Screening and characterization of vaginal Lactobacillus strains as probiotic candidates

Screening and characterization of vaginal Lactobacillus strains as probiotic candidates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Screening and characterization of vaginal Lactobacillus strains as probiotic candidates Chennuo Zhu, Baoqi Huang, Changhao Yu, Yuqing Che, Dan Wu, Yue Wu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1868131/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lactobacillus iners is often associated with increased risk of adverse gynecological and obstetric outcomes, such as vaginal dysbiosis and bacterial vaginosis (BV). Lactobacillus strains from the vagina of healthy women were screened for inhibitory activity against L. iners. Four active strains were identified, whereby L. gasseri H87 exhibited the highest inhibitory activity against L. iners , followed by L. rhamnosus H23. All four strains inhibited the growth of potential pathogens of the gastrointestinal, urogenital and reproductive tract, and were capable of co-aggregating with the cells of the tested pathogens to varying degrees. H23 and H78 exhibited survival rates above 80% under in vitro conditions simulating the vaginal, gastric and intestinal environment. The four strains showed good auto-aggregation of more than 30%. H23 and H87 also exhibited good co-aggregation of more than 30% with Listeria monocytogenes and Escherichia coli. Safety assessment showed that the four strains had the usual antibiotic susceptibility profile and did not produce hemolysins. Considering that H87 exhibited a better performance as a probiotic, we investigated the antibacterial substance produced by H87, and identified a bacteriocin with a molecular weight of 6.5 kDa. Based on strain characteristics and beneficial properties, we confirmed its ability to prevent/treat vaginal dysbiosis and maintain a healthy vaginal ecosystem. Therefore, we believe that H87 is a promising candidate for human trials. probiotics bacteriocin vaginal microbiome Lactobacillus iners Lactobacillus gasseri Figures Figure 1 Figure 2 Figure 3 Introduction The species composition of vaginal microbiota determines states of eubiosis versus dysbiosis and plays an important role in women’s reproductive health [1]. The vaginal microbiota were clustered into five bacterial community state types (CSTs). CST I ( L. crispatus dominated), CST II ( L. gasseri dominated) and CST V ( L. jensenii dominated) vaginal microbiota can suppress the growth of urogenital pathogens by producing lactic acid, H 2 O 2 , and bacteriocins. Conversely, CST III vaginal microbiota ( L. iners dominated) are often related to dysbiosis (pH > 4.5), leading to a less stable microflora that is more likely to deteriorate into a diseased state (CST IV). Many anaerobic bacteria (such as Gardnerella vaginalis ) belonging to CST IV are potential causes of bacterial vaginosis (BV) in women of reproductive age [2,3]. BV is caused by the disturbances and dysbiosis of vaginal microbiota, which is characterized by an increase in the relative abundance of facultative or obligate anaerobic bacteria and a decrease of beneficial Lactobacillus species. Globally, 23 to 29% of women are affected by BV, whereby 84% report no symptoms [4]. Although BV is usually asymptomatic, it is significantly associated with miscarriage, preterm birth, low birth weight and other gynecological diseases and adverse pregnancy outcomes [5,6]. Amoxicillin, clindamycin, and metronidazole are frequently used to treat BV, but recurrence is common. In addition, antibiotic treatment kills beneficial vaginal bacteria and thereby disturbs the microbiome, resulting in increased antibiotic resistance and fungal infection [7]. Conversely, the presence of H 2 O 2 -producing Lactobacillus species, such as L. crispatus , L. jensenii and L. gasseri , has beneficial effects such as higher live birth rates after in vitro fertilization and lower rates of BV or preterm birth [8]. However, it is not clear whether Lactobacillus species that do not produce H 2 O 2 , mainly represented by L. iners , have the same effect. A number of Lactobacillus species have become a hotspot of research due to their excellent biological activity, and some were gradually developed into probiotic products that are widely accepted by consumers. A recent systematic review confirmed that L. rhamnosus , L. reuteri , L. gasseri and many other Lactobacillus species have been widely utilized in commercial products[9]. Primary metabolites of lactic acid bacteria, such as lactic acid, as well as intermediate metabolites such as H 2 O 2 , or secondary metabolites such as bacteriocins, all have significant antibac terial activity. Bacteriocins are polypeptides synthesized by ribosomes, which commonly inhibit closely related bacteria in order to maintain the dynamic balance of the local habitat. Bacteriocins are commonly classified into three classes [10]. Class I is mainly composed of enzymatically modified small peptides that introduce non-coding amino acids as active parts during biosynthesis. Class II contains unmodified small thermostable peptides of less than 10 kDa, which are further divided into four subgroups: Class IIa (pediocin-like bacteriocins), IIb (two-peptide bacteriocins), IIc (leaderless bacteriocins), and IId (linear non-pediocin-like single-peptide bacteriocins). Class III includes unmodified bacteriocins larger than 10 kDa with a bacteriolytic or non-bacteriolytic mechanism. The application of bacteriocins generated by lactic acid bacteria in food preservation has been widely studied. However, bacteriocins have not been fully developed as antibacterial agents for treating infectious diseases in humans [11]. Although numerous bacteriocin-producing microorganisms isolated from the human body and clinical samples have been identified in recent years, few of their antibacterial activities have been described in detail. Due to the predominance of Lactobacillus species in the vagina of healthy women, it is worthwhile to evaluate and characterize the bacteriocins produced by these microbiota to improve vaginal health. This study investigated the inhibitory effects of Lactobacillus species isolated from the vagina of healthy women on L. iners . Characteristics such as low-pH tolerance, survival in simulated vaginal fluid, gastric juice and intestinal fluid, as well as aggregation ability, safety, and antimicrobial potential indicate that L gasseri H87 is a promising candidate probiotic. We also preliminarily characterized the properties of the antibacterial substances produced by H87 [12]. Results Screening of Lactobacillus strains for antimicrobial activity. A total of 350 isolates were obtained from vaginal swabs of healthy Chinese women. The antibacterial activity of cell-free supernatants (CFS) from isolated strains against L. iners was tested using the cylinder-plate method. Supernatants were obtained from cultures with comparable cell densities and pH. The CFS of only four strains (1.1%) were able to inhibit L. iners , including H23, H87, H198, H271. They were identified as L. rhamnosus , L. gasseri , L. paragasseri and L. reuteri by 16S rRNA gene sequencing, respectively. As shown in Table 1.,the CFS of H87 exhibited the most potent inhibitory activity with an inhibition zone of 14.23 mm, followed by those of H23, H198 and H271. None of the other isolated strains showed significant antibacterial activity (＜3 mm). Table 1. Antibacterial activities of Lactobacillus strains against L. iners Strain Inhibition zone (mm) L. rhamnosus H23 15.31±0.29 L. gasseri H87 19.23±0.33 L. paragasseri H198 7.06±0.41 L. reuteri H271 4.25±0.31 Data are presented as the means ± SD of three replicates. The Lactobacillus strains without detectable antibacterial activity (inhibition zone < 3 mm), are not shown in the table. Inhibition profile of the selected Lactobacillus strains. Several microorganisms associated with gastrointestinal, urogenital and reproductive tract infection were tested for their sensitivity to inhibition by the 4 selected Lactobacillus strains (Table 2). Each of the 4 selected Lactobacillus spp. inhibited at least one of the pathogenic indicator strains, albeit at different inhibition levels. Notably, H87 exhibited activity against all tested gastrointestinal, urogenital and reproductive tract pathogens. The antimicrobial activity of H23 and H87 selectively inhibited pathogenic bacteria, while retaining most of the tested beneficial vaginal Lactobacillus strains and probiotics. Table 2. Inhibitory spectrum of Lactobacillus strains Indicator strain Inhibition zone H23 H87 H198 H271 Potential gastrointestinal, urogenital and reproductive tract pathogens L. iners +++ +++ ++ + G. vaginalis - ++ - - A. vaginae - + + - E. coli ++ +++ +++ ++ Ent. faecalis ++ +++ + - Strep. agalactiae + +++ - - Staph. aureus - +++ + + P. aeruginosa - +++ + + Lis. monocytogenes - + - - Vaginal Lactobacillus species L. rhamnosus ++ + L. crispatus - - + + L. jensenii - - L. reuteri - - + Intestinal probiotic L. bulgaricus - - + - +++ : 11＜x≤15,++ : 7＜x≤11,+ :3＜x≤7, - ：x≤3 Lactobacillus , Enterococcus , Staphylococcus , Streptococcus , and Listeria Survival under in vitro conditions simulating the vaginal environment. The residual viability of H23, H87, H19 and H271 was 80%, 85%, 83% and 88% after 48 h of incubation at pH 3.5. Similarly, the residual viability of H23 and H271 after 48 h of incubation at pH 4.0 was 88% and 95%, respectively. No significant changes of viability (101% and 102%) were noticed when H87 and H198 were cultivated at pH 4.0 for 48 h (Fig. 1). All the cultures showed more than 100% relative viability after 48 h at pH 4.5 and 6.5. Survival under in vitro conditions simulating the gastric and intestinal environments. H23 and H87 exhibited 98 and 94% survival after 48h at pH 3. Which was reduced to 80 and 85% at pH 2, respectively. This is typical of the gastric environment. By contrast, the residual viability of H198 and H271 was less than 50% at both pH 2 and 3. In the presence of 0.3% bile salts, which simulate the intestinal environment, H23, H87, H198 and H271 exhibited 95, 96, 88 and 93% survival after 48 h of incubation, respectively. In the presence of 0.5% bile salts, the survival was reduced to 90, 92, 72 and 88%, respectively. The survival of H23 and H87 was reduced to 86, 90, 65, and 82% after 48 h of incubation in the simulated intestinal fluid (SIF). Aggregation ability. The results of the auto-aggregation and co-aggregation assays of the vaginal Lactobacillus strains are shown in Table 3. The auto-aggregation values ranged from 33 to 61%, indicating that the 4 Lactobacillus strains have good auto-aggregation abilities. A broad range of variation in co-aggregation with pathogens was detected. H23 and H87 showed high co-aggregation with E. coli and Listeria monocytogenes , exhibiting values higher than 30%. By contrast, H198 exhibited low co-aggregation with E. coli , reaching only 19 %, while H271 exhibited no co-aggregation with L. monocytogenes (Table 3). Overall, H23 and H87 showed good aggregation abilities Table 3. Adhesion ability and safety evaluation L. rhamnosus H23 L. gasseri H87 L. paragasseri H198 L. reuteri H271 Auto-aggregation (%) 43±2 58±2 61±2 33±1 Co-aggregation with Listeria monocytogenes (%) 55±2 61±3 40±1 N Co-aggregation with Escherichia coli 32±1 51±1 19±1 42±2 Hemolytic activity γ-hemolysis γ-hemolysis γ-hemolysis γ-hemolysis Ampicillin (4 mg/L) S S S S Kanamycin (64 mg/L) R R R R Streptomycin (64 mg/L) R R R R Tetracycline (32 mg/L) S S S S Chloramphenicol (8 mg/L) S S R S Metronidazole (128 mg/L) S R R S S: sensitive, R: resistance Safety Evaluation. In terms of safety, none of the strains showed hemolytic activity. H23 and H87 were sensitive to all tested antibiotics. H198 was not sensitive to chloramphenicol and H271 was not sensitive to kanamycin. Identification of the antibacterial substance produced by H87. Compared with other strains, H87 had the following advantages: (1) it inhibited the highest proportion of the tested pathogenic bacteria that attack the gastrointestinal, urogenital and reproductive tract, (2) it survived under in vitro conditions simulating the vaginal, gastric and intestinal environments (survival rate＞80% in all cases), (3) it exhibited good auto-aggregation and co-aggregation (>40%), (4) it can be preliminarily considered safe (no hemolysis) and controllable (no drug resistance). H87 therefore exhibited the desired characteristics of probiotics, which prompted us to further investigate the properties of antibacterial substances produced by H87. The results of the experiments excluding the effects of H 2 O 2 and lactic acid are shown in Fig. 3. After the addition of NaOH, the inhibition zone appeared only somewhat smaller, indicating that organic acids were not the main bacteriostatic substance. In addition, strain H87 still had antibacterial activity after the addition of catalase, suggesting that H 2 O 2 is also not the main antibacterial substance. Subsequently, a protease hydrolysis test was performed on the antibacterial substance produced by H87, and the results showed that the substance could be completely hydrolyzed by pepsin and proteinase K, or partially hydrolyzed by trypsin (Fig. 3). Thus, it was demonstrated that the antibacterial substance produced by strain H87 was a peptide. Ultrafiltration membranes with molecular weight cut off (MWCO) values of 3, 10, 30, 50 and 100 kDa were used to separate the bacteriocin produced by H87. Then, a concentrated solution was obtained by centrifugation. The antibacterial activity of the bacteriocin fractions was analyzed and compared after ultrafiltration. The results indicated that the concentrated solution of the MWCO above 10 kDa has no antibacterial activity, while the concentrated solution of the MWCO above 3 kDa has the highest antibacterial activity, and the filtrate of the MWCO of 3 kDa has a lower antibacterial activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the active fractions showed that a band with an approximate molecular weight of 6.5 kDa was present both in the concentrated solution of the MWCO of 3 kDa and the filtrate of the MWCO 3 kDa. Usually, about 70% of the target protein can be intercepted by the off-line ultrafiltration tube, and the rest of the target protein will be passed into the filtrate. Therefore, the antibacterial activity assay and electrophoresis results indicated that the bacteriocin produced by L. gasseri H87 is most likely a single peptide with a molecular weight of approximately 6.5 kDa. Discussion At present, there are few commercial probiotic products that can be directly applied to the vagina [13]. Studies have shown that host-specific microorganisms have a high colonization ability and high survival rate in the host environment. Accordingly, they can produce specific physiological effects. Therefore, it is urgent to develop host-specific probiotics. In this study, we studied the low-pH tolerance, survival under simulated vaginal, gastric and intestinal conditions of four Lactobacillus strains, while also assessing their aggregation ability, safety, and antibacterial potential. Finally, we preliminarily characterized the properties of the antibacterial substance produced by the most effective strain. To treat BV, probiotics can be directly applied to the vagina to directly control the re-colonization of Lactobacillus species [14]. In addition, BV can also be treated with oral probiotics. However, in order to achieve the desired clinical therapeutic effect, probiotics need to be strictly transferred to the colonization site. Researchers generally use the method of recovering specific microorganisms from fecal samples to demonstrate the success of the transfer [15]. Previous studies have shown that immune responses, stool consistency and the density of vaginal Lactobacillus can be improved by probiotics [16,17]. Probiotics applied to the vagina have good efficacy, but oral administration is more convenient [18]. Thus, we investigated the survival of the 4 selected vaginal Lactobacillus strains in the simulated vaginal fluid (SVF), simulated gastric juice (SGJ) and simulated intestinal fluid (SIF) to select promising Lactobacillus strains to be used both as vaginal capsules and dietary supplements. The main components of vaginal fluid are carbohydrates, proteins and a small amount of lipids, and it is a mixture of secretions from the local glands and organs [19]. Under normal conditions, it has an acidic pH of 3.8-4.4. The survival rate of probiotics under such complex and acidic conditions indicates the viability of this strain in the vagina. In this study, the survival rate of the four strains was 88-100% at pH 4.5 and 80-88% at pH 4.0, which indicated that the four strains were resistant to acidic conditions (pH≤4.0), and can adapt to the vaginal environment. The high acidity of gastric juice, pepsin and proteases in the intestinal fluid, as well as bile acid salts in the duodenum can inhibit foreign bacteria. Conversely, bacteria can maintain a sufficiently high viability to exert a probiotic effect only when they have high resistance to the factors mentioned above. As shown in Fig. 2, strains H23 and H87 exhibited high tolerance to acidic pH with survival rates above 80%, indicating that they can adapt to acidic gastric juice with a pH value as low as 2. At the same time, the survival of H23, H87 and H271 also remained above 80% in the presence of bile salts. Similarly, the survival of H23, H87 and H271 remained above 80% in the SIF environment. Thus, the strains H23 and H87 can adapt to gastric and intestinal environments. Auto-aggregation and co-aggregation are important properties of probiotics. They can reflect the ability of biofilm formation on host cells (for instance, intestinal epithelial, vaginal and ectocervical epithelial cells), while the co-aggregation of probiotics with intestinal pathogenic bacteria is helpful for antibacterial substances to kill pathogenic bacteria. We found that H271 has a lower auto-aggregation capacity and lower co-aggregation capacity with pathogens compared to other tested Lactobacillus strains. H198 showed the highest auto-aggregation, as well as moderate levels of co-aggregation with Gram-positive L. monocytogenes , but lower co-aggregation with Gram-negative E. coli. H87 exhibited the second highest auto-aggregation, while having the highest co-aggregation with both of the above-mentioned pathogens. By contrast, H23 showed low co-aggregation with E. coli. The safety assessment showed that none of the four strains produced hemolysin, which may cause harmful effects to the host. These results indicated that all four strains are likely safe for humans. In addition, all strains showed different degrees of drug sensitivity. However, all strains showed resistance to kanamycin and streptomycin. This was consistent with the intrinsic resistance to aminoglycoside antibiotics found in lactic acid bacteria [20]. In addition, the resistance of H87 and H198 to metronidazole is commonly used for the treatment of bacterial vaginosis to ensure their survival during treatment. In sum, L. gasseri H87 showed distinct advantages compared with the other tested strains. L. gasseri is an autochthonous microorganism of the normal human flora [21], and one of the dominant Lactobacillus species in the vaginal ecosystem, together with L. jensenii , L. iners and L. crispatus [22-24]. L. gasseri is a well-known symbiont of the vaginal mucosa, and is negatively correlated with BV [25-27]. Some strains of this species show promising potential as probiotics to maintain vaginal homeostasis [28-30]. H87 inhibited a number of potential gastrointestinal, urogenital and reproductive tract pathogens, including species of Lactobacillus, Enterococcus, Staphylococcus, Streptococcus, and Listeria . GasE and GasT were purified from the supernatants of L. gasseri EV1461 and SBT2055, respectively. The amino acid sequence of the mature active peptide GasE differed in only one amino acid from that of GasT, and their molecular weight was 5 kDa [31]. L. gasseri produces bacteriocin, which belongs to Class IIb and is composed of two different peptides, and their genes are adjacent to each other in the same operon [32]. Examples include GasE/GaeX from L. gasseri EV1461, GasT/GatX from L. gasseri SBT2055, Acd221B/Acd221β from L. gasseri LF221, and GasK7A/ B (GaeA) from L. gasseri K7. The molecular weight of the bacteriocin obtained from H87 was approximately 6.5 kDa, which is consistent with previous reports. In addition, H87 exhibited antibacterial activity against strains of Lactobacillus, Enterococcus, Staphylococcus, Streptococcus, and Listeria , which is also consistent with the literature. Therefore, it is possible that the bacteriocin produced by H87 is homologous to GasT and GasE. H87 was resistant to metronidazole and strongly inhibited L. iners . The reason is related to vaginal imbalance and recurrence of BV, but its pathogenesis is still unclear. Therefore, in order to better understand the pathogenesis, it is meaningful and useful to develop inhibitors that are active against L. iners. Metronidazole is a commonly used antibiotic for the treatment of BV, but L. iners is resistant to metronidazole. Therefore, probiotics that can inhibit L. iners and are resistant to metronidazole have become a focus of research. Metronidazole combined with probiotics can be used in the treatment of BV, and it can also be used to prevent its recurrence. Therefore, the discovery of H87 may improve the treatment of BV. Methods Bacterial strains, growth media, and culture conditions. L. gasseri H87 was isolated from human vaginal fluid and was preserved in the China Center for Type Culture Collection (CCTCC) under the accession number M 2018477. The vaginal Lactobacillus strains and L. iners ATCC 55195 were grown in Man-Rogosa-Sharpe (MRS) medium at 37°C under aerobic conditions. Gardnerella vaginalis was grown anaerobically on chocolate agar plates at 37°C. Escherichia coli, Enterococcus faecalis , Streptococcus agalactiae , Staphylococcus aureus, Pseudomonas aeruginosa and Listeria monocytogenes were grown aerobically on Luria-Bertani (LB) agar plates at 37°C. All strains were maintained as frozen stocks at -80°C in their respective culture media with the addition of 15 to 20% (vol/vol) sterile glycerol. Preparation of Lactobacillus cell suspensions. If not indicated otherwise, cell suspensions were prepared by growing Lactobacillus strains at 37°C for 24 h under aerobic conditions in MRS medium. The cells were pelleted by centrifugation (5000 g, 15 min, 4 °C), washed twice with phosphate-buffered saline (PBS; 0.1 M, pH 7.2, containing 0.85 % (w/v) NaCl) and resuspended in phosphate buffer (0.1 M, pH 7) to obtain a cell suspension with OD 600 = 1, approximately 10 9 CFU/mL. Sampling and isolation of vaginal Lactobacillus strains. The study protocol was approved by the ethics review board of the Second Affiliated Hospital of Nanjing Medical University. We have obtained written informed consent from all study participants. All the procedures were performed in accordance with the Declaration of Helsinki and relevant policies in China. Lactobacilli were isolated from the vaginal microbiome of asymptomatic Chinese women who were invited to participate in the study during their routine gynecological consultations. MRS agar plates with 0.05% of L-cysteine supplementation were incubated anaerobically at 37 °C for 24~48 h. Individual colonies were randomly selected and purified before storing at −80 °C with 20% glycerol. Cell-free supernatants from the isolated Lactobacillus strains were screened for antibacterial activity against L. iners ATCC 55195 using the cylinder-plate method [33]. The L. iners suspension was diluted to 10 7 CFU/mL, mixed with the melted solid culture medium cooled to 40°C. and poured into a plate to solidify. An Oxford cup was gently placed on the solidified plate. After 200 μL of the lactic acid bacteria fermentation supernatant was added to the cup, the system was incubated at 37℃ for 24 h. The antimicrobial activity was evaluated by measuring the diameter of the transparent inhibition zone against the test strain. In vitro antibiotic activity assay. Antibiotic activities of isolated strains were determined using the cylinder-plate method as described before [33]. Tolerance to simulated vaginal fluid (SVF) at low pH. One-milliliter overnight-grown bacterial cultures were separately combined with 100 mL of simulated vaginal fluid (SVF) prepared as described by Ahire et al. [3] (pH adjusted with lactic acid), incubated anaerobically at 37 °C and the OD 600 measured after 48h. Bacteria grown in MRS were included as a control. Tolerance to simulated gastric juice (SGJ) and simulated intestinal fluid (SIF) . A sample comprising 100 μl of cell suspension was mixed with 1 ml of SGJ or SIF, and incubated at 37 °C for 4 h. The resistance was determined by measuring the survival rate percentage (SR %), based on the initial (0 h) and final (4 h) number of viable cells enumerated on MRS agar plates after 48 h. The SGJ was composed of 0.3% pepsin and 0.5% NaCl, with pH adjusted to 2 or 3 adjusted with 1 M HCl. The SIF was composed of 0.1 % pancreatin, 0.5 % bile salts, 0.5 % NaCl, 0.4 % phenol, and pH 8 adjusted with 1 M NaOH [12,34]. Tolerance to bile . Lactobacillus cells (10 9 CFU/mL) were used to inoculate 5 mL of MRS modified with bile salts (0.3 or 0.5 %) at a ratio of 5% (v/v), and incubated at 37 °C for 4 h. Based on the initial (0 h) and final (3 h) number of viable cells enumerated on MRS agar plates after 48 h, the resistance was determined by measuring the survival rate percentage (SR %). Auto-aggregation assay. The auto-aggregation assay was performed as described by Pithva et al. [12]. A cell suspension (2 ml) was vortexed for 10 s and incubated at 37 °C. Aliquots of 0.1 ml were collected from the upper surface at regular time intervals and mixed with 0.9 ml PBS, followed by measurement of the optical density at 600 nm. The auto-aggregation (%) was calculated as [(OD 0 − OD t )/OD 0 ] × 100, where OD 0 represents the optical density at 0 h and OD t represents the optical density of the cell suspension at 24h. Co-aggregation assay. Equal volumes of cell suspensions (1 ml = 10 9 cfu/ml) of Lactobacillus and pathogenic indicator strains were mixed, and then incubated at 37 °C. The control contained 2 ml of pure bacterial or yeast cell suspension of the indicator strain. The OD 600 of the suspensions was measured at the indicated time intervals. The co-aggregation (%) was calculated using the equation [(OD pat + OD Lacto )/2 − OD mix ] / [(OD pat + OD Lacto )/2] × 100 . The OD pat and OD Lacto represent the optical densities of the Lactobacillus sp. and the indicator strain, while OD mix represents the optical density of the mixture of Lactobacillus sp. andthe indicator strain after 24 h. Hemolytic activity. The hemolytic activity was assessed on blood agar plates containing sheep blood according to a modification of the method reported by Pino et al. [34].Briefly, Lactobacillus strains were streaked onto blood agar plates containing sheep blood, and incubated at 37 °C for 24 h under anaerobic conditions. The hemolytic activity was visually detected and distinguished as β-hemolysis, α-hemolysis, or γ-hemolysis based on the appearance of a clear zone, green halo or no zones around colonies, respectively. Bacillus cereus was used as a positive control. Antibiotic susceptibility. The antibiotic resistance pattern of Lactobacillus strains was assessed according to the standard protocol of the European Food Safety Authority [35,36]. Identification of the antibacterial substance. In order to exclude the inhibitory effect of organic acids, cell-free supernatants (CFS) were adjusted to pH 6.5 using NaOH. To clarify whether the detected antimicrobial activity is caused by the production of H 2 O 2 , 2600 IU/ml of catalase was added to 1 ml CFS of LAB, and incubated for 24 h at 30 °C. In addition, CFS (pH 6.0) of selected Lactobacillus isolates were incubated in MRS broth at 30 °C for 24 h and tested for their sensitivity to proteolytic enzymes in order to determine the if the substance responsible of the antimicrobial activity of the bacteria is a polypeptide. One milliliter of CFS was treated for 2 h at 30 °C with 1 mg/ml final concentration of pepsin, trypsin, or proteinase K, respectively. The remaining antimicrobial activity was assessed using the cylinder-plate method with L. iners as the indicator strain. Untreated cell-free supernatants were used as controls. For the bile salt hydrolysis test, fresh cultures were streaked onto MRS agar plates containing 0.5% (w/v) taurodeoxycholic acid. The hydrolysis effect was represented by different colony morphology (partial hydrolysis recorded as 1) from the control MRS plates, after 48 h of anaerobic incubation at 37 °C [18]. Bile salt hydrolase (BSH) activity was determined using the method Caggia et al. reported previously [37]. The appearance of a precipitate around colonies was considered as a positive sign. Based on the confluence of the precipitate, each strain was classified as ‘+++’ for heavy; ‘++’ for intermediate; ‘+’ for low; and ‘−’ for no precipitation. SDS-PAGE. During the purification process, the RPC-FPLC eluted fractions of GasE were analyzed in duplicate by Tris-Tricine SDS-PAGE with an 18 % acrylamide resolving gel [38]. After electrophoresis at 100 mV for 2 h, one gel was silver-stained, while the other was used to detect the inhibitory activity in an overlay assay as described previously [39]. Conclusions L. gasseri H87 showed a good ability to survive at low pH in SVF, SGJ and SIF. The aggregation and antimicrobial abilities of the strain also indicated that it can displace pathogens from the vaginal environment. Finally, a bacteriocin with an approximate size of 6.5 kDa was purified from the culture supernatant of H87. We believe that this strain may be useful to prevent/treat vaginal dysbiosis and maintain a healthy vaginal ecosystem as a vaginal probiotic. Declarations Data availability All data is included in the text. The raw data of this article will be made available by the authors, without undue reservation, to any qualified researcher. References García-Velasco, J.A., Menabrito, M. & Catalán, I.B. What Fertility Specialists Should Know about the Vaginal Microbiome: A Review. 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Vaginal Lactobacillus microbiota of healthy women in the late first trimester of pregnancy. BJOG-Int J Obstet Gy. 114 ,1402–1407. https://doi.org/10.1111/j.1471-0528.2007.01412.x ( 2010 ). Jespers, V. et al. Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests. BMC Microbiol. 83 ,175–181. https://doi.org/10.1186/1471-2180-12-83 ( 2012 ). Tamrakar, R. et al. Association between Lactobacillus species and bacterial vaginosis-related bacteria, and bacterial vaginosis scores in pregnant Japanese women. BMC Infect Dis. 128 ,35–42. https://doi.org/10.1186/1471-2334-7-128 ( 2007 ). Strus, M., Brzychczy-Wloch, M., Gosiewski, T., Kochan , P. & Heczko , P.B . The in vitro effect of hydrogen peroxide on vaginal microbial communities. Fems Immunol Med Mic. 48 ,56–63. https://doi.org/10.1111/j.1574-695X.2006.00120.x ( 2006 ). Larsson, P.G., Stray-Pedersen, B. & Ryttig, K.R. Human lactobacilli as supplementation of clindamycin to patients with bacterial vaginosis reduce the recurrence rate; a 6-month, double-blind, randomized, placebo-controlled study. BMC Womens Health. , 3 ,65–72. https://doi.org/10.1186/1472-6874-8-3 ( 2008 ). Ehrström, S. et al. Lactic acid bacteria colonization and clinical outcome after probiotic supplementation in conventionally treated bacterial vaginosis and vulvovaginal candidiasis. Microbes Infect. 12 ,691–699. https://doi.org/10.1016/j.micinf.2010.04.010 (2010). aldonado-Barragán, A., Caballero-Guerrero, B., Martín, V., Ruiz-Barba , J.L. & Rodríguez , J.M. Purification and genetic characterization of gassericin E, a novel co-culture inducible bacteriocin from Lactobacillus gasseri EV1461 isolated from the vagina of a healthy woman. BMC Microbiol. 37 ,42–51. https://doi.org/10.1186/s12866-016-0663-1 ( 2016 ). Chikindas, M.L., Weeks, R., Drider, D., Chistyakov , V.A. & Dicks , L.M. Functions and emerging applications of bacteriocins. Curr Opin Biotech. 49 ,23–28. https://doi.org/10.1016/j.copbio.2017.07.011 (2017). Wang, Y., Lu, Z.X., Wu, H. & Lv, F.X. Study on the antibiotic activity of microcapsule curcumin against foodborne pathogens. Int J Food Microbiol. 136 ,71–74. https://doi.org/10.1016/j.ijfoodmicro.2009.09.001 (2009). Pino, A., Bartolo, E., Caggia, C., Cianci, A. & Randazzo , C.L . Detection of vaginal lactobacilli as probiotic candidates. Sci Rep. 9 , 3355. https://doi.org/10.1038/s41598-019-40304-3 ( 2009 ) . EFSA. EFSA Panel on Additives and Products or Substances used in Animal Feed, Scientific Opinion on the safety and efficacy of bentonite as a technological feed additive for all species. EFSA J. 13 ,4010. https://doi.org/10.2903/j.efsa.2015.4010 (2015). EFSA. Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA J. 10 ,2740. https://doi.org/10.2903/j.efsa.2012.2740 (2012). Caggia, C., Angelis, M.D., Pitino, I., Pino, A., Pinoa , C. & Randazzoa , L. Probiotic features of Lactobacillus strains isolated from Ragusano and Pecorino Siciliano cheeses. Food Microbiol. 50 ,109–117. https://doi.org/10.1016/j.fm.2015.03.010 (2015). Schägger, H. Tricine–SDS-PAGE. Nat Protoc. 1 ,16–22. https://doi.org/10.1038/nprot.2006.4 ( 2006 ) . Jiménez-Díaz, R., Rios-Sánchez, R.M., Desmazeaud, M., Ruiz-Barba , J.L. & Piard , J.C. Plantaricins S and T, two new bacteriocins produced by Lactobacillus plantarum LPCO10 isolated from a green olive fermentation. Appl Environ Microbiol. 59 ,1416–1424. https://doi.org/10.1128/aem.59.5.1416-1424.1993 (1993). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1868131","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":123943124,"identity":"100947b7-07e6-41b3-9637-6151073c7e8a","order_by":0,"name":"Chennuo Zhu","email":"","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chennuo","middleName":"","lastName":"Zhu","suffix":""},{"id":123943126,"identity":"ebd0f849-e760-464c-bc78-cdbb98d64c8c","order_by":1,"name":"Baoqi Huang","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Baoqi","middleName":"","lastName":"Huang","suffix":""},{"id":123943128,"identity":"d67336d8-ea18-4d12-832a-1b9c2e26388a","order_by":2,"name":"Changhao Yu","email":"","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Changhao","middleName":"","lastName":"Yu","suffix":""},{"id":123943131,"identity":"1771cf84-d999-4155-a7f5-cf90aea4e3d0","order_by":3,"name":"Yuqing Che","email":"","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuqing","middleName":"","lastName":"Che","suffix":""},{"id":123943134,"identity":"644cbb9f-1eea-4ccd-9794-8b76e81a2e3a","order_by":4,"name":"Dan Wu","email":"","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Wu","suffix":""},{"id":123943138,"identity":"291834fe-93af-4df6-987a-b01a801763b0","order_by":5,"name":"Yue Wu","email":"","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Wu","suffix":""},{"id":123943141,"identity":"37871176-1b10-4959-92a1-d943dd95d474","order_by":6,"name":"Ping Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYNACAwYGfgaGBBCTsYFoLZINpGkB6ToAoQlrkW/vPfy6oOCO3ebbDc8e8zDYyG44wPzsAV7Dz5xLs55h8Cx5250D6cY8DGnGGw6wmRvg1SKRY2bMY3A42exGQpo0D8PhxA0HeNgk8DpsBlSL8Qywlv+EtTDcyDF+DNRiZyAB1nKAsBaDM2fMmGcYHE6QADpMco5BsvHMw2xm+B3W3mP8ueDPYXv+GTlpEm8q7GT7jjc/w+8wBgY2aSCR2MDAkwCOUwZmAupBSj4DCXsGBvYDhNWOglEwCkbBiAQAWMxIK7HjANAAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing Normal University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Song","suffix":""},{"id":123943144,"identity":"25938536-89e8-4a52-baf2-fa0a404060d9","order_by":7,"name":"Ye Zhao","email":"","orcid":"","institution":"Nanjing Tech University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2022-07-18 03:59:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1868131/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1868131/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24528223,"identity":"f2fa22eb-beea-4c13-8c55-1a41eb7720c2","added_by":"auto","created_at":"2022-07-29 18:44:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82837,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of \u003cem\u003eLactobacillus\u003c/em\u003e strains under artificial conditions simulating the vaginal environment with low pH (3.5, 4.0, 4.5, 6.5, adjusted with lactic acid) after 0, 24, and 48 h of anaerobic incubation.\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1868131/v1/420fc81c968d7803838c2e59.jpeg"},{"id":24528228,"identity":"4168844d-f0e2-49c3-a6f6-baf4cc25a800","added_by":"auto","created_at":"2022-07-29 18:44:14","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63189,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival under in vitro conditions that simulate the gastric (simulated gastric juice: SGJ) and intestinal (simulated intestinal fluid; SIF) environment.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-1868131/v1/79f45a5119a7debeaed809a0.jpeg"},{"id":24528225,"identity":"aca67cd4-df4c-49db-a062-58ce8e8eae01","added_by":"auto","created_at":"2022-07-29 18:44:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":729187,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the antibacterial substance (A) and SDS-PAGE of the purified bacteriocin (B). Lines 1 and 2, the filtrate of the MWCO of 3 kDa; lines 3 and 4, the concentrate of the MWCO of 3 kDa Lane M, molecular weight marker.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1868131/v1/7b385f82fd31aa254c8ec744.png"},{"id":28544512,"identity":"5fb5c80d-9580-448d-acec-c453ae27f006","added_by":"auto","created_at":"2022-11-02 05:44:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1578135,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1868131/v1/c1727485-4ad7-46df-9ad7-859836897b12.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening and characterization of vaginal Lactobacillus strains as probiotic candidates","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe species composition of vaginal microbiota determines states of eubiosis versus dysbiosis and plays an important role in women\u0026rsquo;s reproductive health [1]. The vaginal microbiota were clustered into five bacterial community state types (CSTs). CST I\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003e\u0026nbsp;L. crispatus\u003c/em\u003e dominated), CST II\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003eL. gasseri\u003c/em\u003e dominated) and CST V\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003e\u0026nbsp;L. jensenii\u003c/em\u003e dominated) vaginal microbiota can suppress the growth of urogenital pathogens by producing lactic acid, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and bacteriocins. Conversely, CST III vaginal microbiota\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003eL. iners\u003c/em\u003e dominated) are often\u0026nbsp;related to dysbiosis (pH \u0026gt; 4.5), leading to a less stable microflora that is more likely to deteriorate into a diseased state (CST IV). Many anaerobic bacteria (such as \u003cem\u003eGardnerella vaginalis\u003c/em\u003e) belonging to CST IV are potential causes of bacterial vaginosis (BV) in women of reproductive age [2,3].\u003c/p\u003e\n\u003cp\u003eBV is caused by the disturbances and dysbiosis of vaginal microbiota, which is characterized by an increase in the relative abundance of facultative or obligate anaerobic bacteria and a decrease of beneficial \u003cem\u003eLactobacillus\u003c/em\u003e species. Globally, 23 to 29% of women are affected by BV, whereby 84% report no symptoms [4]. Although BV is usually asymptomatic, it is significantly associated with miscarriage, preterm birth, low birth weight and other gynecological diseases and adverse pregnancy outcomes [5,6]. Amoxicillin, clindamycin, and metronidazole are frequently used to treat BV, but recurrence is common. In addition, antibiotic treatment kills beneficial vaginal bacteria and thereby disturbs the microbiome, resulting in increased antibiotic resistance and fungal infection [7]. Conversely, the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-producing \u003cem\u003eLactobacillus\u003c/em\u003e species, such as \u003cem\u003eL. crispatus\u003c/em\u003e, \u003cem\u003eL. jensenii\u003c/em\u003e and \u003cem\u003eL. gasseri\u003c/em\u003e, has beneficial effects such as higher live birth rates after in vitro fertilization and lower rates of BV or preterm birth [8]. However, it is not clear whether \u003cem\u003eLactobacillus\u003c/em\u003e species that do not produce H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, mainly represented by \u003cem\u003eL. iners\u003c/em\u003e, have the same effect.\u003c/p\u003e\n\u003cp\u003eA number of \u003cem\u003eLactobacillus\u003c/em\u003e species have become a hotspot of research due to their excellent biological activity, and some were gradually developed into probiotic products that are widely accepted by consumers. A recent systematic review confirmed that \u003cem\u003eL. rhamnosus\u003c/em\u003e, \u003cem\u003eL. reuteri\u003c/em\u003e, \u003cem\u003eL. gasseri\u003c/em\u003e and many other \u003cem\u003eLactobacillus\u003c/em\u003e species have been widely utilized in commercial products[9]. Primary metabolites of lactic acid bacteria, such as lactic acid, as well as intermediate metabolites such as H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, or secondary metabolites such as bacteriocins, all have significant antibac terial activity. Bacteriocins are polypeptides synthesized by ribosomes, which commonly inhibit closely related bacteria in order to maintain the dynamic balance of the local habitat. Bacteriocins are commonly classified into three classes [10]. Class I is mainly composed of enzymatically modified small peptides that introduce non-coding amino acids as active parts during biosynthesis. Class II contains unmodified small thermostable peptides of less than 10 kDa, which are further divided into four subgroups: Class IIa (pediocin-like bacteriocins), IIb (two-peptide bacteriocins), IIc (leaderless bacteriocins), and IId (linear non-pediocin-like single-peptide bacteriocins). Class III includes unmodified bacteriocins larger than 10 kDa with a bacteriolytic or non-bacteriolytic mechanism.\u003c/p\u003e\n\u003cp\u003eThe application of bacteriocins generated by lactic acid bacteria in food preservation has been widely studied. However, bacteriocins have not been fully developed as antibacterial agents for treating infectious diseases in humans [11]. Although numerous bacteriocin-producing microorganisms isolated from the human body and clinical samples have been identified in recent years, few of their antibacterial activities have been described in detail. Due to the predominance of \u003cem\u003eLactobacillus\u003c/em\u003e species in the vagina of healthy women, it is worthwhile to evaluate and characterize the bacteriocins produced by these microbiota to improve vaginal health.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study investigated the inhibitory effects of \u003cem\u003eLactobacillus\u003c/em\u003e species isolated from the vagina of healthy women on \u003cem\u003eL. iners\u003c/em\u003e. Characteristics such as low-pH tolerance, survival in simulated vaginal fluid, gastric juice and intestinal fluid, as well as aggregation ability, safety, and antimicrobial potential indicate that \u003cem\u003eL gasseri\u003c/em\u003e H87 is a promising candidate probiotic. We also preliminarily characterized the properties of the antibacterial substances produced by H87 [12].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eScreening of Lactobacillus strains for antimicrobial activity.\u003c/strong\u003e\u0026nbsp; A total of 350 isolates were obtained from vaginal swabs of healthy Chinese women. The antibacterial activity of cell-free supernatants (CFS) from isolated strains against \u003cem\u003eL. iners\u003c/em\u003e was tested using the cylinder-plate method. Supernatants were obtained from cultures with comparable cell densities and pH.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe CFS of only four strains (1.1%) were able to inhibit\u003cem\u003e\u0026nbsp;L. iners\u003c/em\u003e, including H23, H87, H198, H271. They were identified as \u003cem\u003eL. rhamnosus\u003c/em\u003e, \u003cem\u003eL. gasseri\u003c/em\u003e, \u003cem\u003eL. paragasseri\u003c/em\u003e and \u003cem\u003eL. reuteri\u003c/em\u003e by 16S rRNA gene sequencing, respectively. As shown in Table 1.,the CFS of H87 exhibited the most potent inhibitory activity with an inhibition zone of 14.23 mm, followed by those of H23, H198 and H271. None of the other isolated strains showed significant antibacterial activity (＜3 mm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Antibacterial activities of \u003cem\u003eLactobacillus\u0026nbsp;\u003c/em\u003estrains against \u003cem\u003eL. iners\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003eInhibition zone (mm)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e\u003cem\u003eL. rhamnosus\u0026nbsp;\u003c/em\u003eH23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e15.31\u0026plusmn;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e\u003cem\u003eL. gasseri\u003c/em\u003e H87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e19.23\u0026plusmn;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e\u003cem\u003eL. paragasseri\u003c/em\u003e H198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e7.06\u0026plusmn;0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e H271\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e4.25\u0026plusmn;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData are presented as the means \u0026plusmn; SD of three replicates.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eLactobacillus\u003c/em\u003e strains without detectable antibacterial activity (inhibition zone \u0026lt; 3 mm), are not shown in the table.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition profile of the selected Lactobacillus strains. \u0026nbsp;\u0026nbsp;\u003c/strong\u003eSeveral microorganisms associated with gastrointestinal, urogenital and reproductive tract infection were tested for their sensitivity to inhibition by the 4 selected \u003cem\u003eLactobacillus\u003c/em\u003e strains (Table 2). Each of the 4 selected \u003cem\u003eLactobacillus\u0026nbsp;\u003c/em\u003espp. inhibited at least one of the pathogenic indicator strains, albeit at different inhibition levels. Notably, H87 exhibited activity against all tested gastrointestinal, urogenital and reproductive tract pathogens. The antimicrobial activity of H23 and H87 selectively inhibited pathogenic bacteria, while retaining most of the tested beneficial vaginal \u003cem\u003eLactobacillus\u003c/em\u003e strains and probiotics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Inhibitory spectrum of \u003cem\u003eLactobacillus\u0026nbsp;\u003c/em\u003estrains\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"26.50862068965517%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"27.801724137931036%\"\u003e\n \u003cp\u003eIndicator strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"45.689655172413794%\"\u003e\n \u003cp\u003eInhibition zone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.488151658767773%\"\u003e\n \u003cp\u003eH23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.222748815165875%\"\u003e\n \u003cp\u003eH87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.222748815165875%\"\u003e\n \u003cp\u003eH198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"26.066350710900473%\"\u003e\n \u003cp\u003eH271\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"9\" valign=\"top\" width=\"26.565874730021598%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ePotential gastrointestinal, urogenital and reproductive tract pathogens\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.861771058315334%\"\u003e\n \u003cp\u003e\u003cem\u003eL. iners\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.526997840172786%\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.583153347732182%\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.583153347732182%\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.879049676025918%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eG. vaginalis\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eA. vaginae\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eEnt. faecalis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eStrep. agalactiae\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eStaph. aureus\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eLis. monocytogenes\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" width=\"26.565874730021598%\"\u003e\n \u003cp\u003eVaginal\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eLactobacillus\u003c/em\u003e species\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.861771058315334%\"\u003e\n \u003cp\u003e\u003cem\u003eL. rhamnosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.526997840172786%\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.583153347732182%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.583153347732182%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.879049676025918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eL. crispatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eL. jensenii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"37.94117647058823%\"\u003e\n \u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.058823529411764%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.411764705882353%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.176470588235293%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.565874730021598%\"\u003e\n \u003cp\u003eIntestinal probiotic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.861771058315334%\"\u003e\n \u003cp\u003e\u003cem\u003eL. bulgaricus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.526997840172786%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.583153347732182%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.583153347732182%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.879049676025918%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e+++ : 11＜x\u0026le;15,++ : 7＜x\u0026le;11,+ :3＜x\u0026le;7, -\u0026nbsp;：x\u0026le;3\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, and \u003cem\u003eListeria\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival under in vitro conditions simulating the vaginal environment. \u0026nbsp;\u003c/strong\u003eThe residual viability of H23, H87, H19 and H271 was 80%, 85%, 83% and 88% after 48 h of incubation at pH 3.5. Similarly, the residual viability of H23 and H271 after 48 h of incubation at pH 4.0 was 88% and 95%, respectively. No significant changes of viability (101% and 102%) were noticed when H87 and H198 were cultivated at pH 4.0 for 48 h (Fig. 1). All the cultures showed more than 100% relative viability after 48 h at pH 4.5 and 6.5.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival under in vitro conditions simulating the gastric and intestinal environments.\u0026nbsp;\u003c/strong\u003e H23 and H87 exhibited 98 and 94% survival after 48h at pH 3. Which was reduced to 80 and 85% at pH 2, respectively. This is typical of the gastric environment. By contrast, the residual viability of H198 and H271 was less than 50% at both pH 2 and 3. In the presence of 0.3% bile salts, which simulate the intestinal environment, H23, H87, H198 and H271 exhibited 95, 96, 88 and 93% survival after 48 h of incubation, respectively. In the presence of 0.5% bile salts, the survival was reduced to 90, 92, 72 and 88%, respectively. The survival of H23 and H87 was reduced to 86, 90, 65, and 82% after 48 h of incubation in the simulated intestinal fluid (SIF).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAggregation ability.\u0026nbsp;\u003c/strong\u003e The results of the auto-aggregation and co-aggregation assays of the vaginal \u003cem\u003eLactobacillus\u003c/em\u003e strains are shown in Table 3. The auto-aggregation values ranged from 33 to 61%, indicating that the 4\u003cem\u003e\u0026nbsp;Lactobacillus\u003c/em\u003e strains have good auto-aggregation abilities. A broad range of variation in co-aggregation with pathogens was detected. H23 and H87 showed high co-aggregation with\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e and \u003cem\u003eListeria monocytogenes\u003c/em\u003e, exhibiting values higher than 30%. By contrast, H198 exhibited low co-aggregation with \u003cem\u003eE. coli\u003c/em\u003e, reaching only 19 %, while H271 exhibited no co-aggregation with \u003cem\u003eL. monocytogenes\u003c/em\u003e (Table 3). Overall, H23 and H87 showed good aggregation abilities\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eAdhesion ability and safety evaluation\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e\u003cem\u003eL. rhamnosus\u003c/em\u003e H23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003e\u003cem\u003eL. gasseri\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eH87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e\u003cem\u003eL. paragasseri\u003c/em\u003e H198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e\u003cem\u003eL. reuteri\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eH271\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003cp\u003eAuto-aggregation (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e43\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003e58\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e61\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e33\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003cp\u003eCo-aggregation with \u003cem\u003eListeria monocytogenes\u0026nbsp;\u003c/em\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e55\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003e61\u0026plusmn;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e40\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003cp\u003eCo-aggregation with \u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e32\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003e51\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e19\u0026plusmn;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e42\u0026plusmn;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003ch4\u003eHemolytic activity\u003c/h4\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e\u0026gamma;-hemolysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003e\u0026gamma;-hemolysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e\u0026gamma;-hemolysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e\u0026gamma;-hemolysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003cp\u003eAmpicillin (4 mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003cp\u003eKanamycin (64 mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003cp\u003eStreptomycin (64 mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003cp\u003eTetracycline (32 mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003cp\u003eChloramphenicol (8 mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.66323024054983%\"\u003e\n \u003cp\u003eMetronidazole (128 mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.18213058419244%\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.38487972508591%\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eS: sensitive, R: resistance\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSafety Evaluation.\u0026nbsp;\u003c/strong\u003e In terms of safety, none of the strains showed hemolytic activity. H23 and H87 were sensitive to all tested antibiotics. H198 was not sensitive to chloramphenicol and H271 was not sensitive to kanamycin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of the antibacterial substance produced by H87.\u003c/strong\u003e\u0026nbsp; \u0026nbsp;Compared with other strains, H87 had the following advantages: (1) it inhibited the highest proportion of the tested pathogenic bacteria that attack the gastrointestinal, urogenital and reproductive tract, (2) it survived under in vitro conditions simulating the vaginal, gastric and intestinal environments (survival rate＞80% in all cases), (3) it exhibited good auto-aggregation and co-aggregation (\u0026gt;40%), (4) it can be preliminarily considered safe (no hemolysis) and controllable (no drug resistance). H87 therefore exhibited the desired characteristics of probiotics, which prompted us to further investigate the properties of antibacterial substances produced by H87.\u003c/p\u003e\n\u003cp\u003eThe results of the experiments excluding the effects of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and lactic acid are shown in Fig. 3. After the addition of NaOH, the inhibition zone appeared only somewhat smaller, indicating that organic acids were not the main bacteriostatic substance. In addition, strain H87 still had antibacterial activity after the addition of catalase, suggesting that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is also not the main antibacterial substance. Subsequently, a protease hydrolysis test was performed on the antibacterial substance produced by H87, and the results showed that the substance could be completely hydrolyzed by pepsin and proteinase K, or partially hydrolyzed by trypsin (Fig. 3). Thus, it was demonstrated that the antibacterial substance produced by strain H87 was a peptide.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUltrafiltration membranes with molecular weight cut off (MWCO) values of 3, 10, 30, 50 and 100 kDa were used to separate the bacteriocin produced by H87. Then, a concentrated solution was obtained by centrifugation. The antibacterial activity of the bacteriocin fractions was analyzed and compared after ultrafiltration. The results indicated that the concentrated solution of the MWCO above 10 kDa has no antibacterial activity, while the concentrated solution of the MWCO above 3 kDa has the highest antibacterial activity, and the filtrate of the MWCO of 3 kDa has a lower antibacterial activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the active fractions showed that a band with an approximate molecular weight of 6.5 kDa was present both in the concentrated solution of the MWCO of 3 kDa and the filtrate of the MWCO 3 kDa. Usually, about 70% of the target protein can be intercepted by the off-line ultrafiltration tube, and the rest of the target protein will be passed into the filtrate. Therefore, the antibacterial activity assay and electrophoresis results indicated that the bacteriocin produced by \u003cem\u003eL. gasseri\u0026nbsp;\u003c/em\u003eH87 is most likely a single peptide with a molecular weight of approximately 6.5 kDa.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAt present, there are few commercial probiotic products that can be directly applied to the vagina\u0026nbsp;[13]. Studies have shown that host-specific microorganisms have a high colonization ability and high survival rate in the host environment. Accordingly, they can produce specific physiological effects. Therefore, it is urgent to develop host-specific probiotics. In this study, we studied the low-pH tolerance, survival under simulated vaginal, gastric and intestinal conditions of four \u003cem\u003eLactobacillus\u003c/em\u003e strains, while also assessing their aggregation ability, safety, and antibacterial potential. Finally, we preliminarily characterized the properties of the antibacterial substance produced by the most effective strain.\u003c/p\u003e\n\u003cp\u003eTo treat BV, probiotics can be directly applied to the vagina to directly control the re-colonization of \u003cem\u003eLactobacillus\u003c/em\u003e species [14]. In addition, BV can also be treated with oral probiotics. However, in order to achieve the desired clinical therapeutic effect, probiotics need to be strictly transferred to the colonization site. Researchers generally use the method of recovering specific microorganisms from fecal samples to demonstrate the success of the transfer [15]. Previous studies have shown that immune responses, stool consistency and the density of vaginal \u003cem\u003eLactobacillus\u003c/em\u003e can be improved by probiotics\u0026nbsp;[16,17].\u0026nbsp;Probiotics applied to the vagina have good efficacy, but oral administration is more convenient [18].\u0026nbsp;Thus, we investigated the survival of the 4 selected vaginal \u003cem\u003eLactobacillus\u003c/em\u003e strains in the simulated vaginal fluid (SVF), simulated gastric juice (SGJ) and simulated intestinal fluid (SIF) to select promising \u003cem\u003eLactobacillus\u003c/em\u003e strains to be used both as vaginal capsules and dietary supplements. The main components of vaginal fluid are carbohydrates, proteins and a small amount of lipids, and it is a mixture of secretions from the local glands and organs\u0026nbsp;[19]. Under normal conditions, it has an acidic pH of 3.8-4.4.\u0026nbsp;The survival rate of probiotics under such complex and acidic conditions indicates the viability of this strain in the vagina. In this study, the survival rate of the four strains was 88-100% at pH 4.5 and 80-88% at pH 4.0, which indicated that the four strains were resistant to acidic conditions (pH\u0026le;4.0), and can adapt to the vaginal environment. The high acidity of gastric juice, pepsin and proteases in the intestinal fluid, as well as bile acid salts in the duodenum can inhibit foreign bacteria. Conversely, bacteria can maintain a sufficiently high viability to exert a probiotic effect only when they have high resistance to the factors mentioned above.\u0026nbsp;As shown in Fig. 2, strains H23 and H87 exhibited high tolerance to acidic pH with survival rates above 80%, indicating that they can adapt to acidic gastric juice with a pH value as low as 2. At the same time, the survival of H23, H87 and H271 also remained above 80% in the presence of bile salts. Similarly, the survival of H23, H87 and H271 remained above 80% in the SIF environment. Thus, the strains H23 and H87 can adapt to gastric and intestinal environments.\u003c/p\u003e\n\u003cp\u003eAuto-aggregation and co-aggregation are important properties of probiotics. They can reflect the ability of biofilm formation on host cells (for instance, intestinal epithelial, vaginal and ectocervical epithelial cells), while the co-aggregation of probiotics with intestinal pathogenic bacteria is helpful for antibacterial substances to kill pathogenic bacteria. We found that H271 has a lower auto-aggregation capacity and lower co-aggregation capacity with pathogens compared to other tested \u003cem\u003eLactobacillus\u003c/em\u003e strains. H198 showed the highest\u0026nbsp;auto-aggregation, as well as\u0026nbsp;moderate levels of co-aggregation\u0026nbsp;with Gram-positive\u003cem\u003e\u0026nbsp;L. monocytogenes\u003c/em\u003e, but lower\u0026nbsp;co-aggregation\u0026nbsp;with Gram-negative\u003cem\u003e\u0026nbsp;E. coli.\u0026nbsp;\u003c/em\u003eH87\u0026nbsp;exhibited the second highest\u0026nbsp;auto-aggregation, while having the highest co-aggregation\u0026nbsp;with both of the above-mentioned pathogens. By contrast, H23 showed low\u0026nbsp;co-aggregation with \u003cem\u003eE. coli.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe safety assessment showed that\u0026nbsp;none of the four strains produced hemolysin, which\u0026nbsp;may cause harmful effects to the host. These results indicated that all four strains are likely\u0026nbsp;safe for humans. In addition, all strains showed different degrees of drug sensitivity.\u0026nbsp;However, all strains showed\u0026nbsp;resistance\u0026nbsp;to kanamycin and streptomycin. This was\u0026nbsp;consistent with the\u0026nbsp;intrinsic\u0026nbsp;resistance to aminoglycoside antibiotics found in lactic acid bacteria [20].\u0026nbsp;In addition, the resistance of H87 and H198 to metronidazole\u0026nbsp;is commonly used for the treatment of bacterial vaginosis to ensure their survival during treatment.\u003c/p\u003e\n\u003cp\u003eIn sum, \u003cem\u003eL. gasseri\u0026nbsp;\u003c/em\u003eH87 showed distinct advantages compared with the other tested strains.\u003cem\u003e\u0026nbsp;L. gasseri\u003c/em\u003e is an autochthonous microorganism of the normal human flora [21], and one of the dominant \u003cem\u003eLactobacillus\u003c/em\u003e species in the vaginal ecosystem, together with \u003cem\u003eL. jensenii\u003c/em\u003e, \u003cem\u003eL. iners\u003c/em\u003e and \u003cem\u003eL. crispatus\u003c/em\u003e [22-24]. \u003cem\u003eL. gasseri\u003c/em\u003e is a well-known symbiont of the vaginal mucosa, and is negatively correlated with BV [25-27]. Some strains of this species show promising potential as probiotics to maintain vaginal homeostasis [28-30].\u003cem\u003e\u0026nbsp;\u003c/em\u003eH87 inhibited a number of potential gastrointestinal, urogenital and reproductive tract pathogens, including species of\u0026nbsp;\u003cem\u003eLactobacillus, Enterococcus, Staphylococcus, Streptococcus,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eListeria\u003c/em\u003e.\u0026nbsp;GasE and GasT were purified from the supernatants of \u003cem\u003eL. gasseri\u003c/em\u003e EV1461 and SBT2055, respectively. The amino acid sequence of the mature active peptide GasE differed in only one amino acid from that of GasT, and their molecular weight was 5 kDa [31]. \u003cem\u003eL. gasseri\u003c/em\u003e produces bacteriocin, which belongs to Class IIb and is composed of two different peptides, and their genes are adjacent to each other in the same operon [32]. Examples include GasE/GaeX from \u003cem\u003eL. gasseri\u003c/em\u003e EV1461, GasT/GatX from \u003cem\u003eL. gasseri\u0026nbsp;\u003c/em\u003eSBT2055, Acd221B/Acd221\u0026beta; from \u003cem\u003eL. gasseri\u0026nbsp;\u003c/em\u003eLF221, and GasK7A/ B (GaeA) from \u003cem\u003eL. gasseri\u0026nbsp;\u003c/em\u003eK7. The molecular weight of the bacteriocin obtained from H87 was approximately 6.5 kDa, which is consistent with previous reports. In addition, H87 exhibited antibacterial activity against\u0026nbsp;strains of\u0026nbsp;\u003cem\u003eLactobacillus, Enterococcus, Staphylococcus, Streptococcus,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Listeria\u003c/em\u003e, which is also consistent with the literature. Therefore, it is possible that the bacteriocin produced by H87 is homologous to GasT and GasE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH87 was resistant to metronidazole and strongly inhibited \u003cem\u003eL. iners\u003c/em\u003e. The reason\u0026nbsp;is related to vaginal imbalance and recurrence of BV, but its pathogenesis is still unclear. Therefore, in order to better understand the pathogenesis, it is meaningful and useful to develop inhibitors that are active against \u003cem\u003eL. iners.\u003c/em\u003e Metronidazole is a commonly used antibiotic for the treatment of BV, but \u003cem\u003eL. iners\u003c/em\u003e is resistant to metronidazole. Therefore, probiotics that can inhibit\u003cem\u003e\u0026nbsp;L. iners\u0026nbsp;\u003c/em\u003eand are resistant to metronidazole have become a focus of research. Metronidazole combined with probiotics can be used in the treatment of BV, and it can also be used to prevent its recurrence. Therefore, the discovery of H87 may improve the treatment of BV.\u003c/p\u003e\n"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial strains, growth media, and culture conditions.\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u0026nbsp;L. gasseri H87\u003c/em\u003e was isolated from human vaginal fluid and was preserved in the China Center for Type Culture Collection (CCTCC) under the accession number M 2018477. The vaginal\u003cem\u003e\u0026nbsp;Lactobacillus\u0026nbsp;\u003c/em\u003estrains and \u003cem\u003eL. iners\u003c/em\u003e ATCC 55195 were grown in Man-Rogosa-Sharpe (MRS) medium at 37°C under aerobic conditions.\u003cem\u003e\u0026nbsp;Gardnerella vaginalis\u003c/em\u003e was grown anaerobically on chocolate agar plates at 37°C.\u0026nbsp;\u003cem\u003eEscherichia coli,\u003c/em\u003e\u003cem\u003e\u0026nbsp;Enterococcus faecalis\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Streptococcus agalactiae\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus,\u003c/em\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Listeria monocytogenes\u003c/em\u003e were grown aerobically on Luria-Bertani (LB) agar plates at 37°C.\u0026nbsp;All strains were maintained as frozen stocks at -80°C in their respective culture media with the addition of 15 to 20% (vol/vol) sterile glycerol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Lactobacillus cell suspensions.\u0026nbsp;\u003c/strong\u003e If not indicated otherwise, cell suspensions were prepared by growing \u003cem\u003eLactobacillus\u0026nbsp;\u003c/em\u003estrains at 37°C for 24 h under aerobic conditions in MRS medium. The cells were pelleted by centrifugation (5000 g, 15 min, 4 °C), washed twice with phosphate-buffered saline (PBS; 0.1 M, pH 7.2, containing 0.85 % (w/v) NaCl) and resuspended in phosphate buffer (0.1 M, pH 7) to obtain a cell suspension with OD \u003csub\u003e600\u003c/sub\u003e = 1, approximately 10\u003csup\u003e9\u003c/sup\u003e CFU/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSampling and isolation of vaginal Lactobacillus strains.\u0026nbsp;\u003c/strong\u003e The study protocol was approved by the ethics review board of the Second Affiliated Hospital of Nanjing Medical University. We have obtained written informed consent from all study participants. All the procedures were performed in accordance with the Declaration of Helsinki and relevant policies in China.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLactobacilli\u003c/em\u003e were isolated from the vaginal microbiome of asymptomatic Chinese women who were invited to participate in the study during their routine gynecological consultations. MRS agar plates with 0.05% of L-cysteine supplementation were incubated anaerobically at 37 °C for 24~48 h. Individual colonies were randomly selected and purified before storing at\u0026nbsp;−80\u0026nbsp;°C with 20% glycerol.\u003c/p\u003e\n\u003cp\u003eCell-free supernatants from the isolated \u003cem\u003eLactobacillus\u0026nbsp;\u003c/em\u003estrains were screened for antibacterial activity against \u003cem\u003eL. iners\u0026nbsp;\u003c/em\u003eATCC 55195 using the cylinder-plate method [33]. The \u003cem\u003eL. iners\u003c/em\u003e suspension was diluted to 10\u003csup\u003e7\u003c/sup\u003e CFU/mL, mixed with the melted solid culture medium cooled to 40°C. and poured into a plate to solidify. An Oxford cup was gently placed on the solidified plate. After 200 μL of the lactic acid bacteria fermentation supernatant was added to the cup, the system was incubated at 37℃ for 24 h. The antimicrobial activity was evaluated by measuring the diameter of the transparent inhibition zone against the test strain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro antibiotic activity assay.\u003c/strong\u003e\u0026nbsp; Antibiotic activities of isolated strains were determined using the cylinder-plate method as described before [33].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTolerance to simulated vaginal fluid (SVF) at low pH.\u0026nbsp;\u003c/strong\u003e One-milliliter overnight-grown bacterial cultures were separately combined with 100 mL of simulated vaginal fluid (SVF) prepared as described by Ahire et al. [3] (pH adjusted with lactic acid), incubated anaerobically at 37 °C and the OD\u003csub\u003e600\u003c/sub\u003e measured after 48h. Bacteria grown in MRS were included as a control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTolerance to simulated gastric juice (SGJ) and simulated intestinal fluid (SIF) .\u003c/strong\u003e\u0026nbsp; A sample comprising 100 μl of cell suspension was mixed with 1 ml of SGJ or SIF, and incubated at 37 °C for 4 h. The resistance was determined by measuring the survival rate percentage (SR %), based on the initial (0 h) and final (4 h) number of viable cells enumerated on MRS agar plates after 48 h.\u003c/p\u003e\n\u003cp\u003eThe SGJ was composed of 0.3% pepsin and 0.5% NaCl, with pH\u0026nbsp;adjusted to 2 or 3 adjusted with 1\u0026nbsp;M HCl. The SIF was composed of 0.1\u0026nbsp;% pancreatin, 0.5\u0026nbsp;% bile salts, 0.5\u0026nbsp;% NaCl, 0.4\u0026nbsp;% phenol, and pH\u0026nbsp;8 adjusted with 1\u0026nbsp;M NaOH [12,34].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTolerance to bile\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;Lactobacillus\u003c/em\u003e cells (10\u003csup\u003e9\u003c/sup\u003e CFU/mL) were used to inoculate 5 mL of MRS modified with bile salts (0.3 or 0.5 %) at a ratio of 5% (v/v), and incubated at 37 °C for 4 h. Based on the initial (0 h) and final (3 h) number of viable cells enumerated on MRS agar plates after 48 h, the resistance was determined by measuring the survival rate percentage (SR %).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuto-aggregation assay.\u0026nbsp;\u003c/strong\u003e The auto-aggregation assay was performed as described by Pithva et al. [12]. A cell suspension (2 ml) was vortexed for 10 s and incubated at 37 °C. Aliquots of 0.1 ml were collected from the upper surface at regular time intervals and mixed with 0.9 ml PBS, followed by measurement of the optical density at 600 nm. The auto-aggregation (%) was calculated as [(OD\u003csub\u003e0\u003c/sub\u003e − OD\u003csub\u003et\u003c/sub\u003e)/OD\u003csub\u003e0\u003c/sub\u003e] × 100, where OD\u003csub\u003e0\u003c/sub\u003e represents the optical density at 0 h and OD\u003csub\u003et\u003c/sub\u003e represents the optical density of the cell suspension at 24h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-aggregation assay. \u0026nbsp;\u003c/strong\u003eEqual volumes of cell suspensions (1\u0026nbsp;ml = 10\u003csup\u003e9\u003c/sup\u003e cfu/ml) of \u003cem\u003eLactobacillus\u003c/em\u003e and pathogenic indicator strains were mixed, and then incubated at 37 °C. The control contained 2 ml of pure bacterial or yeast cell suspension of the indicator strain. The OD\u003csub\u003e600\u003c/sub\u003e of the suspensions was measured at the indicated time intervals. The co-aggregation (%) was calculated using the equation [(OD\u003csub\u003epat\u003c/sub\u003e + OD\u003csub\u003eLacto\u003c/sub\u003e)/2 − OD\u003csub\u003emix\u003c/sub\u003e] / [(OD\u003csub\u003epat\u003c/sub\u003e + OD\u003csub\u003eLacto\u003c/sub\u003e)/2] × 100 . The OD\u003csub\u003epat\u003c/sub\u003e and OD\u003csub\u003eLacto\u003c/sub\u003e represent the optical densities of the \u003cem\u003eLactobacillus\u003c/em\u003e sp. and the indicator strain, while OD\u003csub\u003emix\u003c/sub\u003e represents the optical density of the mixture of \u003cem\u003eLactobacillus\u003c/em\u003e sp. andthe indicator strain after 24 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHemolytic activity.\u0026nbsp;\u003c/strong\u003e The hemolytic activity was assessed on blood agar plates containing sheep blood according to a modification of the method reported by Pino et al. [34].Briefly, \u003cem\u003eLactobacillus\u003c/em\u003e strains were streaked onto blood agar plates containing sheep blood, and incubated at 37 °C for 24 h under anaerobic conditions. The hemolytic activity was visually detected and distinguished as β-hemolysis, α-hemolysis, or γ-hemolysis based on the appearance of a clear zone, green halo or no zones around colonies, respectively. \u003cem\u003eBacillus cereus\u003c/em\u003e was used as a positive control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibiotic susceptibility.\u0026nbsp;\u003c/strong\u003e The antibiotic resistance pattern of\u003cem\u003e\u0026nbsp;Lactobacillus\u003c/em\u003e strains was assessed according to the standard protocol of the European Food Safety Authority [35,36].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of the antibacterial substance. \u0026nbsp;\u003c/strong\u003eIn order to exclude the inhibitory effect of organic acids, cell-free supernatants (CFS) were adjusted to pH 6.5 using NaOH. To clarify whether the detected antimicrobial activity is caused by the production of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 2600 IU/ml of catalase was added to 1 ml CFS of LAB, and incubated for 24 h at 30 °C. In addition, CFS (pH 6.0) of selected \u003cem\u003eLactobacillus\u003c/em\u003e isolates were incubated in MRS broth at 30 °C for 24 h and tested for their sensitivity to proteolytic enzymes in order to determine the if the substance responsible of the antimicrobial activity of the bacteria is a polypeptide. One milliliter of CFS was treated for 2 h at 30 °C with 1 mg/ml final concentration of pepsin, trypsin, or proteinase K, respectively. The remaining antimicrobial activity was assessed using the cylinder-plate method with \u003cem\u003eL. iners\u003c/em\u003e as the indicator strain. Untreated cell-free supernatants were used as controls.\u003c/p\u003e\n\u003cp\u003eFor the bile salt hydrolysis test, fresh cultures were streaked onto MRS agar plates containing 0.5% (w/v) taurodeoxycholic acid. The hydrolysis effect was represented by different colony morphology (partial hydrolysis recorded as 1) from the control MRS plates, after 48 h of anaerobic incubation at 37\u0026nbsp;°C [18].\u003c/p\u003e\n\u003cp\u003eBile salt hydrolase (BSH) activity was determined using the method Caggia et al. reported previously [37]. The appearance of a precipitate around colonies was considered as a positive sign. Based on the confluence of the precipitate, each strain was classified as ‘+++’ for heavy; ‘++’ for intermediate; ‘+’ for low; and ‘−’ for no precipitation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSDS-PAGE.\u003c/strong\u003e\u0026nbsp; During the purification process, the RPC-FPLC eluted fractions of GasE were analyzed in duplicate by Tris-Tricine SDS-PAGE with an 18 % acrylamide resolving gel [38]. After electrophoresis at 100 mV for 2 h, one gel was silver-stained, while the other was used to detect the inhibitory activity in an overlay assay as described previously [39].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e\u003cem\u003eL. gasseri\u003c/em\u003e H87 showed a good ability to survive at low pH in SVF, SGJ and SIF. The aggregation and antimicrobial abilities of the strain also indicated that it can displace pathogens from the vaginal environment. Finally, a bacteriocin with an approximate size of 6.5 kDa was purified from the culture supernatant of H87. We believe that this strain may be useful to prevent/treat vaginal dysbiosis and maintain a healthy vaginal ecosystem as a vaginal probiotic.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data is included in the text. The raw data of this article will be made available by the authors, without undue reservation, to any qualified researcher.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a-Velasco, J.A., Menabrito, M. \u0026amp; Catal\u0026aacute;n, I.B. What Fertility Specialists Should Know about the Vaginal Microbiome: A Review. 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Appl Environ Microbiol. \u003cstrong\u003e59\u003c/strong\u003e,1416\u0026ndash;1424. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aem.59.5.1416-1424.1993\u003c/span\u003e\u003c/span\u003e (1993).\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"probiotics, bacteriocin, vaginal microbiome, Lactobacillus iners, Lactobacillus gasseri","lastPublishedDoi":"10.21203/rs.3.rs-1868131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1868131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eLactobacillus iners\u003c/em\u003e is often associated with increased risk of adverse gynecological and obstetric outcomes, such as vaginal dysbiosis and bacterial vaginosis (BV). \u003cem\u003eLactobacillus\u003c/em\u003e strains from the vagina of healthy women were screened for inhibitory activity against\u003cem\u003e L. iners.\u003c/em\u003e Four active strains were identified, whereby \u003cem\u003eL. gasseri\u003c/em\u003e H87 exhibited the highest inhibitory activity against \u003cem\u003eL. iners\u003c/em\u003e, followed by \u003cem\u003eL. rhamnosus\u003c/em\u003e H23. All four strains inhibited the growth of potential pathogens of the gastrointestinal, urogenital and reproductive tract, and were capable of co-aggregating with the cells of the tested pathogens to varying degrees. H23 and H78 exhibited survival rates above 80% under in vitro conditions simulating the vaginal, gastric and intestinal environment. The four strains showed good auto-aggregation of more than 30%. H23 and H87 also exhibited good co-aggregation of more than 30% with \u003cem\u003eListeria monocytogenes\u003c/em\u003e and \u003cem\u003eEscherichia coli.\u003c/em\u003e Safety assessment showed that the four strains had the usual antibiotic susceptibility profile and did not produce hemolysins. Considering that H87 exhibited a better performance as a probiotic, we investigated the antibacterial substance produced by H87, and identified a bacteriocin with a molecular weight of 6.5 kDa. Based on strain characteristics and beneficial properties, we confirmed its ability to prevent/treat vaginal dysbiosis and maintain a healthy vaginal ecosystem. Therefore, we believe that H87 is a promising candidate for human trials.\u003c/p\u003e","manuscriptTitle":"Screening and characterization of vaginal Lactobacillus strains as probiotic candidates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-29 18:44:12","doi":"10.21203/rs.3.rs-1868131/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f39a6e37-dd67-4e3a-a161-ad49d0271c2e","owner":[],"postedDate":"July 29th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-11-02T05:44:24+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-29 18:44:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1868131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1868131","identity":"rs-1868131","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}