구연산 및 제3인산나트륨과 probiotics에 의한 세균성 질증 원인균들에 대한 성장 억제 효과

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Growth Inhibition Effect on Bacterial Vaginosis Causative Bacteria by Citric Acid, Trisodium Phosphate and Probiotics

Sihn Wan-Jin
Advisor: Prof. Yang, Nam-Woong
Department of Alternative
Complementary Medicine,
Graduate School Chosun University

Objective: Bacterial vaginosis is the most frequent vaginal disease in sexually active women and apt to relapse. We have no therapeutic means for perfect cure of bacterial vaginosis yet. This research aims to develop a new therapeutic means of bacterial vaginosis using the mixture of probiotics, citric acid (CA), trisodium phosphate (TSP) and antibiotics.
Methods: 16S rRNA partial sequencing was used for identification of two Gram(+) bacillus strains isolated from a healthy woman vagina. Probiotic effects of both strains was measured by the similar one to antibiotic disc diffusion method. The growth inhibition effect of the mixture of citric acid and trisodium phosphate on bacterial vaginosis causative bacteria and probiotics was measured by spread plate method. Antibiotic disc diffusion method was used for the selection of antibiotic susceptible to Gardnerella vaginalis but resistant to probiotics.
Results: Two Gram(+) bacilli were identified as Steroidobacter denitrificans (YH1) and Lactobacillus crispatus (YH2), respectively. Probiotic effect of S. denitrificans YH1 is stronger than L. crispatus YH2. The colony counts of G. vaginalis (ATCC 14018) was reduced to 6.0 x 105/㎖ after 24 h and to zero after 48 h incubation from inoculating counts of 2.3 x 106 in media containing CA 250㎎ and TSP 550㎎. The growth of Peptostreptococcus asaccharolyticus (KCTC 3321) was completely inhibited in media containing CA 500㎎ and TSP 200㎎ after 24 h incubation. The colony counts of Bacteroides fragilis (ATCC 25285) was reduced to 2.3 x 102/㎖ from inoculating counts of 7.0 x 108 after 48 h incubation in the same media with P. asaccharolyticus. The colony counts of Mobiluncus mulieris (ATCC 35239) was reduced to 1.5 x 103/㎖ from inoculating counts of 3.2 x 108 after 24 h incubation in the same media with P. asaccharolyticus. The colony counts of S. denitrificans YH1 was decreased to 6.7 x 105/㎖ from inoculating counts of 3.4 x 107 after 24 h incubation but increased to 1.8 x 107/㎖ after 48 h. The colony counts of L. crispatus YH2 was decreased to 3.5 x 105/㎖ from inoculating counts of 2.7 x 106 after 24 h incubation but increase to 4.6 x 106/㎖ after 48 h. The antibiotic, which was susceptible to G. vaginalis but resistant to both probiotics, was amoxicillin, and the inhibition zone diameter was 24㎜.
Conclusion: The results of this study suggest that the proper combination of citric acid, trisodium phosphate, S. denitrificans, L. crispatus and amoxicillin would be most likely a new therapeutic means for the perfect cure of bacterial vaginosis.

Key words: bacterial vaginosis, probiotics, citric acid, trisodium phosphate
Alternative Title
Growth Inhibition Effect on Bacterial Vaginosis Causative Bacteria by Citric Acid,Trisodium Phosphate and Probiotics
Alternative Author(s)
Sihn wan jin
일반대학원 보완대체의학과
Table Of Contents
목 차

표 목차 iii

도 목차 ⅳ
I. 서 론 3
II. 재료 및 방법 5
A. 건강한 여성에서 분리된 그람 양성 간균의 동정 5
B. G.vaginalis의 배양을 위한 사람 적혈구 용해액의 제조 5
C. 실험에 사용된 세균성 질증의 원인 균주들 5
D. 한국 미생물 보존센터에서 동정된 steroidobacter denitrificans YH1과 Lactobacillus crispatus YH2의 probiotic 효과 6
1. G.vaginalis에 대한 probiotic 효과 6
2. B.fragils, M. mulieris, P.asaccharolyticus에 대한 6
E. Citric acid 와 trisodium phosphate 에 의한 7
1. G.vaginalis에 대한 성장 억제 7
2. B.fragils, M. mulieris, P.asaccharolyticus에대한 성장 억제 7
3. Probiotics 에 대한 성장 억제 8
F. G.vaginalis 표준균주와 probiotics들의 항균제 감수성 검사 8

III. 결과 9
A. 한국 미생물 보존센타(KCCM)의 균주 동정 결과 9
B. Steroidobacter denitrificans YH1과 Lactobacillus crispatus YH2의 probiotic 효과 11
C. Citric acid와 trisodium phosphate에 의한 세균성 질증 원인균들의 성장 억제 효과 12

1. G.vaginalis에 대한 성장 억제 효과 12
2. P.asaccharolyticus에 대한 성장 억제 효과 14
3. B.fragilis 에 대한 성장 억제 효과 16
4. M. mulieris 에 대한 성장 억제 효과 18
5. S. denitrificans YH1에 대한 성장 억제 효과 20
6. L. crispatus YH2에 대한 성장 억제 효과 22
7. G.vaginalis 표준균주와 probiotics들의 24

Ⅳ. 고 찰 25
Ⅴ. 결 론 28
참 고 문 헌 29

표 목차

Table 1. Mean diameters of growth inhibition zones by S. denitrificans YH1 and
L. crispatus YH2 strains 11
Table 2. Growth rate of G. vaginalis according to media supplements 13
Table 3. Growth rate of P. asaccharolyticus according to media supplements 15
Table 4. Growth rate of B. fragilis according to media supplements 17
Table 5. Growth rate of M. mulieris according to media supplements 19
Table 6. Growth rate of S. denitrificans YH1 according to media supplements 21
Table 7. Growth rate of L. crispatus YH2 according to media supplements 23
Table 8. Growth inhibition zone diameters of G. vaginalis (ATCC 14018) and probiotics by disc diffusion method 24

도 목차

Figure 1. Base sequence of S. denitrificans YH1 strain has the 99% similarity to accession No. EF605262 strain of KCCM gene bank

Figure 2. Base sequence of L. crispatus YH2 strain has the 99% similarity to accession No. AF257097 strain of KCCM gene bank

Figure. 3. Logarithmic counts of Gardnerella vaginalis in each media

Figure 4. Logarithmic counts of Peptostreptococcus asaccharolyticus in each media

Figure 5. Logarithmic counts of Bacteroides fragilis in each media

Figure 6. Logarithmic counts of Mobiluncus mulieris in each media

Figure 7. Logarithmic counts of Steroidobascter denitrificans YH1 in each media

Figure 8. Logarithmic counts of Lactobacillus crispatus YH2 in each media
신완진. (2014). 구연산 및 제3인산나트륨과 probiotics에 의한 세균성 질증 원인균들에 대한 성장 억제 효과.
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