Lactobacillus crispatus 7-4 Mitigates Salmonella typhimurium-Induced Enteritis via the γ­Glutamylcysteine-Mediated Nrf2 Pathway.

Salmonella typhimurium (S. typhimurium) constitutes a major public health concern. We have previously proven that Lactobacillus crispatus 7-4 (L. crispatus 7-4) can inhibit the growth of S. typhimurium and thus can be used as a biocontrol strategy to suppress foodborne S. typhimurium infections. However, the inhibitory effect and in-depth mechanism of L. crispatus 7-4 remain to be elucidated. In this study, we found that L. crispatus 7-4 can protect against S. typhimurium-induced ileum injury by promoting intestinal barrier integrity, maintaining intestinal mucosal barrier homeostasis, and reducing intestinal inflammatory response. Furthermore, we demonstrated that this probiotic strain can increase the abundance of Lactobacillus spp. to maintain microbial homeostasis and simultaneously increase the amount of γ­glutamylcysteine (γ-GC) by activating the glutathione metabolic pathway. The increased γ-GC promoted the transcription of Nrf2 target genes, thereby improving the host antioxidant level, reducing reactive oxygen species (ROS) accumulation, and removing pro-inflammatory cytokines. In other words, L. crispatus 7-4 could activate the enterocyte Nrf2 pathway by improving γ-GC to protect against S. typhimurium-induced intestinal inflammation and oxidative damage.

Vaginal microbes alter epithelial transcriptome and induce epigenomic modifications providing insight into mechanisms for susceptibility to adverse reproductive outcomes.

The cervicovaginal microbiome is highly associated with women's health, with microbial communities dominated by Lactobacillus species considered optimal. Conversely, a lack of lactobacilli and a high abundance of strict and facultative anaerobes, including Gardnerella vaginalis, have been associated with adverse reproductive outcomes. However, how host-microbial interactions alter specific molecular pathways and impact cervical and vaginal epithelial function remains unclear. Using RNA-sequencing, we characterized the in vitro cervicovaginal epithelial transcriptional response to different vaginal bacteria and their culture supernatants. We showed that G. vaginalis upregulates genes associated with an activated innate immune response. Unexpectedly, G. vaginalis specifically induced inflammasome pathways through activation of NLRP3-mediated increases in caspase-1, IL-1ß and cell death, while live L. crispatus had minimal transcriptomic changes on epithelial cells. L. crispatus culture supernatants resulted in a shift in the epigenomic landscape of cervical epithelial cells that was confirmed by ATAC-sequencing showing reduced chromatin accessibility. This study reveals new insights into host-microbe interactions in the lower reproductive tract and suggests potential therapeutic strategies leveraging the vaginal microbiome to improve reproductive health.

The microbiota of sexual intercourse and its effect on prostatitis.

It is estimated that microorganisms colonize 90% of the body surface. In some tracts, such as the genitourinary tract, the microbiota varies throughout life, influenced by hormonal stimulation and sexual practices. This study evaluated the semen differences and presence of Lactobacillus crispatus, Lactobacillus iners, Gardnerella vaginalis and Atopobium vaginae in semen samples from patients with symptoms of chronic prostatitis and men asymptomatic for urogenital infections. Fifty-three semen samples were included: 22 samples from men with symptoms of chronic prostatitis and 31 asymptomatic men (control group). In addition to the presence of L. crispatus, L. iners, G. vaginalis and A. vaginae, semen parameters, total antioxidant capacity of seminal plasma, prostatic antigen and some proinflammatory cytokines were evaluated in each semen sample. Volunteers with symptoms of chronic prostatitis presented a lower percentage of sperm morphology (4.3% vs. control group 6.0%, p = 0.004); in the semen samples of volunteers in the group asymptomatic for urogenital infections, microorganisms associated with the vaginal microbiota were detected more frequently. The presence of bacteria in the vaginal microbiota can also benefit male reproductive health, which undergoes various modifications related to lifestyle habits that are susceptible to modification. Microorganisms associated with the vaginal microbiota, such as L. crispatus, L. iners, G. vaginalis and A. vaginae, may have a protective role against the development of male genitourinary diseases such as prostatitis.

Synergistic Inhibition of Synbiotic Cultures among Lactobacilli and Plant Extracts against Vaginal Discharge Causing Candida albicans.

Vulvovaginal candidiasis (VVC) is the most common cause of vaginal discharge among women. The present study aimed to investigate the synergistic anticandidal effect of lactobacillus cultures supplemented with plant extracts. Among 600 isolates of lactic acid bacteria, 41 isolates exhibited inhibitory activity against Candida albicans ATCC10231. Six out of 41 cell-free supernatants demonstrated the most potent antibacterial and anticandidal activities. They also inhibited the clinical isolates of C. albicans, causing VVC and non-C. albicans. The synergistic effect between Lactobacillus crispatus 84/7 and Limosilactobacillus reuteri 89/4 was demonstrated by the lowest fractional inhibitory concentration index (FICI = 0.5). The synbiotic culture of bacterial combination, cultured with Jerusalem artichoke (H. tuberosus) extract, also exhibited the strongest inhibition against the tested C. albicans. Biofilm formation decreased after 12 h of incubation in the selected cell-free supernatants of this synbiotic culture. The anticandidal activity of crude extracts was lost after treatment with proteinase K and trypsin but not with heating conditions, suggesting that it may be a heat-stable substance. In conclusion, the combination of L. crispatus 84/7 and L. reuteri 89/4 with H. tuberosus may be a promising candidate for inhibiting Candida infection and biofilm formation, with the potential use as ingredients in vaginal biotherapeutic products.

Draft genome sequences of three Lactobacillus crispatus strains, isolated from the female urinary tract.

Lactobacillus crispatus is a frequent member of the female urogenital microbiota. Here, we present the draft genome assemblies of three L. crispatus strains: UMB4356, UMB5661, and UMB6244. All strains were isolated from voided urine samples from females with type 2 diabetes.

Gonorrhea caused due to antimicrobial-resistant bacteria Neisseria gonorrhoeae treated using probiotic peptide.

Neisseria gonorrhea is a sexually transmitted disease from gonorrhea that lacks treatment; despite the urgency, the absence of adequate drugs, lack of human correlates of protection, and inadequate animal models of infection have delayed progress toward the prevention of gonococcal infection. Lactobacillus crispatus is a lactic acid bacterium typically found in the human vaginal microbiota. Peptides from L. crispatus have shown a potential therapeutic option for targetting N. gonorrhea. Bioinformatics analysis is important for speeding up drug target acquisition, screening refinement, and evaluating adverse effects and drug resistance prediction. Therefore, this study identified an antimicrobial peptide from the bacteriocin immunity protein (BIP) of L. crispatus using the bioinformatics tool and Collection of Antimicrobial Peptide (CAMPR3). Based on the AMP score and highest ADMET properties, the peptide SM20 was chosen for docking analysis. SM20 was docked against multiple proteins from the genome of the AMR bacterium N. gonorrhea using an online tool; protein-peptide interactions were established and visualized using the PyMol visualizing tool. Molecular docking was carried out using the CABSdock tool, and multiple conformations were obtained against the membrane proteins of N. gonorrhoea. The peptide SM20 exhibited higher docking scores and ADMET properties. Therefore, SM20 could be further encapsulated with cellulose; it can be applied topically to the genital tract to target N. gonorrhea infection. The controlled release of the antimicrobial peptide from the gel can provide sustained delivery of the treatment, increasing its efficacy and reducing the risk of resistance development.

Anti-inflammatory effects of extracellular vesicles and cell-free supernatant derived from Lactobacillus crispatus strain RIGLD-1 on Helicobacter pylori-induced inflammatory response in gastric epithelial cells in vitro.

Helicobacter pylori infection is the major risk factor associated with the development of gastric cancer. Currently, administration of standard antibiotic therapy combined with probiotics and postbiotics has gained significant attention in the management of H. pylori infection. In this work, the immunomodulatory effects of Lactobacillus crispatus-derived extracellular vesicles (EVs) and cell-free supernatant (CFS) were investigated on H. pylori-induced inflammatory response in human gastric adenocarcinoma (AGS) cells. L. crispatus-derived EVs were isolated by ultracentrifugation and physically characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Furthermore, the protein content of L. crispatus-derived EVs was also evaluated by SDS-PAGE. Cell viability of AGS cells exposed to varying concentrations of EVs and CFS was assessed by MTT assay. The mRNA expression of IL-1ß, IL-6, IL-8, TNF-α, IL-10, and TGF-ß genes was determined by RT-qPCR. ELISA was used for the measurement of IL-8 production in AGS cells. In addition, EVs (50 µg/mL) and CFS modulated the H. pylori-induced inflammation by downregulating the mRNA expression of IL-1ß, IL-6, IL-8, and TNF-α, and upregulating the expression of IL-10, and TGF-ß genes in AGS cells. Furthermore, H. pylori-induced IL-8 production was dramatically decreased after treatment with L. crispatus-derived EVs and CFS. In conclusion, our observation suggests for the first time that EVs released by L. crispatus strain RIGLD-1 and its CFS could be recommended as potential therapeutic agents against H. pylori-triggered inflammation.

Lactobacillus crispatus CCFM1339 Inhibits Vaginal Epithelial Barrier Injury Induced by Gardnerella vaginalis in Mice.

The vaginal epithelial barrier, which integrates mechanical, immune, chemical, and microbial defenses, is pivotal in safeguarding against external pathogens and upholding the vaginal microecological equilibrium. Although the widely used metronidazole effectively curtails Gardnerella vaginalis, a key pathogen in bacterial vaginosis, it falls short in restoring the vaginal barrier or reducing recurrence rates. Our prior research highlighted Lactobacillus crispatus CCFM1339, a vaginally derived Lactobacillus strain, for its capacity to modulate the vaginal epithelial barrier. In cellular models, L. crispatus CCFM1339 fortified the integrity of the cellular monolayer, augmented cellular migration, and facilitated repair. Remarkably, in animal models, L. crispatus CCFM1339 substantially abated the secretion of the barrier disruption biomarker E-cadherin (from 101.45 to 82.90 pg/mL) and increased the anti-inflammatory cytokine IL-10 (35.18% vs. the model), consequently mitigating vaginal inflammation in mice. Immunological assays in vaginal tissues elucidated increased secretory IgA levels (from 405.56 to 740.62 ng/mL) and curtailed IL-17 gene expression. Moreover, L. crispatus CCFM1339 enhanced Lactobacilli abundance and attenuated Enterobacterium and Enterococcus within the vaginal microbiome, underscoring its potential in probiotic applications for vaginal barrier regulation.

Diverse mechanisms control amino acid-dependent environmental alkalization by Candida albicans.

Candida albicans has the capacity to neutralize acidic growth environments by releasing ammonia derived from the catabolism of amino acids. The molecular components underlying alkalization and its physiological significance remain poorly understood. Here, we present an integrative model with the cytosolic NAD+-dependent glutamate dehydrogenase (Gdh2) as the principal ammonia-generating component. We show that alkalization is dependent on the SPS-sensor-regulated transcription factor STP2 and the proline-responsive activator Put3. These factors function in parallel to derepress GDH2 and the two proline catabolic enzymes PUT1 and PUT2. Consistently, a double mutant lacking STP2 and PUT3 exhibits a severe alkalization defect that nearly phenocopies that of a gdh2-/- strain. Alkalization is dependent on mitochondrial activity and in wild-type cells occurs as long as the conditions permit respiratory growth. Strikingly, Gdh2 levels decrease and cells transiently extrude glutamate as the environment becomes more alkaline. Together, these processes constitute a rudimentary regulatory system that counters and limits the negative effects associated with ammonia generation. These findings align with Gdh2 being dispensable for virulence, and based on a whole human blood virulence assay, the same is true for C. glabrata and C. auris. Using a transwell co-culture system, we observed that the growth and proliferation of Lactobacillus crispatus, a common component of the acidic vaginal microenvironment and a potent antagonist of C. albicans, is unaffected by fungal-induced alkalization. Consequently, although Candida spp. can alkalinize their growth environments, other fungal-associated processes are more critical in promoting dysbiosis and virulent fungal growth.

Vaginal prevention of Candida albicans: synergistic effect of lactobacilli and mannan oligosaccharides (MOS).

Vulvovaginal candidiasis (VVC) affects approximately 30-50% of women at least once during their lifetime, causing uncomfortable symptoms and limitations in their daily quality of life. Antifungal therapy is not very effective, does not prevent recurrencies and usually causes side effects. Therefore, alternative therapies are urgently needed. The goal of this work was to investigate the potential benefits of using mannan oligosaccharides (MOS) extracts together with a Lactobacillus sp. pool, composed by the most significant species present in the vaginal environment, to prevent infections by Candida albicans. Microbial growth of isolated strains of the main vaginal lactobacilli and Candida strains was assessed in the presence of MOS, to screen their impact upon growth. A pool of the lactobacilli was then tested against C. albicans in competition and prophylaxis studies; bacterial and yeast cell numbers were quantified in specific time points, and the above-mentioned studies were assessed in simulated vaginal fluid (SVF). Finally, adhesion to vaginal epithelial cells (HeLa) was also evaluated, once again resorting to simultaneous exposure (competition) or prophylaxis assays, aiming to measure the effect of MOS presence in pathogen adherence. Results demonstrated that MOS extracts have potential to prevent vaginal candidiasis in synergy with vaginal lactobacilli, with improved results than those obtained when using lactobacilli alone. KEY POINTS: Potential benefits of MOS extracts with vaginal lactobacilli to prevent C. albicans infections. MOS impacts on growth of vaginal lactobacilli pool and C. albicans in SVF. MOS extracts in synergy with L. crispatus inhibit C. albicans adhesion in HeLa cells.

Relationship between vaginal microbiota and chorioamnionitis: A prospective cohort study.

OBJECTIVE: This study aimed to determine the association between vaginal microbiota and chorioamnionitis and its predictive value. METHODS: Thirty pregnant women in their third trimester were prospectively recruited. The participants were categorized into three groups based on their clinical manifestations and placental pathology: the clinical chorioamnionitis group (IP group), the asymptomatic histological chorioamnionitis group (CP group), and the healthy control group (CN group). Basic data and medical history were collected from each participant. Vaginal samples were collected before delivery and analyzed using microbial diversity sequencing. RESULTS: No significant differences were observed in age, body mass index, and education among the groups (P > 0.05). However, the IP group exhibited higher rates of low birth weight (60 % vs 20 % vs 0 %, P = 0.008) and respiratory distress syndrome (50 % vs 20 % vs 0 %, P = 0.003) compared with the CP and CN groups. The Shannon index [2.09 (1.16-3.86) vs 0.84 (0.19-1.11) vs 0.44 (0.25-0.85), P = 0.009] and Simpson index [0.70 (0.41-0.81) vs 0.26 (0.04-0.39) vs 0.11 (0.05-0.29), P = 0.010] in the IP group were higher than those in the CN and CP groups. ß diversity analysis indicated that the microbial community structure differed among the three groups, with a 14.1 % variation associated with group differences (P = 0.002). At the genus level, the random forest model revealed that Lactobacillus, Dialister, Prevotella, Ligilactobacillus, and Anaerococcus had Gini indexes higher than 1. Further, linear discriminant analysis (LDA) demonstrated that the abundance of Lactobacillus crispatus in the IP group was lower than in the CN group (LDA >4.0, mean relative abundance 9.19 % vs 54.40 %, P = 0.031). The logistic regression analysis indicated that a decreased abundance of L. crispatus was associated with an increased risk of clinical chorioamnionitis. CONCLUSIONS: The reduction of L. crispatus and increasing trend of specific anaerobic groups are associated with the onset of chorioamnionitis, suggesting their potential value in chorioamnionitis identification. The vaginal microbiota could serve as a useful biomarker for predicting future disease and tailoring surveillance efforts. Additionally, it may present a viable target for developing prevention and therapeutic strategies.

A combined molecular approach utilizing microbial DNA and microRNAs in a qPCR multiplex for the classification of five forensically relevant body fluids.

Body fluid identification is an essential step in the forensic biology workflow that can assist DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the specificity that molecular techniques can achieve; therefore, molecular methods, including microRNA (miRNA) and microbial signature characterization, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase the discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance (blood and semen). This project synergized the benefits of microRNAs and microbial DNA to identify multiple body fluids using DNA extracts. A reverse transcription (RT)-qPCR duplex targeting miR-891a and let-7g was validated, and miR-891a differential expression was significantly different between blood and semen. The miRNA duplex was incorporated into a previously reported qPCR multiplex targeting 16S rRNA genes of Lactobacillus crispatus, Bacteroides uniformis, and Streptococcus salivarius to presumptively identify vaginal/menstrual secretions, feces, and saliva, respectively. The combined classification regression tree model resulted in the presumptive classification of five body fluids with 94.6% overall accuracy, now including blood and semen identification. These results provide proof of concept that microRNAs and microbial DNA can classify multiple body fluids simultaneously at the quantification step of the current forensic DNA workflow.

Effect of Lactobacillus crispatus on Female Reproductive Tract Health and Its Antibacterial and Anti-inflammatory Mechanisms / 中山大学学报(医学科学版)

Lactobacilli are important colonizing bacteria in female reproductive tract， among which Lactobacillus crispatus is closely associated with reproductive tract health and plays a crucial role in maintaining the vaginal microbiota balance. A decrease in the abundance of Lactobacillus crispatus may be correlated with various female reproductive tract diseases， such as bacterial vaginosis and vulvovaginal candidiasis， and it can even lead to adverse pregnancy outcomes such as infertility and embryo arrest. This article provides an overview of the basic characteristics of Lactobacillus crispatus， relationship between the decrease of its quantity and reproductive tract diseases， its ability to inhibit pathogenic bacteria such as Candida albicans and Chlamydia trachomatis， and the anti-inflammatory effects of Lactobacillus crispatus. The aim is to provide references for the use of Lactobacillus crispatus in vaginal therapeutics.

The mobilome of Lactobacillus crispatus M247 includes two novel genetic elements: Tn7088 coding for a putative bacteriocin and the siphovirus prophage ΦM247.

Lactobacillus crispatus is a member of the vaginal and gastrointestinal human microbiota. Here we determined the complete genome sequence of the probiotic strain M247 combining Nanopore and Illumina technologies. The M247 genome is organized in one circular chromosome of 2 336 109 bp, with a GC content of 37.04 % and 2303 ORFs, of which 1962 could be annotated. Analysis of the M247 mobilome, which accounts for 14 % of the whole genome, revealed the presence of: (i) Tn7088, a novel 14 105 bp long integrative and mobilizable element (IME) containing 16 ORFs; (ii) ΦM247, a novel 42 510 bp long siphovirus prophage containing 52 ORFs; (iii) three clustered regularly interspaced short palindromic repeats (CRISPRs); and (iv) 226 insertion sequences (ISs) belonging to 14 different families. Tn7088 has a modular organization including a mobilization module encoding FtsK homologous proteins and a relaxase, an integration/excision module coding for an integrase and an excisionase, and an adaptation module coding for a class I bacteriocin and homologous to the listeriolysin S (lls) locus of Listeria monocytogenes. Genome-wide homology search analysis showed the presence of Tn7088-like elements in 12 out of 23 L. crispatus complete public genomes. Mobilization and integration/excision modules are essentially conserved, while the adaptation module is variable since it is the target site for the integration of different ISs. Prophage ΦM247 contains genes for phage structural proteins, DNA replication and packaging, lysogenic and lytic cycles. ΦM247-like prophages are present in seven L. crispatus complete genomes, with sequence variability mainly due to the integration of ISs. PCR and sequencing showed that the Tn7088 IME excises from the M247 chromosome producing a circular form at a concentration of 4.32×10-5 copies per chromosome, and reconstitution of the Tn7088 chromosomal target site occurred at 6.65×10-4 copies per chromosome. The ΦM247 prophage produces an excised form and a reconstituted target site at a level of 3.90×10-5 and 2.48×10-5 copies per chromosome, respectively. This study identified two novel genetic elements in L. crispatus. Tn7088 represents the first example of an IME carrying a biosynthetic gene cluster for a class I bacteriocin in L. crispatus.

Protective Properties of S-layer Protein 2 from Lactobacillus crispatus 2029 against Candida albicans Infections.

Previously, the protective role of the S-layer protein 2 (Slp2) of the vaginal Lactobacillus crispatus 2029 (LC2029) strain against foodborne pathogens Campylobacter jejuni, Salmonella enterica serovar Enteritidis, and Escherichia coli O157:H was demonstrated. We demonstrate the new roles of the Slp2-positive LC2029 strain and soluble Slp2 against C. albicans infections. We show that LC2029 bacteria can adhere to the surface of the cervical epithelial HeLa cells, prevent their contact with C. albicans, and block yeast transition to a pathogenic hyphal form. Surface-bound Slp2 provides the ability for LC2029 to co-aggregate with various C. albicans strains, including clinical isolates. C. albicans-induced necrotizing epithelial damage is reduced by colonization with the Slp2-positive LC2029 strain. Slp2 inhibits the adhesion of various strains of C. albicans to different human epithelial cells, blocks yeast transition to a pathogenic hyphal form, and prevents the colonization and pathogenic infiltration of mucosal barriers. Only Slp2 and LC2029 bacteria stimulate the production of protective human ß-defensin 3 in various epithelial cells. These findings support the anti-Candida albicans potential of the probiotic LC2029 strain and Slp2 and form the basis for further research on their ability to prevent and manage invasive Candida infections.

Vaginal Microbiota Molecular Profiling in Women with Bacterial Vaginosis: A Novel Diagnostic Tool.

Bacterial vaginosis (BV) is a most common microbiological syndrome. Multiplex next-generation sequencing (NGS) or molecular tests allow a complete and accurate vaginal microbiota profiling in order to determine the primary causative agent. Due to the high costs and limited availability of NGS, the multiplex real-time PCR draws more attention. The present study aimed to evaluate the microbial composition and dominant lactobacilli species in non-pregnant women with bacterial vaginosis using a multiplex RT-PCR test and determine its diagnostic significance. In total, 331 women complaining of vaginal discharge were included. BV was confirmed upon clinical examination and Nugent criteria. A real-time PCR test was carried out with a new Femoflor test, which identifies opportunistic bacteria, STD pathogens, and some viruses. According to the results, the rate of lactobacilli is significantly reduced in BV-affected patients when compared to healthy women. Moreover, the rate of L. crispatus significantly decreases, while the rate of L. iners remains high. Among obligate anaerobic bacteria, Gardnerella vaginalis was the most prevalent in women with BV. The Femoflor test demonstrated high sensitivity and specificity for diagnosing BV. Moreover, the test allows the identification of infection in women with intermediate vaginal microbiota, as well as STD pathogens, and viruses. Thus, the application of real-time PCR tests can be effectively used in vaginal microbiota evaluation in women with BV, intermediate vaginal microbiota, and healthy women. In addition, this test may be used as an alternative to the Amsel criteria and Nugent scoring method in diagnosing BV.

Vaginal microbes alter epithelial transcriptomic and epigenomic modifications providing insight into the molecular mechanisms for susceptibility to adverse reproductive outcomes.

The cervicovaginal microbiome is highly associated with women's health with microbial communities dominated by Lactobacillus spp. being considered optimal. Conversely, a lack of lactobacilli and a high abundance of strict and facultative anaerobes including Gardnerella vaginalis, have been associated with adverse reproductive outcomes. However, the molecular pathways modulated by microbe interactions with the cervicovaginal epithelia remain unclear. Using RNA-sequencing, we characterize the in vitro cervicovaginal epithelial transcriptional response to different vaginal bacteria and their culture supernatants. We showed that G. vaginalis upregulated genes were associated with an activated innate immune response including anti-microbial peptides and inflammasome pathways, represented by NLRP3-mediated increases in caspase-1, IL-1ß and cell death. Cervicovaginal epithelial cells exposed to L. crispatus showed limited transcriptomic changes, while exposure to L. crispatus culture supernatants resulted in a shift in the epigenomic landscape of cervical epithelial cells. ATAC-sequencing confirmed epigenetic changes with reduced chromatin accessibility. This study reveals new insight into host-microbe interactions in the lower reproductive tract and suggest potential therapeutic strategies leveraging the vaginal microbiome to improve reproductive health.

Study of the effect of Lactobacillus crispatus FSCDJY67L3 on Helicobacter Pylori eradication: a double-blind randomized controlled clinical trial.

Helicobacter pylori (H. pylori) is a gram-negative bacterium exhibiting high pathogenicity. Traditional antibiotic treatments are considered ineffective as the H. pylori resistance has increased. Recently, a quadruple therapy strategy of probiotics and antibiotics to eliminate H. pylori was proposed. Probiotics play a therapeutic role as supplements in this process. The present research screened a probiotic strain (Lactobacillus crispatus FSCDJY67L3) that co-aggregates strongly with H. pylori. L. crispatus FSCDJY67L3 was demonstrated to significantly reduce H. pylori load (14C breath test) in clinical trials with H. pylori-positive patients. The Gastrointestinal Symptom Rating Scale (GSRS) score decreased, indicating improvement in the gastrointestinal discomfort of patients. Furthermore, L. crispatus FSCDJY67L3 showed no change in the structure of the intestinal flora of patients. Routine blood indices and blood biochemical indices related to liver and kidney function were also not affected in the patients. Therefore, L. crispatus FSCDJY67L3 may be used clinically as a supplement for the treatment of H. pylori. Clinical Trial Registration: https://www.chictr.org.cn/, Chinese Clinical Trial Registry (ChiCTR2100053710).

Mesh and layered electrospun fiber architectures as vehicles for Lactobacillus acidophilus and Lactobacillus crispatus intended for vaginal delivery.

Bacterial vaginosis (BV) is a recurrent condition that affects millions of women worldwide. The use of probiotics is a promising alternative or an adjunct to traditional antibiotics for BV prevention and treatment. However, current administration regimens often require daily administration, thus contributing to low user adherence and recurrence. Here, electrospun fibers were designed to separately incorporate and sustain two lactic acid producing model organisms, Lactobacillus crispatus (L. crispatus) and Lactobacillus acidophilus (L. acidophilus). Fibers were made of polyethylene oxide and polylactic-co-glycolic acid in two different architectures, one with distinct layers and the other with co-spun components. Degradation of mesh and layered fibers was evaluated via mass loss and scanning electron microscopy. The results show that after 48 h and 6 days, cultures of mesh and layered fibers yielded as much as 108 and 109 CFU probiotic/mg fiber in total, respectively, with corresponding daily recovery on the order of 108 CFU/(mg·day). In addition, cultures of the fibers yielded lactic acid and caused a significant reduction in pH, indicating a high level of metabolic activity. The formulations did not affect vaginal keratinocyte viability or cell membrane integrity in vitro. Finally, mesh and layered probiotic fiber dosage forms demonstrated inhibition of Gardnerella, one of the most prevalent and abundant bacteria associated with BV, respectively resulting in 8- and 6.5-log decreases in Gardnerella viability in vitro after 24 h. This study provides initial proof of concept that mesh and layered electrospun fiber architectures developed as dissolving films may offer a viable alternative to daily probiotic administration.

Role of D(-)-Lactic Acid in Prevention of Chlamydia trachomatis Infection in an In Vitro Model of HeLa Cells.

A vaginal microbiota dominated by certain Lactobacillus species may have a protective effect against Chlamydia trachomatis infection. One of the key antimicrobial compounds produced is lactic acid, which is believed to play a central role in host defense. Lactobacillus strains producing the D(-)-lactic acid isomer are known to exert stronger protection. However, the molecular mechanisms underlying this antimicrobial action are not well understood. The aim of this study was to investigate the role of D(-)-lactic acid isomer in the prevention of C. trachomatis infection in an in vitro HeLa cell model. We selected two strains of lactobacilli belonging to different species: a vaginal isolate of Lactobacillus crispatus that releases both D(-) and L(+) isomers and a strain of Lactobacillus reuteri that produces only the L(+) isomer. Initially, we demonstrated that L. crispatus was significantly more effective than L. reuteri in reducing C. trachomatis infectivity. A different pattern of histone acetylation and lactylation was observed when HeLa cells were pretreated for 24 h with supernatants of Lactobacillus crispatus or L. reuteri, resulting in different transcription of genes such as CCND1, CDKN1A, ITAG5 and HER-1. Similarly, distinct transcription patterns were found in HeLa cells treated with 10 mM D(-)- or L(+)-lactic acid isomers. Our findings suggest that D(-) lactic acid significantly affects two non-exclusive mechanisms involved in C. trachomatis infection: regulation of the cell cycle and expression of EGFR and α5ß1-integrin.