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.

Lactobacilluscrispatus dominance in the vaginalmicrobiome reduces the occurrence of spontaneouspreterm birth in women with a short cervical length / Dominância de Lactobacillus crispatus no microbioma vaginal reduz a ocorrênciade parto prematuro espontâneo em mulheres com encurtamento do colo uterino

Introduction: The majority of pregnant women with a short cervix will deliver at term and, thus, may unnecessarily receive advanced monitoring and treatment. It is still necessary to define more accurately which sub-population of women with a short cervix is at elevated risk for early delivery. Objective: To determine if vaginal microbiome composition influenced the rate of spontaneous preterm birth in women with a short cervical length. Methods: In an exploratory, observational prospective study, vaginal secretions were obtained from 591 women at 21­24 week gestation. Vaginal microbiome composition was determined by analyzing the V1­V3 region of the bacterial 16S ribosomal RNA gene. Results: Lactobacillus crispatus was numerically dominant in the vagina in 41.7% of subjects, followed by L. iners in 32% and Gardnerella vaginalis in 12%. In women whose cervix was ≤25mm, the sensitivity to predict an spontaneous preterm birth was 11.8%. However, when L. crispatus was not the dominant vaginal bacterium, this sensitivity increased to 81.8%. Similarly, in women with a cervical length ≤30mm, the sensitivity to predict an spontaneous preterm birth increased from 21.7 to 78.3% when L. crispatus was not the dominant vaginal bacterium.In women with a prior spontaneous preterm birth and a cervix ≤25 or ≤30mm, L. crispatus dominance was also associated with a reduced rate of spontaneous preterm birth in the current pregnancy (p<0.001). Conclusion: In pregnant women with a cervix ≤25mm or ≤30mm, the risk for an spontaneous preterm birth is increased if L. crispatus is not dominant in the vagina.

Introdução: A maioria das mulheres grávidas com colo do útero curto dará à luz a termo e, portanto, pode receber desnecessariamente monitoramento e tratamento avançados. Permanece a necessidade de definir com mais precisão qual subpopulação de mulheres com colo do útero curto está em risco elevado de parto prematuro. Objetivo: Determinar se a composição do microbioma vaginal influenciou a taxa de parto prematuro espontâneo em mulheres com colo curto. Métodos: Em um estudo prospectivo exploratório observacional, os conteúdos vaginais foram obtidos de 591 mulheres com 21­24 semanas de gestação. A composição do microbioma vaginal foi determinada pela análise da região V1­V3 do gene de RNA ribossômico bacteriano 16S. Resultados: Lactobacilluscrispatus foi numericamente dominante na vagina em 41,7% dos indivíduos, seguido por L. iners em 32% e Gardnerella vaginalis em 12%. Em mulheres cujo colo do útero era <25 mm, a sensibilidade para prever uma taxa de parto prematuro espontâneo foi de 11,8%. No entanto, quando L. crispatus não era a bactéria vaginal dominante, essa sensibilidade aumentou para 81,8%. Da mesma forma, em mulheres com comprimento cervical <30 mm, a sensibilidade para prever uma taxa de parto prematuro espontâneo aumentou de 21,7 para 78,3% quando L. crispatus não era a bactéria vaginal dominante. Em mulheres com taxa de parto prematuro espontâneo anterior e colo do útero <25 ou <30 mm, a dominância de L. crispatus também foi associada a uma taxa reduzida de taxa de parto prematuro espontâneo na gravidez atual (p<0,001). Conclusão: Em mulheres grávidas com colo do útero <25 ou <30 mm, o risco de parto prematuro espontâneo é aumentado se L. crispatus não for dominante na vagina.

Identifying probiotic characteristics of Lactobacillus crispatus isolated from the vagina

Lactobacilli prevent overproduction of pathogenic microorganisms and contribute protecting vaginal microbiota. Many probiotic microorganisms are categorized as Lactic Acid Bacteria. In this study, it was aimed identifying probiotic characteristics of Lactobacillus crispatus isolated from the vagina of a healthy woman. For this purpose, lactic acid, hydrogen peroxide and proteolytic activity quantities and auto-aggregation, co-aggregation and hydrophobicity abilities of Lactobacillus crispatus, which has been isolated and identified by 16s rRNA sequence analysis, were determined. Additionally, bile salt and acid resistance, along with antibiotic susceptibility of Lactobacillus crispatus were analyzed by the end of 3 hours. Lactic acid, hydrogen peroxide and proteolytic activity quantities of Lactobacillus crispatus were measured 2.275%, 0.334±0.075 µg/mL and 2.131±0,000 mg/mL respectively. The findings include existence of co-aggregation and auto-aggregation ability, but not hydrophobicity. By the end of 3 hours, the viability was preserved in 0.1% and 0.3% bile salt medium and, at pH 3. L. crispatus exhibited resistance to methicillin, metronidazole, oxacillin, and sulfamethoxazole + trimethoprim, but the bacteria exhibited susceptibility to tested the other antibiotics. This study will make an important contribution to the literature about probiotic characteristics of L. crispatus and our strain isolated from the vagina might be considered as a candidate probiotic.

Scanning electron microscopic observation of interactions of vaginal epithelial cells with Candida albicans and Lactobacillus crispatus / 中华妇产科杂志

Objective To investigate interactions of vaginal epithelial cells (VEC) with Candida albicans and the protective effect of Lactobacillus crispatus on VEC. Methods VK2/E6E7 VEC cell line was cultured. Candida albicans and Lactobacillus crispatus suspension were prepared respectively. Ultrastructural morphological changes of VEC infected with Candida albicans and treated with Lactobacillus crispatus were observed by scanning electron microscope (SEM). Results Candida albicans invaded into VEC mainly through endocytosis after an early infection of 6 hours. Pseudopods of VEC increased, contacted and covered the surface of pseudohyphae. After 12 hours infection, active penetration developed into the predominant invasion mechanism. Candida albicans directly penetrated through VEC, destroyed the surface structures, and even led to cell death. A reduction of pseudopods on VEC was observed. After co-incubation with Lactobacillus crispatus, SEM revealed a morphological recovery of VEC and destruction of pseudohyphae. Conclusions Candida albicans invade VEC mainly through endocytosis and active penetration. Lactobacillus crispatus could utilize occupying effect to exert protective effect on VEC during the infection with Candida albicans.

Scanning electron microscopic observation of interactions of vaginal epithelial cells with Candida albicans and Lactobacillus crispatus / 中华妇产科杂志

Objective To investigate interactions of vaginal epithelial cells (VEC) with Candida albicans and the protective effect of Lactobacillus crispatus on VEC. Methods VK2/E6E7 VEC cell line was cultured. Candida albicans and Lactobacillus crispatus suspension were prepared respectively. Ultrastructural morphological changes of VEC infected with Candida albicans and treated with Lactobacillus crispatus were observed by scanning electron microscope (SEM). Results Candida albicans invaded into VEC mainly through endocytosis after an early infection of 6 hours. Pseudopods of VEC increased, contacted and covered the surface of pseudohyphae. After 12 hours infection, active penetration developed into the predominant invasion mechanism. Candida albicans directly penetrated through VEC, destroyed the surface structures, and even led to cell death. A reduction of pseudopods on VEC was observed. After co-incubation with Lactobacillus crispatus, SEM revealed a morphological recovery of VEC and destruction of pseudohyphae. Conclusions Candida albicans invade VEC mainly through endocytosis and active penetration. Lactobacillus crispatus could utilize occupying effect to exert protective effect on VEC during the infection with Candida albicans.

The Growth Inhibition Effect on the Causative Bacteria of Bacterial Vaginosis by Bacterial Strains Isolated from the Vagina of a Healthy Woman

Two Gram-positive rod strains isolated from the healthy vagina of a woman were tested for the possibility as probiotics. One strain was identified as Steroidobacter denitrificans (YH1) and the other as Lactobacillus crispatus (YH2) by 16S rRNA partial sequencing. The Casman agar and Man-Rogosa-Sharpe (MRS) agar were mixed in same quantity, supplemented with 5% human rbc lysate (CMB agar). The Wilkins-Chalgren agar and MRS agar were mixed in same quantity (WCM agar). Gardnerella vaginalis was cultured in Casman broth, supplemented with 5% human rbc lysate and 1,000 x-diluted with normal saline. Bacteroides fragilis, Mobiluncus mulieris and Peptostreptococcus asaccharolyticus were cultured in Wilkins-Chalgren anaerobe broth and 2,000x-diluted. S. denitrificans YH1 and L. crispatus YH2 were cultured in MRS broth anaerobically and 100x-diluted. The diluted suspensions of B. fragilis, M. mulieris and P. asaccharolyticus were inoculated on WCM agar and G. vaginalis on CMB agar by cotton swabs. Ten microl aliquots of YH1 and YH2 were inoculated on the center of WCM agar and CMB agar. The growth inhibition zone diameters of B. fragilis, G. vaginalis, M. mulieris and P. asaccharolyticus by YH1 were 35 mm, 35 mm, 25 mm and 60 mm. The inhibition diameters by YH2 were 25 mm, 30 mm, 20 mm and 40 mm, respectively. These results implicate that S. denitrificans YH1 can be the stronger probiotics for the treatment of bacterial vaginosis than L. crispatus, compared inhibition zone diameters by YH1 and YH2.

Microbiologic and Molecular Genetic Analysis of Lactobacillus spp. Isolated from Vagina of Korean Women and A Pilot Clinical Study on the Treatment of Vaginitis using the Best Lactobacillus Strain KLB 46 / 대한산부인과학회잡지

OBJECTIVE: The aim of this study was to analize the biologic and molecular genetic characteristics of Lactobacillus spp. isolated from vagina of Korean healthy women and to perform a pilot clinical study on the treatment of vaginitis using the best Lactobacillus strain KLB 46. METHODS: Vaginal specimens were obtained from 205 Korean women with healthy vaginal ecosystems. 155 lactobacillus strains were used for catalase test, cell surface hydrophobicity (CSH) test and antimicrobial activity test. Also we identified the species prevalence by PCR-RFLP analysis of 16S rDNA gene and performed the minimal inhibitory concentrationtest using14 antibiotics to determine their patterns of antibiotic susceptibility. A pilot clinical trial of 8 bacterial vaginosis, 1 trichomonas vaginitis and 6 atrophic vaginitis using Lactobacillus crispatus KLB 46 was done by vaginal instillation (10(10)-10(11) cells/mL). RESULTS: 155 lactobacilli strains were isolated and all isolates showed high CSH and 61% of the isolates had higher CSH. L. acidophilus and L. crispatus were the most prevalent species by PCR-RFLP analysis of their 16S rDNA gene. And PCR-RFLP analysis suggested that KLB 46 was L. crispatus. The clinical study showed that the vaginal epithelial adherence rate was high as 77% for bacterial vaginosis and 33.3% for atrophic vaginitis. CONCLUSION: The predominating lactobacilli were L. crispatus and L. acidophilus in Korean women's vaginas. Although further study would be needed, L. crispatus KLB 46 is a good candidate of suitable probiotics for bacterial vaginosis.