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Introduction: In healthy women, lactobacilli play a crucial role in maintaining the microbial homeostasis of the vaginal niche. In case of bacterial vaginosis (BV), a condition characterised by a depletion of lactobacilli and an increasing number of anaerobes, a higher risk of urogenital and sexually transmitted infections (STIs) is reported. The vaginal environment of healthy and BV-positive women have been extensively studied, leading to the identification of the microbial species dominating these opposite conditions and to the description of specific metabolic profiles. Besides that, less is known about the vaginal microbiome in case of STIs, as Chlamydia trachomatis (CT) infections. The aim of this study was to analyse the composition of the endogenous microbiota and the metabolic signatures of the vaginal niche in 3 different conditions: healthy, BV and CT infections. Methods: From July 2016, all the pre-menopausal women attending the STI Outpatients Clinic of Sant’Orsola-Malpighi Hospital in Bologna (Italy) and meeting one of the following criteria were enrolled: presence of vaginal symptoms or presence of risk factors for CT infection. Patients with vaginal candidiasis were excluded. For all the patients, a vaginal swab was collected for molecular CT detection (Versant CT/GC DNA 1.0 Assay; Siemens), whereas Amsel criteria were used for BV assessment. Moreover, for each woman, an additional vaginal swab stored in saline was collected and centrifuged. Cell pellets were examined with a DNA-microarray platform including 17 probe sets specific for the most representative vaginal bacterial groups and with a quantitative real-time PCR targeting 16s rRNA gene of Gardnerella vaginalis (GV). Cell-free supernatants were used for metabolomic analysis by means of 1H-NMR spectroscopy. NMR spectra were recorded with an AVANCE spectrometer (Bruker). Similarities among microbial and metabolic profiles of samples were investigated by means of a principal component analysis (PCA). Differences in GV DNA loads and metabolites concentrations were analysed by ANOVA test. The study was approved by the Hospital Ethical Committee. Results: Among all the women enrolled, 25 were considered healthy, 18 received a diagnosis of BV and 22 were positive for CT. PCA revealed that the vaginal microbiome of healthy and BV-subjects were clearly distinct and that CT-positive women were more similar to healthy women rather than to BV-positives, both for microbial composition and for metabolic profile. The mean GV DNA load was significantly different between the groups (p=0.03): healthy and CT positive women showed similar and lower mean loads compared to BV group. At a metabolic level, significantly higher concentrations of formate, ethanolamine and methylamine were found in BV-patients, while tryptophan and lactate were more present in healthy and CT-positive women. Conclusion: Specific microbial and metabolic signatures characterise different clinical conditions of the vaginal tract. In this context, CT-positive women are definitely more similar to healthy than BV-subjects.

Introduction: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Mycoplasma genitalium (MG) represent the most common agents of bacterial sexually transmitted infections (STIs), worldwide. In women, uro-genital infections caused by these microorganisms are often asymptomatic and, left untreated, can lead to several sequelae. Nucleic acid amplification techniques (NAATs) have become the reference methods for the diagnosis, thanks to the suitability for different specimens and the outstanding sensitivity and specificity. The aim of this study was to assess the performance of Aptima Assays for CT, NG and MG detection in a group of selected women, by a head-to-head comparison with other NAATs. Moreover, an evaluation about the suitability for the Aptima assays with one of the most used swab collection device (E-Swab; Copan) was carried out. Methods: Routinely, all the women attending the STI Outpatients Clinic of Sant’Orsola-Malpighi Hospital of Bologna (Italy) complaining of genital STI-related symptoms or reporting unsafe intercourse, are managed as follows. After a clinical visit, a sample of first-void urines and a vaginal swab collected in E-swab, are obtained for CT, NG and MG detection. A duplex real-time PCR (Versant CT/GC DNA 1.0 assay; Siemens) is used for CT and NG detection, while, MG presence is investigated by a home-made PCR, starting from the remaining eluate of Versant PCR plate. From January 2016, a total of 100 patients were selected and their samples were also tested with Aptima assays. Previously frozen samples were thawed and transferred to the suitable collection devices for Aptima assays: in particular, 2 ml of urines and 100 µl of vaginal E-swab were used. All the specimens were processed by Aptima Combo2® for CT and NG detection and by the Aptima® Mycoplasma genitalium assay for MG infection diagnosis. These assays were run on Panther system (Hologic). A comparison between the different molecular methods, stratified by type of sample and microorganism, was conducted. Results: In the group of 100 women selected, 25 patients were positive for CT, 4 for NG and 6 for MG. One case of CT-NG and two cases of CT-MG co-infections were found. Interestingly, more than 50% of CT-positive women were completely asymptomatic. By the routine tests, all positive cases were simultaneously found both on the urine sample and on the vaginal swab, except for 3 CT, 1 NG and 1 MG infections, detected only on the vaginal swab. A complete concordance with Aptima assays, both for the type of sample and microorganism was found, with only one discordant result (a CT case detected by Versant on urines and vaginal swab, found by Aptima only on urines). Any interference due to the different liquid components of E-Swab was excluded. Conclusion: Given the outstanding performance, Aptima assays can represent an excellent choice for CT, NG and MG molecular detection. Moreover, it is noteworthy that Aptima assays allow testing of specimens collected by E-Swab, enabling the possibility to use the same sample for both NG molecular detection and culture.

Backgrounds: The vaginal microbiota of healthy women is generally dominated by Lactobacillus spp., which are known to protect the female genital tract from microbial dysbiosis and pathogen overgrowth. Alterations of the vaginal microbiota composition are found in bacterial vaginosis (BV). Lactobacilli have also been hypothesized to protect women from sexually transmitted diseases, including Chlamydia trachomatis (CT) infection, which represents the most common bacterial sexually transmitted infection worldwide. Both BV and CT infection can lead to severe sequelae and complications, including preterm labor and delivery, and tubal infertility. Objectives: The aim of this study is to analyse the composition of the endogenous microbiota and the metabolic profiles of the vaginal niche in three different conditions: healthy, BV and CT infection. Methods: Vaginal swabs were obtained from 66 women, belonging to three groups (healthy, BV and CT-infected women). The microbial composition of the samples was determined by using a microarray-based tool (VaginArray) targeting the most representative bacterial groups of the vaginal ecosystem, together with a quantitative real-time PCR for Gardnerella vaginalis. The metabolic profiles of the vaginal specimens were assessed by 1H-NMR. Conclusions: From our results the microbial signature of BV-affected women is clearly different from that found in healthy subjects, CT-infected women are characterized by a microbiota similar to the healthy condition. The metabolomics approach evidenced that BV-samples are characterized by significant variations in organic acids, aminoacids, short chain fatty acids concentrations. CT-infected women metabolome resembles that of healthy subjects, nevertheless significant variations in biogenic amines content were underlined.

Introduction: Lactobacilli play a fundamental role in maintaining the ecological equilibrium of the vaginal niche, preventing the overgrowth of endogenous microorganisms and impeding the colonisation of pathogens. Although many studies have focused on the mechanisms displayed by lactobacilli in counteracting several urogenital pathogens, a few data are available on the interaction between lactobacilli and Chlamydia trachomatis (CT). The aim of this study was therefore to assess the in vitro activity of different vaginal Lactobacillus strains against CT infectivity, investigating two different fractions of bacteria (cell pellets and cell-free supernatants), by three different mechanisms of action (competition, exclusion and displacement). Methods: A total of 17 Lactobacillus strains, isolated from vaginal swabs of healthy premenopausal women and belonging to L. crispatus (BC1; BC3-BC8), L. gasseri (BC9-BC14), L. vaginalis (BC16-BC17) and L. plantarum (BC18-19) species, were included in the study. The capacity of lactobacilli cell pellets (CP) and cell-free supernatants (CFS) to interfere with CT adhesion and entry in HeLa epithelial cells was evaluated, by means of competition, exclusion and displacement mechanisms. In particular, lactobacilli fractions corresponding to 5×107 colony forming units (CFU) were incubated with 5×103 CT elementary bodies (EBs) of strain GO/86, (serotype D), following different timelines. CT infection was evaluated by counting chlamydia inclusion forming units (IFUs) by direct immunofluorescence. Moreover, on the basis of CT infectivity interference results, 5 lactobacilli were selected for dose-effect assays and the same experiments were repeated, using CP or CFS fractions with 5×106 and 5×105 lactobacilli cells, in order to verify if a dose-dependent activity was present. Finally, the capacity of Lactobacillus strains to adhere to HeLa cells was assessed as well: results were read at light-microscopy and HeLa cells were scored for the presence and number of lactobacilli attached. Results: We found that lactobacilli cell pellets were the most active fraction in counteracting CT infectivity, particularly by means of an exclusion strategy, and that L. crispatus was the most effective species, even though a strain-specific activity was detected. Moreover, the anti-chlamydial activity was not correlated with the level of lactobacilli adhesion on epithelial cells and it was significantly maintained with low numbers of lactobacilli, although in presence of a dose-response effect. Conclusion: We identified specific vaginal Lactobacillus strains (L. crispatus BC4, L. crispatus BC5, L. crispatus BC7, L. gasseri BC14 and L. plantarum BC19) able to interfere with CT EBs adhesion and entry in epithelial cells and we were able to shed light on the mechanisms displayed by lactobacilli in counteracting CT infectivity. A major potential application lies on the use of these Lactobacillus strains as probiotics for the prophylaxis and/or adjuvant therapy of CT infections.

Background: In healthy pre-menopausal women, lactobacilli play a crucial role in maintaining the ecological equilibrium of the vaginal niche. In case of bacterial vaginosis (BV), a condition characterized by a depletion of vaginal lactobacilli and an increasing number of anaerobes, a higher risk of urogenital and sexually transmitted infections (STIs) acquisition is reported. The vaginal environment of healthy and BV-positive women has been extensively studied by molecular approaches, in order to identify the microbial species dominating these opposite conditions. Moreover, many studies have addressed the composition of vaginal metabolites produced by microbes and host cells, revealing BV-specific metabolic profiles. Besides that, less is known about the vaginal metabolome in case of STIs, as Chlamydia trachomatis (CT) infections. The aim of this study was therefore to characterize and compare the vaginal metabolic profiles in different conditions (healthy, BV, CT infections), by means of 1H-NMR spectroscopy. Material/methods: From July 2016, all the pre-menopausal women attending the STI Outpatients Clinic of Sant’Orsola-Malpighi Hospital in Bologna (Italy) and meeting one of the following criteria were enrolled: presence of vulvo-vaginal symptoms or presence of risk factors for CT infection. Patients with vulvo-vaginal candidiasis were excluded from this study. For all the patients, after a clinical visit, a vaginal swab was collected for molecular CT detection (Versant CT/GC DNA 1.0 Assay; Siemens), whereas Amsel criteria were used for BV assessment. Moreover, for each woman, an additional vaginal swab stored in saline was collected for metabolomic analysis: after centrifugation, 1 ml of cellsfree supernatant was added to 160 μL of a D2O solution of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt 6.25 mM. 1H-NMR spectra were recorded with an AVANCE spectrometer (Bruker). Similarities among the metabolic profiles of samples were investigated by means of a principal component analysis (PCA). Differences in metabolites concentrations were analysed using ANOVA test based on Tukey contrast. The study was approved by the Hospital Ethical Committee. Results: Among all the women enrolled, 25 were considered healthy, 18 received a diagnosis of BV and 22 were positive for CT. PCA analysis confirmed that BV-metabolic profile was reasonably distinct from the one of healthy women. The metabolome of CT-positive women resembled more the one of healthy women rather than that of BV-positives. Comparing the metabolite composition of the three groups, statistically significant differences were found. As an example, higher concentrations of formate, trimethylamine, ethanolamine and methylamine were found in BV-patients, while tryptophan, lactate and phenyl-propionate were more present in healthy women. In CT-positive women, concentrations of these metabolites were similar to healthy women or intermediate between healthy and BV groups. Conclusions: Specific metabolic signatures characterize different clinical conditions of the vaginal tract. At a metabolic level, CT-positive women are more similar to healthy than BV-subjects.

Background: Gardnerella vaginalis (GV) is a facultative anaerobic, pleomorphic, Gram-negative to Gram-variable bacterium. GV is the most common species found in bacterial vaginosis (BV), a condition characterized by a depletion of vaginal lactobacilli and an overgrowth of anaerobes. Despite being the most prevalent and virulent species found in BV, GV can also be a part of the vaginal microbiota in a significant proportion of healthy women. Besides this aspect, less is known about the role of GV in other clinical conditions affecting the genital tract of women, such as vaginal candidiasis and Chlamydia trachomatis (CT) infections. The aim of this study was therefore to evaluate and compare GV load in the vaginal tract in 4 different conditions: healthy, BV, vaginal candidiasis and CT infection. Material/methods: From July 2016, all the pre-menopausal women attending the STI Outpatients Clinic of Sant’Orsola-Malpighi Hospital in Bologna (Italy) and meeting one of the following criteria were enrolled: presence of vulvo-vaginal symptoms (as vaginal discharge, abnormal bleeding, itching) or presence of risk factors for CT infection (age < 25 years, multiple sex partners, unsafe intercourse). Exclusion criteria comprised the use of any antibiotics or vaginal medications in the past month, the use of estro-progestinic products and the presence of chronic diseases. For all the patients, after a clinical visit, a vaginal swab for molecular CT detection (Versant CT/GC DNA 1.0 Assay; Siemens) was collected. Moreover, Amsel criteria were used for BV assessment and a microscopic examination of secretions were performed for vaginal candidiasis. For each patient, starting from the remaining eluate of vaginal swab of Versant PCR plate, a quantitative real-time PCR targeting 16s rRNA gene of GV was performed. Results were expressed as GV DNA copies/reaction. Statistical comparisons of mean GV DNA load between groups were performed with one-way ANOVA. The study was approved by the Hospital Ethical Committee. Results: Among all the women enrolled for the study, 24 were considered healthy (any symptoms and no microbiological positivity), 16 received a diagnosis of candidiasis, 18 of BV and 23 were positive for CT. The mean GV DNA load was significantly different between the groups (P = 0.0186). Healthy and CT positive women showed similar and lower mean loads (195274 and 162627 copies, respectively) compared to BV and candidiasis groups (562807 and 3.4 × 106 copies). Conclusions: Vaginal GV load varies significantly considering different clinical conditions: CT positive women have low level of GV DNA comparable to healthy subjects, while women with candidiasis show even higher level of GV DNA than women with BV. Further studies are needed to better understand the role of GV and if the variations in GV levels precede or come after the onset of these conditions.

Background: Lactobacilli represent a wide range of bacterial species with several implications for the human host. They play a crucial role in maintaining the equilibrium of different biological niches and are essential for fermented food production and probiotic formulation. Despite the consensus about the ‘health-promoting’ significance of Lactobacillus genus, its species identification still poses several difficulties. Since traditional genotypic methods are relatively expensive and do not relate to the physiological properties, new approaches are needed for lactobacilli identification. The aim of this study was to assess the integration of different methods, genotypic (16S rRNA gene sequencing), proteomic (MALDI-TOF MS) and metabolomic (1H-NMR), for the taxonomic characterization of Lactobacillus species. Material/methods: A total of 40 Lactobacillus strains, isolated from clinical specimens, included in probiotics or belonging to collections of microorganisms were used in this study. The genotypic identification at species level was obtained by 16S rRNA gene sequencing. A phylogenetic tree based on 16S rDNA sequences was created using MEGA 6 software. MALDI-TOF MS analysis was performed after protein extraction starting from bacterial cell pellets, using a Bruker Microflex instrument. For species identification, spectra collected were compared with the ones of the reference database. A clustering analysis was performed by the generation of a score-oriented dendrogram, starting from the main spectrum profiles (MSPs) of each strain with MALDI Biotyper 3.1 software. 1H-NMR metabolomic analysis was performed using an AVANCE III spectrometer (Bruker), both on the intracellular metabolome, after appropriate bacterial lysis, and on the extracellular supernatants, after an overnight growth in culture broth. Differences in the metabolome composition were calculated by means of an unpaired Wilcoxon test Results: 16S rRNA gene sequencing led to the following identifications: 7 L. crispatus, 7 L. gasseri, 5 L. acidophilus, 5 L. delbrueckii, 2 L. vaginalis, 2 L. reuteri, 6 L. plantarum, 1 L. pentosus, 2 L. rhamnosus, 2 L. casei/paracasei and 1 L. brevis. For species level identification, an excellent agreement between MALDI-TOF and the genotypic analysis was found (97.5%), with only one discordant result. Moreover, the phylogenetic tree based on 16S rRNA gene sequences and the MALDI-TOF dendrogram were highly similar. The metabolomic approach led to identify a panel of molecules whose variations were strictly associated with the taxonomy. As an example, considering the extracellular metabolome, L. crispatus showed the highest glucose consumption (P=1 × 10-3), whereas L. acidophilus species was characterized by the highest-level production of acetone and pyruvate (P=3 × 10-4 and P=1 × 10-3). Conclusions: MALDI-TOF MS is an excellent method for lactobacilli species identification and could represent a simple and reliable tool for their taxonomic characterization. Metabolomic analysis could be useful in correlating lactobacilli taxonomy with biological properties, as their anti-microbial activity or fermentation capacity for food production.

Purpose: The first aim of this study was to investigate the probiotic potential of Bifidobacterium breve BC104, isolated from a vaginal swab1, by assessing its antimicrobial activity against bacteria and fungi responsible for urogenital diseases (Candida spp., C. trachomatis, E. coli, E. faecalis, E. faecium, S. faecium, E. hirae, S. aureus and P. mirabilis) and against gastrointestinal pathogens (E. coli enterotoxigentic, S. typhimurium and Y. enterocolitica). To provide functional benefits to the gut and urogenital tract, oral probiotics must survive passage through the acidic stomach and colonize colon. Thus, tolerance to bile salts and low pH, as well as adhesion to Caco-2 and HeLa cells, were investigated. The second aim was to develop an oral gastro-resistant and mucoadhesive formulation able to favour bacterial survival during storage and following administration and bacterial intestinal and vaginal colonization. Methods: Antifungal activity was tested against Candida spp. by broth micro-dilution method. The anti-Chlamydia activity was measured as reduction in Chlamydia infectivity to HeLa cells treated with BC104. The antibacterial activity against the other pathogens was tested by using agar spot test2. The capability of Bifidobacterium to adhere to Caco-2 and HeLa cells was assessed by microscopy after Giemsa staining. To mimic gastric and intestinal conditions, Bifidobacterium cells were suspended in simulated gastric juice (pH 2) for three hours and then in simulated intestinal fluid (pH 7) for other three hours. Aliquots were taken every hours to estimate viability over time3. The oral formulation was obtained by spray-drying followed by prilling. Firstly, the overnight culture of BC104 was suspended in prebiotic media, containing an encapsulating agent (reconstituted skim milk 10%) and prebiotic (inulin 5%, oligofructose 5% and ascorbic acid 2.5%)4. Spray-drying process was performed at constant inlet temperature of 120°C. Secondly, gastro-resistant mucoadhesive beads were obtained by dripping and cooling of molten lauric acid (75%, p/p) containing CMC (10%, p/p) and spray-dried microcapsules (15%, p/p). Viability after the manufacturing procedure was evaluated as well as BC104 release from the beads. Results: B. breve BC104 exhibited a strong fungistatic activity against C. albicans and was able to reduce Chlamydia infectivity to HeLa cells. Moreover, BC104 exerted a good antibacterial activity towards all other pathogens examined, in particular against Escherichia coli, Staphylococcus aureus and Yersinia enterocolitica. BC104 showed a good ability to adhere to Caco-2 but not to HeLa cells, accordingly to the fact that bifidobacteria are a minor component of vaginal ecosystem, while they are essential in maintaining gastrointestinal homeostasis. Even after three hours at pH 2 and further exposure to the shift of pH under intestinal condition, BC104 maintained elevated cell concentration (> 105 CFU/mL), showing an intrinsic ability to resist to gastric acidity. Microcapsules containing B. breve showed high survival after spray drying (1010 CFU/g) and after 4 weeks of storage at 4°C. BC104 maintained also a good viability after incorporation inside beads (1.7*107 CFU/g). Conclusions: The results show that B. breve BC104 possess a good antimicrobial activity and can be deliver in oral gastro-resistant formulation. 1 Parolin, C., Marangoni, A., Laghi, L., Foschi, C., Ñahui Palomino, R.A., Calonghi, N., Cevenini, R., Vitali, B. Isolation of Vaginal Lactobacilli and Characterization of Anti-Candida Activity. PLoS One 2015, 10(6), e0131220. 2 Schillinge,r U., Lücke, F.K. Antibacterial activity of Lactobacillus sake isolated from meat. Appl Environ Microbiol 1989, 55(8), 1901-6. 3 Fernández, M.F., Boris, S., Barbés, C. Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. J Appl Microbiol 2003, 94(3), 449-55. 4 Fritzen-Freire, C.B., Prudencio, E.S., Amboni, R.D.M.C., Pinto, S.S., Negrao-Murakami, A.N., Murakami, F. S. Microencapsulation of bifidobacteria by spray drying in the presence of prebiotics. Food Research Internationa 2011, 45, 306-312.

Objectives. Lactobacilli are the dominant bacteria of healthy vaginal microbiota and their principal function is to maintain an environment that restricts the growth of pathogenic and opportunistic microorganisms, as fungi belonging to the genus Candida. Lactobacilli form a critical line of defence against potential pathogens by lowering the environmental pH through lactic acid production and producing antimicrobial compounds, or through competitive exclusion. Anyway, the mechanisms underlying antifungal activity against Candida spp. are still not fully understood. In this study, the potential activity against Candida spp. of different strains of vaginal lactobacilli was analysed, focusing on hydrogen peroxide generation, lactic acid production and antimicrobial supernatant fluids activity. Methods. Seventeen strains of lactobacilli were isolated from vaginal swabs collected from pre-menopausal healthy women. They were taxonomically identified by sequencing the 16S ribosomal RNA gene. Hydrogen peroxide generation was tested in a semi-quantitative assay on de Man, Rogosa, Sharpe (MRS) agar plates containing tetramethylbenzidine and horseradish peroxidase in anaerobic conditions. Isolates were scored as low, medium and high producing strains. Lactic acid production was measured in cell free supernatants of Lactobacillus cultures by 1H-nuclear magnetic resonance (NMR) analysis. Lactobacillus culture supernatants were tested for their fungistatic or fungicidal activity against 9 Candida strains isolated from vaginal swabs submitted to the Microbiology Laboratory of Sant’Orsola-Malpighi University Hospital of Bologna for routine diagnostic procedures, belonging to C. albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, and C. lusitaniae species. The in vitro activity of free-cell supernatants was determined by broth microdilution assay in accordance with EUCAST guidelines. To determine if Lactobacillus strains supernatants had a killing effect, samples from wells exhibiting less than 50% of growth were taken and spotted onto SD agar plates. Fungicidal activity was defined as a ≥3 log10 reduction from the starting inoculum. Results. The Lactobacillus isolates were taxonomically identified as follows: 8 strains of L. crispatus (BC1-BC8), 6 strains of L. gasseri (BC9-BC14), and 3 strains of L. vaginalis (BC15-BC17). All Lactobacillus strains exhibited a good generation of hydrogen peroxide, while the production of lactic acid, even if recorded for all the strains tested, showed concentrations ranging from 4.8 to 50.9 mM. When the anti-fungal activity of Lactobacillus was assessed, L. crispatus supernatants were the most effective, especially versus C. albicans and C. lusitaniae. None of the Lactobacillus strains was able to interfere with C. krusei and C. parapsilosis. Detailed results of fungistatic or fungicidal activity are shown in figure 1. Conclusion. A major potential application of this study concerns the identification of active Lactobacillus strains that could be administered as probiotics for prophylaxis and/or adjuvant therapy of vulvovaginal candidiasis. Further studies are ongoing to elucidate the mechanisms by which lactobacilli exert their protective functions against Candida.

Background: Chlamydia trachomatis (CT) is the agent of the most prevalent bacterial sexually transmitted infection (STI) worldwide. Genotyping of CT strains is important to monitor contact tracing and to enable association with epidemiological and clinical findings. The aim of this study was to assess CT infection prevalence and investigate serovar distribution in a group of high-risk subjects compared to general population in the North of Italy. Material/methods: From January 2011 to May 2014, data about all the consecutive clinical samples submitted to the Microbiology Laboratory of St.Orsola Hospital of Bologna for CT detection were collected. The specimens were obtained from patients attending the STI Outpatients Clinic of the Hospital (high-risk population) or from subjects attending gynecologic clinics or general practitioners offices (low-risk population). All the specimens were tested by the Versant CT/GC DNA 1.0 Assay (Siemens), a duplex real-time PCR simultaneously revealing the presence of CT and Neisseria gonorrhoeae (GC) DNA. In case of a CT positive result, molecular genotyping based on omp1 gene semi-nested PCR followed by RFLP analysis was performed. Results: During the study period, a total of 9315 samples, including 8918 specimens from urogenital sites and 397 from extra-genital localizations (pharyngeal and rectal swabs) were collected from 8906 patients. Subjects attending the STI Outpatients Clinic (N=4081; 2195 males) were younger compared to unselected patients (N=4825; 621 males) (mean age: 32 vs 36; P

Background: Chlamydia trachomatis (CT) is the most frequent bacterial sexually transmitted infection (STI) worldwide. The human vaginal microbioma plays a key-role in preventing a large number of urogenital diseases, including STIs. These protective functions are mainly attributed to Lactobacillus spp. dominating the vaginal niche of healthy women. Up to now only few studies have investigated the possible interaction between lactobacilli and CT. The aim of our study was to investigate anti-bacterial activity of 17 vaginal lactobacilli isolates against elementary bodies (EBs) of CT in relation to their metabolic profiles. Material/methods: Seventeen Lactobacillus strains, isolated from vaginal swabs of healthy premenopausal women and cultured in MRS broth were used: in particular, 8 strains of L. crispatus (BC1-8), 6 of L. gasseri (BC9-14) and 3 of L. vaginalis (BC15-17). After turbidimetric determination of cell concentration, cultures were centrifuged to separate cell pellets (CP) from cell-free supernatants (CFS). All CP and CFS corresponding to 2.5×108 , 2.5×107 and 2.5×105 CFU were mixed with 5×103 IFU of EBs of CT serotype D. pH were measured in the final volume; EBs diluted in PBS were used as controls. Tubes were incubated for 7, 15 and 60 minutes and then centrifuged. Supernatants were used to infect confluent Hela cells in single tubes containing sterile coverslips. After 48h of incubation at 37°C we evaluated CT infectivity by direct immunofluorescence (Meridian). IFUs were counted in 30 randomly chosen 200× microscopic fields and compared with controls. Statistical analyses were performed using GraphPad Prism software, applying Wilcoxon test. 1H-NMR analysis of metabolic profiles of CFS was conducted on AVANCE spectrometer (Bruker); signals were assigned comparing their chemical shifts and multiplicity with Chenomx software data bank (Chenomx, ver8.02). Statistical analysis of 1H-NMR data was performed using R computational software, applying Wilcoxon test. Results: CT infectivity was significantly reduced only by CFS (p=0.038), in contrast to CP (p=0.425). CSF at the highest concentration showed the strongest anti-chlamydial activity. Under these conditions CFS were found to have pH0.6). Comparing metabolic profiles of the 3 groups we identified one metabolite produced in significantly higher concentration in active group: orotic acid (p=0.005). Anyway, when tested alone at the concentration of 0,03mM, the highest concentration found among active CFS, it showed no anti-chlamydial activity. Conclusions: Our results suggest a possible role of vaginal lactobacilli in the inhibition of CT infectivity, exercised by their metabolites with synergic functions. Anti-chlamydial activity is highly dependent on acid microenvironment.