jbv Journal of Bacteriology and Virology J Bacteriol Virol 1598-2467 2093-0249 Journal of Bacteriology and Virology 10.4167/jbv.2015.45.3.228 jbv-45-228 Original Article The Growth Inhibition Effect on the Bacterial Vaginosis Causative Bacteria by Citric Acid and Trisodium Phosphate YangNam-Woong 1 ∗ SihnWan-Jin 2 Department of Microbiology, College of Medicine, Chosun University, Gwangju, Korea Department of Alternative Complementary Medicine, Graduate School Chosun University, Gwangju, Korea Corresponding author: Nam-Woong Yang. Department of Microbiology, College of Medicine, Chosun University, 309 Pilmun-daero Dong-gu Gwangju 501-759, Korea. Phone: +82-62-230-6350, Fax: +82-62-232-3125, e-mail: nwyang@chosun.ac.kr

This study was supported by research fund from Chosun University, 2014.

 22015 922015 453 228234 30052015 16072015 27072015 Copyright © 2015 Journal of Bacteriology and Virology 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract

Bacterial vaginosis (BV) is the most frequent vaginal disease being apt to relapse. The growth inhibition effect of the mixture of citric acid (CA) and trisodium phosphate (TSP) on BV causative bacteria and probiotics was measured. Gardnerella vaginalis was reduced to zero in WCCT-1 (CA 0.25% and TSP 0.55% in Wilkins-Chalgren broth), 2.0 × 104/ml in WCCT-2 (CA 0.5% and TSP 0.8% in WC), and 3.3 × 103/ml in WCCT-3 (CA 1.0% and TSP 2.6% in WC) comparing with 1.3 × 105/ml in WC after 48 h. Bacteroides fragilis was reduced to 6.0 × 103/ml in WCCA (CA 0.34% in WC), 2.3 × 102/ml in WCCT (CA 0.5% and TSP 0.2% in WC), 7.0 × 103/ml in WCHCl (HCl in WC) after 48 h. Mobiluncus mulieris was reduced to 1.08 × 104/ml in WCCA, 1.03 × 103/ml in WCCT, and 10 ea/ml in WCHCl after 48 h. Peptostreptococcus asaccharolyticus was completely inhibited in WCCA, WCCT, and WCHCl after 24 h. Probiotics, Steroidobacter denitrificans YH1 (3.4 × 107/ml) and Lactobacillus crispatus YH2 (2.7 × 106/ml), grew to 1.25 × 108/ml and 2.6 × 107/ml in MRSCA (CA 1.0% in MRS), 1.8 × 107/ml and 4.6 × 106/ml in MRSCT (CA 1.5% and TSP 0.58% in MRS), 1.2 × 108/ml and 2.3 × 107/ml in MRSHCl after 48 h, respectively. These results mean that the CA-TSP mixture can be used as the useful vaginal pH controller, growth inhibitor on BV causative bacteria, and an efficient means for settlement of probiotics.

 Bacterial vaginosis Citric acid Trisodium phosphate Probiotics REFERENCES Dawson SG Harris JR Gardnerella vaginalis and nonspecific vaginitis Br J Hosp Med 198329 2837 Joesoef MR Schmid G Bacterial vaginosis Clin Evid 200513 196878 Sobel JD Bacterial vaginosis –an ecologic mystery Ann Intern Med 1989111 5513 Spiegel CA Bacterial vaginosis Clin Microbiol Rev 1991485-502 Borchardt KA Adly BS Smith RF Eapen J Beal CB Importance of Gardnerella vaginalis as an aetiological agent in bacterial vaginosis Genitourin Med 198965 285 Sihn WJ Yang NW The growth inhibition effect on the causative bacteria of bacterial vaginosis by bacterial strains isolated from the vagina of a healthy woman J Bacteriol Virol 201444 24451 Yang NW Lim Y Sin SH Drug-resistant profiles of clinical isolates of Gardnerella vaginalis on Columbia agar base supplemented with human erythrocyte lysate and horse serum Infect chemother 200335 8690 Yang NW Kim JM Choi GJ Jang SJ Development and evaluation of the quick anaero-system: A new disposable anaerobic culture system Korean J Lab Med 201030 1337 Hillier SL Nugent RP Eschenbach DA Krohn MA Gibbs RS Martin DH Association between bacterial vaginosis and preterm delivery of a low-birth-weight infant. The vaginal infections and prematurity study group N Engl J Med 1995333 173742 Watts DH Eschenbach DA Kenny GE Early postpartum endometritis: the role of bacteria, genital mycoplasmas, and Chlamydia trachomatis Obstet Gynecol 198973 5260 Mania-Pramanik J Kerkar SC Salvi VS Bacterial vaginosis: a cause of infertility? Int J STD AIDS 200920 77881 Menard JP Mazouni C Salem-Cherif I Fenollar F Raoult D Boubli L High vaginal concentrations of Atopobium vaginae and Gardnerella vaginalis in women undergoing preterm labor Obstet Gynecol 2010115 13440 Atassi F Brassart D Grob P Graf F Servin AL Lactobacillus strains isolated from the vaginal microbiota of healthy women inhibit Prevotella bivia and Gardnerella vaginalis in coculture and cell culture FEMS Immunol Med Microbiol 200648 42432 Skarin A Sylwan J Vaginal lactobacilli inhibiting growth of Gardnerella vaginalis, Mobiluncus and other bacterial species cultured from vaginal content of women with bacterial vaginosis Acta Pathol Microbial Immunol Scand B 198694 399403 Sethi S Das A Sharma M Inhibition of Gardnerella vaginalis by lactobacilli Int J Gynecol Obstet 200693 1589 Anukam KC Osazuwa E Osemene GI Ehigiagbe F Bruce AW Reid G Clinical study comparing probiotic Lactobacillus GR-1 and RC-14 with metronidazole vaginal gel to treat symptomatic bacterial vaginosis Microbes Infect 20068 27726 Saunders S Bocking A Challis J Reid G Effect of Lactobacillus challenge on Gardnerella vaginalis biofilms Colloids Surf B Biointerfaces 200755 13842 Machado A Jefferson KK Cerca N Interactions between Lactobacillus crispatus and bacterial vaginosis (BV)-associated bacterial species in initial attachment and biofilm formation Int J Mol Sci 201314 1200412 Ray B Sandine WE Acetic, propionic, and lactic acids of starter culture bacteria as biopreservatives Ray B. Baeschel M. Boca Raton, FL, USA CRC Press 1991 Capita R Alonso-Calleja C Prieto M García-Fernández Mdel C Moreno B Effectiveness of trisodium phosphate against Listeria monocytogenes on excised and non-excised chicken skin J Food Prot 200366 614 Ismail SA Dea T Abd El-Rahman H Yassien MA Beuchat LR Effectiveness of immersion treatments with acids, trisodium phosphate, and herb decoctions in reducing populations of Yarrowia lipolytica and naturally occurring aerobic microorganisms on raw chicken Int J Food Microbiol 200164 139 Tables

Growth rate of G. vaginalis according to media supplements

Media\hr 241 48
WCCT-12 6.0 × 105/ml NG
WCCT-23 4.2 × 105/ml 2.0 × 104/ml
WCCT-34 4.2 × 105/ml 3.3 × 103/ml
Control5 1.29 × 108/ml 1.3 × 105/ml

Counts of inittial bacterial inoculation are 2.3 × 106/ml

WC broth conntaining 0.25% CA and 0.55% TSP, pH 6.27

WC broth conntaining 0.5% CA and 0.8%% TSP, pH 6.27

WC broth conntaining 1% CA and 2.6% TTSP, pH 6.27

WC broth conntrol, pH 6.92

Growth rate of B. fragilis according to media suppleme

Media/hr 241 48
WCCA2 1.6 × 105/ml 6.0 × 103/ml
WCCT3 1.3 × 104/ml 2.3 × 102/ml
WCHCl4 6.2 × 105/ml 7.0 × 103/ml
Control5 2.2 × 109/ml 1.4 × 109/ml

Counts of initial bacterial inoculation are 7.0 × 108/ml

WC broth containing 0.34% CA, pH 4.2

WC broth containing 0.5% CA and 0.2% TSP, pH 4.2

WC broth controlled by 5 N HCl to pH 4.2

WC broth control, pH 6.92

Growth rate of M. mulieris according to media sup plements

Media/hr 241 48
WCCA2 7.6 × 104/ml 1.08 × 104/ml
WCCT3 1.5 × 103/ml 1.03 × 103/ml
WCHCl4 30 ea/ml 10 ea/ml
Control5 3.6 × 108/ml 4.6 × 109/ml

Counts of initial bacterial inoculation are 3.2 × 108/ml

WC broth containing 0.34% CA, pH 4.2

WC broth containing 0.5% CA and 0.2% TSP, pH 4.

WC broth controlled by 5 N HCl to pH 4.2

WC broth control, pH 6.92

Growth rate of P. asaccharolyti supplements

Media/hr 241 48
WCCA3 NG2 NG
WCCT4 NG NG
WCHCl5 NG NG
Control6 5.5 × 106/ml 1.0 × 107/ml

Amounts of initial bacterial inoculation are 3.0 × 106/ml

No growth

WC broth containing 0.34% CA, pH 4.2

WC broth containing 0.5% CA and 0.2% TSP, pH 4.2

WC broth controlled by 5 N HCl to pH 4.2

WC broth control, pH 6.92

Growth supplements rate of S. denitrificans YH1 according to media

Media/hr 241 48
MRSCA2 6.8 × 106/ml 1.25 × 108/ml
MRSCT3 6.7 × 105/ml 1.8 × 107/ml
MRSHCl4 1.2 × 108/ml 1.2 × 108/ml
MRS5 1.9 × 108/ml 2.0 × 108/ml

Counts of initial bacterial inoculation are 3.4 × 107/ml

MRS broth containing 1% CA, pH 4.2

MRS broth containing 1.5% CA and 0.6% TSP pH 4.2

MRS broth controlled to pH 4.2 by 5 N HCl

MRS broth control, pH 5.72

Growth rate of L. crispatus YH2 according to media supplements

Media/hr 241 48
MRSCA2 2.5 × 103/ml 2.6 × 107/ml
MRSCT3 3.5 × 105/ml 4.6 × 106/ml
MRSHCl4 1.33 × 106/ml 2.3 × 107/ml
MRS5 3.0 × 107/ml 7.0 × 106/ml

Amounts of initial bacterial inoculation are 2.7 × 106/ml

MRS broth containing 1% CA, pH 4.2

MRS broth containing 1.5% CA and 0.6% TSP pH 4.2

MRS broth controlled by 5 N HCl to pH 4.2

MRS broth control, pH 5.7