic> model. These findings could pave the way for novel strategies to prevent or counteract chlamydial infection. Among them, we can hypothesize the development of new probiotic formulations based on <italic>L. crispatus</italic> species, the use lactobacilli-derived postbiotics, or the application of vaginal gels containing lactic acid (<xref ref-type="bibr" rid="B35">Pendharkar et&#xa0;al., 2023</xref>; <xref ref-type="bibr" rid="B41">Shen et&#xa0;al., 2023</xref>; <xref ref-type="bibr" rid="B42">Spaggiari et&#xa0;al., 2023</xref>). Finally, we can speculate that the practice of vaginal microbiome transplantation (VMT) from a donor with a flora dominated by <italic>L. crispatus</italic> could be of aid in preventing CT infections, by the restoration of a vaginal healthy state (<xref ref-type="bibr" rid="B22">Ma et&#xa0;al., 2019</xref>).</p>
</sec>
<sec id="s5" sec-type="data-availability">
<title>Data availability statement</title>
<p>The datasets presented in this study can be found in online repositories and/or in the <xref ref-type="supplementary-material" rid="SM2"><bold>Supplementary Material</bold></xref>. The names of the repository/repositories and accession number(s) can be found in the article.</p>
</sec>
<sec id="s6" sec-type="ethics-statement">
<title>Ethics statement</title>
<p>The studies involving humans were approved by Bioethics Committee of the University of Bologna (protocol number 0122421). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.</p>
</sec>
<sec id="s7" sec-type="author-contributions">
<title>Author contributions</title>
<p>SM: Writing &#x2013; original draft, Investigation, Data curation, Formal analysis. CaC: Writing &#x2013; original draft, Investigation, Data curation, Formal analysis, Software. MD: Writing &#x2013; original draft, Formal analysis. LL: Writing &#x2013; original draft, Formal analysis, Resources. TC: Writing &#x2013; original draft, Formal analysis, Resources. ClC: Writing &#x2013; original draft, Formal analysis, Resources. CF: Conceptualization, Writing &#x2013; original draft, Supervision, Validation. MS: Investigation, Writing &#x2013; original draft, Data curation, Supervision. AM: Conceptualization, Writing &#x2013; original draft, Supervision, Validation, Resources.</p>
</sec>
</body>
<back>
<sec id="s8" sec-type="funding-information">
<title>Funding</title>
<p>The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.</p>
</sec>
<ack>
<title>Acknowledgments</title>
<p>We would like to thank Dr. Laura Dionisi, Dr. Egle Pizzirani and Dr. Anna Chiara Lombardi for providing excellent technical support during the study.</p>
</ack>
<sec id="s9" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="s10" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s11" sec-type="supplementary-material">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fcimb.2024.1403782/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fcimb.2024.1403782/full#supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="DataSheet_1.xlsx" id="SM1" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet"/>
<supplementary-material xlink:href="DataSheet_2.docx" id="SM2" mimetype="application/vnd.openxmlformats-officedocument.wordprocessingml.document"/>
</sec>
<ref-list>
<title>References</title>
<ref id="B1">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Abbai</surname> <given-names>N. S.</given-names>
</name>
<name>
<surname>Reddy</surname> <given-names>T.</given-names>
</name>
<name>
<surname>Ramjee</surname> <given-names>G.</given-names>
</name>
</person-group> (<year>2016</year>). <article-title>Prevalent bacterial vaginosis infection - a risk factor for incident sexually transmitted infections in women in Durban, South Africa</article-title>. <source>Int. J. STD AIDS</source> <volume>27</volume>, <fpage>1283</fpage>&#x2013;<lpage>1288</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1177/0956462415616038</pub-id>
</citation>
</ref>
<ref id="B2">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Argentini</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Fontana</surname> <given-names>F.</given-names>
</name>
<name>
<surname>Alessandri</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Lugli</surname> <given-names>G. A.</given-names>
</name>
<name>
<surname>Mancabelli</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Ossiprandi</surname> <given-names>M. C.</given-names>
</name>
<etal/>
</person-group>. (<year>2022</year>). <article-title>Evaluation of modulatory activities of <italic>lactobacillus crispatus</italic> strains in the context of the vaginal microbiota</article-title>. <source>Microbiol. Spectr.</source> <volume>10</volume>, <elocation-id>e0273321</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/spectrum.02733-21</pub-id>
</citation>
</ref>
<ref id="B3">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Callahan</surname> <given-names>B. J.</given-names>
</name>
<name>
<surname>McMurdie</surname> <given-names>P. J.</given-names>
</name>
<name>
<surname>Rosen</surname> <given-names>M. J.</given-names>
</name>
<name>
<surname>Han</surname> <given-names>A. W.</given-names>
</name>
<name>
<surname>Johnson</surname> <given-names>A. J.</given-names>
</name>
<name>
<surname>Holmes</surname> <given-names>S. P.</given-names>
</name>
</person-group> (<year>2016</year>). <article-title>DADA2: High-resolution sample inference from Illumina amplicon data</article-title>. <source>Nat. Methods</source> <volume>13</volume>, <fpage>581</fpage>&#x2013;<lpage>583</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/nmeth.3869</pub-id>
</citation>
</ref>
<ref id="B4">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Carter</surname> <given-names>K. A.</given-names>
</name>
<name>
<surname>Fischer</surname> <given-names>M. D.</given-names>
</name>
<name>
<surname>Petrova</surname> <given-names>M. I.</given-names>
</name>
<name>
<surname>Balkus</surname> <given-names>J. E.</given-names>
</name>
</person-group> (<year>2023</year>). <article-title>Epidemiologic evidence on the role of lactobacillus iners in sexually transmitted infections and bacterial vaginosis: A series of systematic reviews and meta-analyses</article-title>. <source>Sex Transm. Dis.</source> <volume>50</volume>, <fpage>224</fpage>&#x2013;<lpage>235</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1097/OLQ.0000000000001744</pub-id>
</citation>
</ref>
<ref id="B5">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ceccarani</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Parolin</surname> <given-names>C.</given-names>
</name>
<name>
<surname>D'Antuono</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Gaspari</surname> <given-names>V.</given-names>
</name>
<name>
<surname>Consolandi</surname> <given-names>C.</given-names>
</name>
<etal/>
</person-group>. (<year>2019</year>). <article-title>Diversity of vaginal microbiome and metabolome during genital infections</article-title>. <source>Sci. Rep.</source> <volume>9</volume>, <fpage>14095</fpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/s41598-019-50410-x</pub-id>
</citation>
</ref>
<ref id="B6">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Chen</surname> <given-names>H.</given-names>
</name>
<name>
<surname>Min</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Wang</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Zhao</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Luo</surname> <given-names>F.</given-names>
</name>
<name>
<surname>Lei</surname> <given-names>W.</given-names>
</name>
<etal/>
</person-group>. (<year>2022</year>). <article-title>Lactobacillus Modulates Chlamydia Infectivity and Genital Tract Pathology in <italic>vitro</italic> and in <italic>vivo</italic>
</article-title>. <source>Front. Microbiol.</source> <volume>13</volume>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fmicb.2022.877223</pub-id>
</citation>
</ref>
<ref id="B7">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Coudray</surname> <given-names>M. S.</given-names>
</name>
<name>
<surname>Madhivanan</surname> <given-names>P.</given-names>
</name>
</person-group> (<year>2020</year>). <article-title>Bacterial vaginosis-A brief synopsis of the literature</article-title>. <source>Eur. J. Obstet. Gynecol. Reprod. Biol.</source> <volume>245</volume>, <fpage>143</fpage>&#x2013;<lpage>148</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.ejogrb.2019.12.035</pub-id>
</citation>
</ref>
<ref id="B8">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Dieterle</surname> <given-names>F.</given-names>
</name>
<name>
<surname>Ross</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Schlotterbeck</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Senn</surname> <given-names>H.</given-names>
</name>
</person-group> (<year>2006</year>). <article-title>Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in 1H NMR metabonomics</article-title>. <source>Anal. Chem.</source> <volume>78</volume>, <fpage>4281</fpage>&#x2013;<lpage>4290</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1021/ac051632c</pub-id>
</citation>
</ref>
<ref id="B9">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Diop</surname> <given-names>K.</given-names>
</name>
<name>
<surname>Diop</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Raoult</surname> <given-names>D.</given-names>
</name>
<name>
<surname>Fournier</surname> <given-names>P. E.</given-names>
</name>
<name>
<surname>Fenollar</surname> <given-names>F.</given-names>
</name>
</person-group> (<year>2016</year>). <article-title>'Olegusella massiliensis' strain KHD7, a new bacterial genus isolated from the female genital tract</article-title>. <source>New Microbes New Infect.</source> <volume>12</volume>, <fpage>56</fpage>&#x2013;<lpage>58</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.nmni.2016.04.008</pub-id>
</citation>
</ref>
<ref id="B10">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Di Pietro</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Filardo</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Simonelli</surname> <given-names>I.</given-names>
</name>
<name>
<surname>Pasqualetti</surname> <given-names>P.</given-names>
</name>
<name>
<surname>Sessa</surname> <given-names>R.</given-names>
</name>
</person-group> (<year>2022</year>). <article-title>Cervicovaginal microbiota composition in <italic>chlamydia trachomatis</italic> infection: A systematic review and meta-analysis</article-title>. <source>Int. J. Mol. Sci.</source> <volume>23</volume>, <elocation-id>9554</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/ijms23179554</pub-id>
</citation>
</ref>
<ref id="B11">
<citation citation-type="web">
<person-group person-group-type="author">
<collab>ECDC</collab>
</person-group>. (<year>2024</year>). <source>Chlamydia Infection&#x2014;Annual Epidemiological Report for 2019</source>. Available online at: <uri xlink:href="https://www.ecdc.europa.eu/en/publications-data/chlamydia-infection-annual-epidemiological-report-2019">https://www.ecdc.europa.eu/en/publications-data/chlamydia-infection-annual-epidemiological-report-2019</uri> (Accessed <access-date>February 2024</access-date>).</citation>
</ref>
<ref id="B12">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Edwards</surname> <given-names>V. L.</given-names>
</name>
<name>
<surname>Smith</surname> <given-names>S. B.</given-names>
</name>
<name>
<surname>McComb</surname> <given-names>E. J.</given-names>
</name>
<name>
<surname>Tamarelle</surname> <given-names>J.</given-names>
</name>
<name>
<surname>Ma</surname> <given-names>B.</given-names>
</name>
<name>
<surname>Humphrys</surname> <given-names>M. S.</given-names>
</name>
<etal/>
</person-group>. (<year>2019</year>). <article-title>The cervicovaginal microbiota-host interaction modulates <italic>chlamydia trachomatis</italic> infection</article-title>. <source>mBio</source> <volume>10</volume>, <fpage>e01548</fpage>&#x2013;<lpage>e01519</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/mBio.01548-19</pub-id>
</citation>
</ref>
<ref id="B13">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Alvisi</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Baldassarre</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Laghi</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Gava</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Mancini</surname> <given-names>I.</given-names>
</name>
<etal/>
</person-group>. (<year>2022</year>). <article-title>Vaginal metabolites in postmenopausal women with or without vulvo-vaginal atrophy at baseline and after ospemifeme and systemic hormone treatment</article-title>. <source>Maturitas</source> <volume>159</volume>, <fpage>7</fpage>&#x2013;<lpage>14</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.maturitas.2021.12.007</pub-id>
</citation>
</ref>
<ref id="B14">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Bortolotti</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Marziali</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Polito</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Marangoni</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Bolognesi</surname> <given-names>A.</given-names>
</name>
</person-group> (<year>2019</year>). <article-title>Survival and death of intestinal cells infected by <italic>Chlamydia trachomatis</italic>
</article-title>. <source>PloS One</source> <volume>14</volume>, <elocation-id>e0215956</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pone.0215956</pub-id>
</citation>
</ref>
<ref id="B15">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Laghi</surname> <given-names>L.</given-names>
</name>
<name>
<surname>D'Antuono</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Gaspari</surname> <given-names>V.</given-names>
</name>
<name>
<surname>Zhu</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Dellarosa</surname> <given-names>N.</given-names>
</name>
<etal/>
</person-group>. (<year>2018</year>). <article-title>Urine metabolome in women with Chlamydia trachomatis infection</article-title>. <source>PloS One</source> <volume>13</volume>, <elocation-id>e0194827</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pone.0194827</pub-id>
</citation>
</ref>
<ref id="B16">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>France</surname> <given-names>M. T.</given-names>
</name>
<name>
<surname>Rutt</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Narina</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Arbaugh</surname> <given-names>S.</given-names>
</name>
<name>
<surname>McComb</surname> <given-names>E.</given-names>
</name>
<name>
<surname>Humphrys</surname> <given-names>M. S.</given-names>
</name>
<etal/>
</person-group>. (<year>2020</year>). <article-title>Complete genome sequences of six <italic>lactobacillus iners</italic> strains isolated from the human vagina</article-title>. <source>Microbiol. Resour. Announc.</source> <volume>9</volume>, <fpage>e00234</fpage>&#x2013;<lpage>e00220</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/MRA.00234-20</pub-id>
</citation>
</ref>
<ref id="B17">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Freeman</surname> <given-names>G. H.</given-names>
</name>
<name>
<surname>Halton</surname> <given-names>J. H.</given-names>
</name>
</person-group> (<year>1951</year>). <article-title>Note on an exact treatment of contingency, goodness of fit and other problems of significance</article-title>. <source>Biometrika.</source> <volume>38</volume>, <fpage>141</fpage>&#x2013;<lpage>149</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1093/biomet/38.1-2.141</pub-id>
</citation>
</ref>
<ref id="B18">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Haggerty</surname> <given-names>C. L.</given-names>
</name>
<name>
<surname>Gottlieb</surname> <given-names>S. L.</given-names>
</name>
<name>
<surname>Taylor</surname> <given-names>B. D.</given-names>
</name>
<name>
<surname>Low</surname> <given-names>N.</given-names>
</name>
<name>
<surname>Xu</surname> <given-names>F.</given-names>
</name>
<name>
<surname>Ness</surname> <given-names>R. B.</given-names>
</name>
</person-group> (<year>2010</year>). <article-title>Risk of sequelae after <italic>Chlamydia trachomatis</italic> genital infection in women</article-title>. <source>J. Infect. Dis.</source> <volume>201 Suppl 2</volume>, <fpage>S134</fpage>&#x2013;<lpage>S155</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1086/652395</pub-id>
</citation>
</ref>
<ref id="B19">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Kneen</surname> <given-names>M. A.</given-names>
</name>
<name>
<surname>Annegarn</surname> <given-names>H. J.</given-names>
</name>
</person-group> (<year>1996</year>). <article-title>Algorithm for fitting XRF, SEM and PIXE X-ray spectra backgrounds</article-title>. <source>Nucl. Instrum Methods Phys. Res. B</source> <volume>109</volume>, <fpage>209</fpage>&#x2013;<lpage>213</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/0168-583X(95)00908-6</pub-id>
</citation>
</ref>
<ref id="B20">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Laghi</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Zagonari</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Patuelli</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Zhu</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Morselli</surname> <given-names>S.</given-names>
</name>
<etal/>
</person-group>. (<year>2021</year>). <article-title>Vaginal metabolic profiles during pregnancy: Changes between first and second trimester</article-title>. <source>PloS One</source> <volume>16</volume>, <elocation-id>e0249925</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pone.0249925</pub-id>
</citation>
</ref>
<ref id="B21">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Liland</surname> <given-names>K. H.</given-names>
</name>
<name>
<surname>Alm&#xf8;y</surname> <given-names>T.</given-names>
</name>
<name>
<surname>Mevik</surname> <given-names>B. H.</given-names>
</name>
</person-group> (<year>2010</year>). <article-title>Optimal choice of baseline correction for multivariate calibration of spectra</article-title>. <source>Appl. Spectrosc</source>. <volume>64</volume>, <fpage>1007</fpage>&#x2013;<lpage>1016</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1366/000370210792434350</pub-id>
</citation>
</ref>
<ref id="B22">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ma</surname> <given-names>D.</given-names>
</name>
<name>
<surname>Chen</surname> <given-names>Y.</given-names>
</name>
<name>
<surname>Chen</surname> <given-names>T.</given-names>
</name>
</person-group> (<year>2019</year>). <article-title>Vaginal microbiota transplantation for the treatment of bacterial vaginosis: a conceptual analysis</article-title>. <source>FEMS Microbiol. Lett.</source> <volume>366</volume>, <elocation-id>fnz025</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1093/femsle/fnz025</pub-id>
</citation>
</ref>
<ref id="B23">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Marangoni</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Fiorino</surname> <given-names>E.</given-names>
</name>
<name>
<surname>Gilardi</surname> <given-names>F.</given-names>
</name>
<name>
<surname>Aldini</surname> <given-names>R.</given-names>
</name>
<name>
<surname>Scotti</surname> <given-names>E.</given-names>
</name>
<name>
<surname>Nardini</surname> <given-names>P.</given-names>
</name>
<etal/>
</person-group>. (<year>2015</year>a). <article-title><italic>Chlamydia pneumoniae</italic> acute liver infection affects hepatic cholesterol and triglyceride metabolism in mice</article-title>. <source>Atherosclerosis</source> <volume>241</volume>, <fpage>471</fpage>&#x2013;<lpage>479</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.atherosclerosis.2015.05.023</pub-id>
</citation>
</ref>
<ref id="B24">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Marangoni</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Nardini</surname> <given-names>P.</given-names>
</name>
<name>
<surname>Compri</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Cevenini</surname> <given-names>R.</given-names>
</name>
</person-group> (<year>2015</year>b). <article-title>Evaluation of the Versant CT/GC DNA 1.0 assay (kPCR) for the detection of extra-genital <italic>Chlamydia trachomatis</italic> and <italic>Neisseria gonorrhoeae</italic> infections</article-title>. <source>PloS One</source> <volume>10</volume>, <elocation-id>e0120979</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pone.0120979</pub-id>
</citation>
</ref>
<ref id="B25">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Marziali</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Marangoni</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Re</surname> <given-names>M. C.</given-names>
</name>
<name>
<surname>Calonghi</surname> <given-names>N.</given-names>
</name>
</person-group> (<year>2020</year>). <article-title>Effect of sugars on <italic>chlamydia trachomatis</italic> infectivity</article-title>. <source>Pathogens</source> <volume>9</volume>, <elocation-id>298</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/pathogens9040298</pub-id>
</citation>
</ref>
<ref id="B26">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Mastromarino</surname> <given-names>P.</given-names>
</name>
<name>
<surname>Di Pietro</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Schiavoni</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Nardis</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Gentile</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Sessa</surname> <given-names>R.</given-names>
</name>
</person-group> (<year>2014</year>). <article-title>Effects of vaginal lactobacilli in <italic>Chlamydia trachomatis</italic> infection</article-title>. <source>Int. J. Med. Microbiol.</source> <volume>304</volume>, <fpage>654</fpage>&#x2013;<lpage>661</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.ijmm.2014.04.006</pub-id>
</citation>
</ref>
<ref id="B27">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>McMurdie</surname> <given-names>P. J.</given-names>
</name>
<name>
<surname>Holmes</surname> <given-names>S.</given-names>
</name>
</person-group> (<year>2013</year>). <article-title>phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data</article-title>. <source>PloS One</source> <volume>8</volume>, <elocation-id>e61217</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pone.0061217</pub-id>
</citation>
</ref>
<ref id="B28">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Menon</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Timms</surname> <given-names>P.</given-names>
</name>
<name>
<surname>Allan</surname> <given-names>J. A.</given-names>
</name>
<name>
<surname>Alexander</surname> <given-names>K.</given-names>
</name>
<name>
<surname>Rombauts</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Horner</surname> <given-names>P.</given-names>
</name>
<etal/>
</person-group>. (<year>2015</year>). <article-title>Human and pathogen factors associated with <italic>chlamydia trachomatis</italic>-related infertility in women</article-title>. <source>Clin. Microbiol. Rev.</source> <volume>28</volume>, <fpage>969</fpage>&#x2013;<lpage>985</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/CMR.00035-15</pub-id>
</citation>
</ref>
<ref id="B29">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Nardini</surname> <given-names>P.</given-names>
</name>
<name>
<surname>&#xd1;ahui Palomino</surname> <given-names>R. A.</given-names>
</name>
<name>
<surname>Parolin</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Laghi</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Cevenini</surname> <given-names>R.</given-names>
</name>
<etal/>
</person-group>. (<year>2016</year>). <article-title><italic>Lactobacillus crispatus</italic> inhibits the infectivity of <italic>Chlamydia trachomatis</italic> elementary bodies, in <italic>vitro</italic> study</article-title>. <source>Sci. Rep.</source> <volume>6</volume>, <elocation-id>29024</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1038/srep29024</pub-id>
</citation>
</ref>
<ref id="B30">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Novak</surname> <given-names>J.</given-names>
</name>
<name>
<surname>Ravel</surname> <given-names>J.</given-names>
</name>
<name>
<surname>Ma</surname> <given-names>B.</given-names>
</name>
<name>
<surname>Ferreira</surname> <given-names>C. S. T.</given-names>
</name>
<name>
<surname>Trist&#xe3;o</surname> <given-names>A. D. R.</given-names>
</name>
<name>
<surname>Silva</surname> <given-names>M. G.</given-names>
</name>
<etal/>
</person-group>. (<year>2022</year>). <article-title>Characteristics associated with Lactobacillus iners-dominated vaginal microbiota</article-title>. <source>Sex Transm. Infect.</source> <volume>98</volume>, <fpage>353</fpage>&#x2013;<lpage>359</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1136/sextrans-2020-054824</pub-id>
</citation>
</ref>
<ref id="B31">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Nugent</surname> <given-names>R. P.</given-names>
</name>
<name>
<surname>Krohn</surname> <given-names>M. A.</given-names>
</name>
<name>
<surname>Hillier</surname> <given-names>S. L.</given-names>
</name>
</person-group> (<year>1991</year>). <article-title>Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation</article-title>. <source>J. Clin. Microbiol.</source> <volume>29</volume>, <fpage>297</fpage>&#x2013;<lpage>301</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/jcm.29.2.297-301.1991</pub-id>
</citation>
</ref>
<ref id="B32">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Parks</surname> <given-names>D. H.</given-names>
</name>
<name>
<surname>ChuvoChina</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Rinke</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Mussig</surname> <given-names>A. J.</given-names>
</name>
<name>
<surname>Chaumeil</surname> <given-names>P. A.</given-names>
</name>
<name>
<surname>Hugenholtz</surname> <given-names>P.</given-names>
</name>
</person-group> (<year>2022</year>). <article-title>GTDB: an ongoing census of bacterial and archaeal diversity through a phylogenetically consistent, rank normalized and complete genome-based taxonomy</article-title>. <source>Nucleic Acids Res.</source> <volume>50</volume>, <fpage>D785</fpage>&#x2013;<lpage>D794</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1093/nar/gkab776</pub-id>
</citation>
</ref>
<ref id="B33">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Parolin</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Laghi</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Zhu</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Banzola</surname> <given-names>N.</given-names>
</name>
<name>
<surname>Gaspari</surname> <given-names>V.</given-names>
</name>
<etal/>
</person-group>. (<year>2018</year>a). <article-title>Insights into vaginal bacterial communities and metabolic profiles of <italic>chlamydia trachomatis</italic> infection: positioning between eubiosis and dysbiosis</article-title>. <source>Front. Microbiol.</source> <volume>9</volume>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fmicb.2018.00600</pub-id>
</citation>
</ref>
<ref id="B34">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Parolin</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Frisco</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Giordani</surname> <given-names>B.</given-names>
</name>
<name>
<surname>Salvo</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Vitali</surname> <given-names>B.</given-names>
</name>
<etal/>
</person-group>. (<year>2018</year>b). <article-title><italic>Lactobacillus crispatus</italic> BC5 Interferes With <italic>Chlamydia trachomatis</italic> Infectivity Through Integrin Modulation in Cervical Cells</article-title>. <source>Front. Microbiol.</source> <volume>9</volume>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fmicb.2018.02630</pub-id>
</citation>
</ref>
<ref id="B35">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pendharkar</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Skafte-Holm</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Simsek</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Haahr</surname> <given-names>T.</given-names>
</name>
</person-group> (<year>2023</year>). <article-title>Lactobacilli and their probiotic effects in the vagina of reproductive age women</article-title>. <source>Microorganisms</source> <volume>11</volume>, <elocation-id>636</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/microorganisms11030636</pub-id>
</citation>
</ref>
<ref id="B36">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Petrova</surname> <given-names>M. I.</given-names>
</name>
<name>
<surname>Lievens</surname> <given-names>E.</given-names>
</name>
<name>
<surname>Malik</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Imholz</surname> <given-names>N.</given-names>
</name>
<name>
<surname>Lebeer</surname> <given-names>S.</given-names>
</name>
</person-group> (<year>2015</year>). <article-title>Lactobacillus species as biomarkers and agents that can promote various aspects of vaginal health</article-title>. <source>Front. Physiol.</source> <volume>6</volume>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fphys.2015.00081</pub-id>
</citation>
</ref>
<ref id="B37">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Raimondi</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Candeliere</surname> <given-names>F.</given-names>
</name>
<name>
<surname>Amaretti</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Morselli</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Gaspari</surname> <given-names>V.</given-names>
</name>
<etal/>
</person-group>. (<year>2021</year>). <article-title>Vaginal and Anal Microbiome during <italic>Chlamydia trachomatis</italic> Infections</article-title>. <source>Pathogens</source> <volume>10</volume>, <elocation-id>1347</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/pathogens10101347</pub-id>
</citation>
</ref>
<ref id="B38">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ravel</surname> <given-names>J.</given-names>
</name>
<name>
<surname>Gajer</surname> <given-names>P.</given-names>
</name>
<name>
<surname>Abdo</surname> <given-names>Z.</given-names>
</name>
<name>
<surname>Schneider</surname> <given-names>G. M.</given-names>
</name>
<name>
<surname>Koenig</surname> <given-names>S. S.</given-names>
</name>
<name>
<surname>McCulle</surname> <given-names>S. L.</given-names>
</name>
<etal/>
</person-group>. (<year>2011</year>). <article-title>Vaginal microbiome of reproductive-age women</article-title>. <source>Proc. Natl. Acad. Sci. U.S.A.</source> <volume>108 Suppl 1</volume>, <fpage>4680</fpage>&#x2013;<lpage>4687</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1073/pnas.1002611107</pub-id>
</citation>
</ref>
<ref id="B39">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Severgnini</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Camboni</surname> <given-names>T.</given-names>
</name>
<name>
<surname>Ceccarani</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Morselli</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Cantiani</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Zagonari</surname> <given-names>S.</given-names>
</name>
<etal/>
</person-group>. (<year>2021</year>). <article-title>Distribution of ermB, ermF, tet(W), and tet(M) Resistance Genes in the Vaginal Ecosystem of Women during Pregnancy and Puerperium</article-title>. <source>Pathogens</source> <volume>10</volume>, <elocation-id>1546</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/pathogens10121546</pub-id>
</citation>
</ref>
<ref id="B40">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Severgnini</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Morselli</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Camboni</surname> <given-names>T.</given-names>
</name>
<name>
<surname>Ceccarani</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Laghi</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Zagonari</surname> <given-names>S.</given-names>
</name>
<etal/>
</person-group>. (<year>2022</year>). <article-title>A deep look at the vaginal environment during pregnancy and puerperium</article-title>. <source>Front. Cell Infect. Microbiol.</source> <volume>12</volume>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fcimb.2022.838405</pub-id>
</citation>
</ref>
<ref id="B41">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Shen</surname> <given-names>X.</given-names>
</name>
<name>
<surname>Xu</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Zhang</surname> <given-names>Z.</given-names>
</name>
<name>
<surname>Yang</surname> <given-names>Y.</given-names>
</name>
<name>
<surname>Li</surname> <given-names>P.</given-names>
</name>
<name>
<surname>Ma</surname> <given-names>T.</given-names>
</name>
<etal/>
</person-group>. (<year>2023</year>). <article-title>Postbiotic gel relieves clinical symptoms of bacterial vaginitis by regulating the vaginal microbiota</article-title>. <source>Front. Cell Infect. Microbiol.</source> <volume>13</volume>. doi:&#xa0;<pub-id pub-id-type="doi">10.3389/fcimb.2023.1114364</pub-id>
</citation>
</ref>
<ref id="B42">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Spaggiari</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Squartini Ramos</surname> <given-names>G. B.</given-names>
</name>
<name>
<surname>Squartini Ramos</surname> <given-names>C. A.</given-names>
</name>
<name>
<surname>Ardizzoni</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Pedretti</surname> <given-names>N.</given-names>
</name>
<name>
<surname>Blasi</surname> <given-names>E.</given-names>
</name>
<etal/>
</person-group>. (<year>2023</year>). <article-title>Anti-candida and anti-inflammatory properties of a vaginal gel formulation: novel data concerning vaginal infection and dysbiosis</article-title>. <source>Microorganisms.</source> <volume>11</volume>, <elocation-id>1551</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/microorganisms11061551</pub-id>
</citation>
</ref>
<ref id="B43">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Tamarelle</surname> <given-names>J.</given-names>
</name>
<name>
<surname>Thi&#xe9;baut</surname> <given-names>A. C. M.</given-names>
</name>
<name>
<surname>de Barbeyrac</surname> <given-names>B.</given-names>
</name>
<name>
<surname>B&#xe9;b&#xe9;ar</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Ravel</surname> <given-names>J.</given-names>
</name>
<name>
<surname>Delarocque-Astagneau</surname> <given-names>E.</given-names>
</name>
</person-group> (<year>2019</year>). <article-title>The vaginal microbiota and its association with human papillomavirus, <italic>Chlamydia trachomatis</italic>, <italic>Neisseria gonorrhoeae</italic> and <italic>Mycoplasma genitalium</italic> infections: a systematic review and meta-analysis</article-title>. <source>Clin. Microbiol. Infect.</source> <volume>25</volume>, <fpage>35</fpage>&#x2013;<lpage>47</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.cmi.2018.04.019</pub-id>
</citation>
</ref>
<ref id="B44">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Valore</surname> <given-names>E. V.</given-names>
</name>
<name>
<surname>Park</surname> <given-names>C. H.</given-names>
</name>
<name>
<surname>Igreti</surname> <given-names>S. L.</given-names>
</name>
<name>
<surname>Ganz</surname> <given-names>T.</given-names>
</name>
</person-group> (<year>2002</year>). <article-title>Antimicrobial components of vaginal fluid</article-title>. <source>Am. J. Obstet. Gynecol.</source> <volume>187</volume>, <fpage>561</fpage>&#x2013;<lpage>568</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1067/mob.2002.125280</pub-id>
</citation>
</ref>
<ref id="B45">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ventrella</surname> <given-names>D.</given-names>
</name>
<name>
<surname>Laghi</surname> <given-names>L.</given-names>
</name>
<name>
<surname>Barone</surname> <given-names>F.</given-names>
</name>
<name>
<surname>Elmi</surname> <given-names>A.</given-names>
</name>
<name>
<surname>Romagnoli</surname> <given-names>N.</given-names>
</name>
<name>
<surname>Bacci</surname> <given-names>M. L.</given-names>
</name>
</person-group> (<year>2016</year>). <article-title>Age-related 1H NMR characterization of cerebrospinal fluid in newborn and young healthy piglets</article-title>. <source>PloS One</source> <volume>11</volume>, <elocation-id>e0157623</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.1371/journal.pone.0157623</pub-id>
</citation>
</ref>
<ref id="B46">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Wiesenfeld</surname> <given-names>H. C.</given-names>
</name>
<name>
<surname>Hillier</surname> <given-names>S. L.</given-names>
</name>
<name>
<surname>Krohn</surname> <given-names>M. A.</given-names>
</name>
<name>
<surname>Landers</surname> <given-names>D. V.</given-names>
</name>
<name>
<surname>Sweet</surname> <given-names>R. L.</given-names>
</name>
</person-group> (<year>2003</year>). <article-title>Bacterial vaginosis is a strong predictor of <italic>Neisseria gonorrhoeae</italic> and <italic>Chlamydia trachomatis</italic> infection</article-title>. <source>Clin. Infect. Dis.</source> <volume>36</volume>, <fpage>663</fpage>&#x2013;<lpage>668</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1086/367658</pub-id>
</citation>
</ref>
<ref id="B47">
<citation citation-type="web">
<person-group person-group-type="author">
<collab>World Health Organization (WHO)</collab></person-group>. (<year>2015</year>). <source>Report on Global Sexually Transmitted Infection Surveillance</source>. Available online at: <uri xlink:href="https://www.who.int/reproductivehealth/publications/rtis/stis-surveillance-2015/en/">https://www.who.int/reproductivehealth/publications/rtis/stis-surveillance-2015/en/</uri> (Accessed <access-date>February 2024</access-date>).</citation>
</ref>
<ref id="B48">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Younes</surname> <given-names>J. A.</given-names>
</name>
<name>
<surname>Lievens</surname> <given-names>E.</given-names>
</name>
<name>
<surname>Hummelen</surname> <given-names>R.</given-names>
</name>
<name>
<surname>van der Westen</surname> <given-names>R.</given-names>
</name>
<name>
<surname>Reid</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Petrova</surname> <given-names>M. I.</given-names>
</name>
</person-group> (<year>2018</year>). <article-title>Women and their microbes: the unexpected friendship</article-title>. <source>Trends Microbiol.</source> <volume>26</volume>, <fpage>16</fpage>&#x2013;<lpage>32</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1016/j.tim.2017.07.008</pub-id>
</citation>
</ref>
<ref id="B49">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zalambani</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Rizzardi</surname> <given-names>N.</given-names>
</name>
<name>
<surname>Marziali</surname> <given-names>G.</given-names>
</name>
<name>
<surname>Foschi</surname> <given-names>C.</given-names>
</name>
<name>
<surname>Morselli</surname> <given-names>S.</given-names>
</name>
<name>
<surname>Djusse</surname> <given-names>M. E.</given-names>
</name>
<etal/>
</person-group>. (<year>2023</year>). <article-title>Role of D(-)-lactic acid in prevention of <italic>chlamydia trachomatis</italic> infection in an <italic>in vitro</italic> model of heLa cells</article-title>. <source>Pathogens</source> <volume>12</volume>, <elocation-id>883</elocation-id>. doi:&#xa0;<pub-id pub-id-type="doi">10.3390/pathogens12070883</pub-id>
</citation>
</ref>
<ref id="B50">
<citation citation-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zozaya-Hinchliffe</surname> <given-names>M.</given-names>
</name>
<name>
<surname>Lillis</surname> <given-names>R.</given-names>
</name>
<name>
<surname>Martin</surname> <given-names>D. H.</given-names>
</name>
<name>
<surname>Ferris</surname> <given-names>M. J.</given-names>
</name>
</person-group> (<year>2010</year>). <article-title>Quantitative PCR assessments of bacterial species in women with and without bacterial vaginosis</article-title>. <source>J. Clin. Microbiol.</source> <volume>48</volume>, <fpage>1812</fpage>&#x2013;<lpage>1819</lpage>. doi:&#xa0;<pub-id pub-id-type="doi">10.1128/JCM.00851-09</pub-id>
</citation>
</ref>
</ref-list>
</back>
</article>
