对阴道加德纳菌、白色假丝酵母菌有抑制作用的人阴道乳酸杆菌的筛选和功能

Initial screening and functional identification of vaginal lactobacilli with the ability of inhibiting Gardnerella vaginalis and Candida albicansor

目的从健康妇女阴道分泌物中分离并筛选出对阴道加德纳菌(Gv)或白色假丝酵母菌(Ca)具有抑菌活性的乳酸杆菌,并观察其功能。方法采集30例健康育龄妇女的阴道分泌物,经数次增菌培养-平板分离后选出疑似乳酸杆菌菌株,采用16S r DNA基因测序法确定菌株类型。采用双层琼脂扩散法评价其中的乳酸杆菌菌株对指示菌(10种细菌和5种假丝酵母菌)的抑菌效果,筛选出具有抑Gv/Ca活性的人阴道乳酸杆菌。采用共孵育实验定量观察筛选出的人阴道乳酸杆菌对Gv/Ca的抑菌活性,同时观察筛选出的乳酸杆菌菌株的产过氧化氢、产乳酸能力,以及自聚合/共聚合能力和生物膜形成能力。结果 30例健康女性阴道分泌物中分离出51株菌,其中23株具有乳酸杆菌特征。16S r DNA基因测序确定其均为乳酸杆菌。双层琼脂扩散实验结果显示,共有8个菌株对Gv/Ca有抑菌作用:包括仅抑制Gv的2号菌株、仅抑制Ca的7、26和44号菌株以及对同时抑制二者的16、19、31和41号菌株。上述8个菌株即为筛选出的有抑Gv/Ca菌活性的人阴道乳酸杆菌。筛选出的8种乳酸杆菌与Gv共培养时,全部菌株均显示了抑菌活性,其中41号菌株完全抑制了Gv的生长;当用乳酸杆菌培养上清液孵育Gv时,全部菌株培养上清液均显示了明显的抑菌活性。当乳酸杆菌与Ca共培养时,全部菌株均显示了抑菌活性;当乳酸杆菌培养上清液孵育Ca时,全部菌株培养上清液均显示了抑菌活性,其中19号菌株完全抑制了Ca的生长。8个乳酸杆菌菌株p H值、乳酸生成量和过氧化氢生成量与阴性对照菌株相比,P均<0.05。其中以19号和41号菌株的产酸和产过氧化氢能力最强,达到了阳性对照菌株水平(P均>0.05)。Gv的自聚合水平为45.25%,而Ca的自聚合水平则高达100%。乳酸杆菌菌株16号(92.57%)、19号(96.32%)、31号(92.16%)和41号(93.52%)均显示了明显的自聚合能力。19号、26号和41号菌株显示了与Ca较强的共聚合能力(定性均为++,定量分别为61.23%、58.72%和54.59%),其他菌株与Ca的共聚合水平为28.53%~44.21%。16号、19号和41号菌株显示了与Gv较强的共聚合能力(定性均为++,定量分别为82.34%、83.41%和93.56%),其他菌株与Ca的共聚合水平为41.33%~46.12%。筛选出的8种乳酸杆菌均能够形成生物膜,其中19号菌株最强,其次是41号菌株。结论成功筛选出8个有抑Gv/Ca活性的人阴道乳酸杆菌菌株。8个菌株的乳酸杆菌均对Gv/Ca有抑菌作用,且有较强的产过氧化氢、乳酸和形成生物膜的能力。其中19、41号菌株具有成为新的阴道益生菌的潜力。

Objective To identify lactobacilli that can inhibit Gardnerella vaginalis（ Gv） and Candida albicansor（ Ca） through isolation and screening from the vaginal secretion of healthy women and to evaluate their properties. Methods Vaginal swab samples were collected from 30 healthy women of childbearing age. The suspected lactobacillus strains were selected after several enriched culture-plate separation,and the strain type was determined by 16 S rDNA sequencing.The inhibitory effect of lactobacillus strains on the indicator bacteria（ 10 kinds of bacteria and 5 kinds of candida albicans）was evaluated by double layer agar diffusion assay,and the human vaginal lactobacillus with inhibitory Gv/Ca activity was screened out. The antimicrobial activity of the screened human vaginal lactobacillus was studied by coincubating Gv and Ca with cultures or supernatants of selected lactobacillus strains. Moreover,the hydrogen peroxide production capacity,the lactic acid production capacity, auto-aggregation and coaggregation and the biofilm formation ability of the lactobacillus strains screened out were observed. Results（1） Fifty-one strains were isolated from the vaginal secretion of 30 healthy women,and 23 strains had the characteristics of lactobacillus. 16 S rDNA sequencing confirmed that all of them were lactobacillus.（2） Eight strains were selected based on their antagonist potential against Gv and/or Ca,including： strain 2 that only inhibited Gv,strains 7,26 and 44 that only inhibited Ca,and strains 16,19,31,and 41 that inhibited both simultaneously. The above 8 strains were screened for the human vagina lactobacillus with inhibitory activity on Gv/Ca. Further,through co-cultivation of the strains and co-culture of the supernatants,strains 2,7,16,31,and 41 all showed antibacterial activity against Gv,and strain 41 completely inhibited the growth of Gv. Similarly,strains 7,16,19,26,31,41 and44 all had antibacterial activity against Ca; strain 19 supernatant completely inhibited the growth of Ca.（3） Statistical difference was found in the pH value,lactic acid generation and H2O2 generation of 8 lactobacillus strains as compared with those of negative controls（ all P〈0. 05）. The strains 19 and 41 had the strongest acid production and H2O2 ability,and reached the level of positive control strains（ both P〉0. 05）.（4） The auto-aggregation ability of Gv was 45. 25%,while that of Ca was as high as 100%. Lactobacillus strains 16（ 92. 57%）,19（ 96. 32%）,31（ 92. 16%） and 41（ 93. 52%）showed significant auto-aggregation ability.（5） Strains 19,26 and 41 showed relatively strong co-aggregation with Ca（ qualitative was ＋ ＋,quantitative was 61. 23%,58. 72% and 54. 59%,respectively）,and the co-aggregation ability of other strains with Ca was 28. 53%-44. 21%. Strains 16,19,and 41 showed relatively strong co-aggregation with Gv（ qualitative was ＋ ＋,quantitative was 82. 34%,83. 41% and 93. 56,respectively）,and the co-aggregation ability of other strains with Gv was 41. 33%-46. 12%.（6）The 8 strains of lactobacillus were able to form biofilms,of which,strain 19 were strongest,followed by strain 41. Conclusions Eight strains of human vaginal lactobacillus with inhibitory activity on Gv/Ca are successfully screened out. All 8 strains of lactobacillus have antibacterial effect on Gv/Ca,and have strong ability to produce hydrogen peroxide,lactic acid,and biofilm formation. Among them,strains 19 and 41 have the potential to become new vaginal probiotics.