应用rep-PCR分型技术筛选潜在治疗性乳杆菌

Selection and genotyping of lactobacillus with potential preventive effect by repetitive element sequence-based PCR analysis

摘要:

分离鉴定阴道弯曲乳酸杆菌并对其进行基因分型分析和产H2O2能力测定，初步筛选具有潜在防治女性生殖道感染的弯曲乳酸杆菌菌株。将健康妇女阴道分泌物接种到de Man，Rogosa and Sharpe (MRS) 培养基，分离培养乳酸杆菌。通过16S rRNA序列进行乳酸杆菌分类鉴定，重复序列片段PCR扩增方法进行弯曲乳酸杆菌的基因分型，并进一步采用pH直接测酸法和辣根过氧化物酶催化四甲基联苯胺与过氧化氢反应显色法检测了10株弯曲乳酸杆菌产酸和产H2O2能力。经过序列比对鉴定，共得到65株乳酸杆菌。其中，弯曲乳杆菌Lactobacillus crispatus 19株，詹氏乳杆菌Lactobacillus jensenii 17株，发酵乳杆菌Lactobacillus fermentum 12株；rep-PCR分型发现不同种类的乳酸杆菌和同一种弯曲乳酸杆菌均表现为不同的带型指纹图；10株弯曲乳酸杆菌均产酸，其中T22-3和T29-5两株弯曲乳酸杆菌产H2O2量最高。结果表明个体阴道内乳酸杆菌分布具有差异，弯曲乳酸杆菌具有种内多样性，产H2O2丰富的T22-3和T29-5两株弯曲乳酸杆菌有可能作为防治女性生殖道感染的有益菌株。

Abstract:

We selected and characterized isolates of Lactobacillus crispatus (L. crispatus) for potential preventing infections of the female reproductive tract. We cultured vaginal swabs from healthy volunteers on de Man, Rogosa and Sharpe (MRS) agar and identified the isolates at the species level by 16S rRNA sequence and genotyped the isolates of Lactobacillus by PCR amplification of repetitive bacterial DNA elements (rep-PCR). Furthermore, 10 L. crispatus strains were assessed for hydrogen peroxide (H2O2) and acid production. Overall 65 isolates were confirmed to be Lactobacillus by sequence analogy, among them 19 were L. crispatus, 17 were Lactobacillus jensenii and 12 were Lactobacillus fermentum. rep-PCR produced specie and strain-specific genomic fingerprints for the Lactobacillus isolates. The selected 10 L. crispatus isolates produced highly acidic environment after growth in MRS. The isolates T22-3 and T29-5 demonstrated high production of H2O2. This study indicated that there are individual differences with vaginal Lactobacillus colonization, and strain diversity within vaginal L. crispatus isolates, T22-3 and T29-5 might be candidates for restoring urogenital health environment in females.