Growth and Acidification by Vaginal Lactobacilli in Anaerobic Liquid Medium Over the pH Range 5.5 – 8.0

Abstract

The flora of the healthy vagina is simple and usually dominated by one or a few species of lactobacilli (LB) [7,10]. LB maintains the acidity of the vagina and are critical for vaginal health. Bacterial vaginosis (BV) is the most prevalent vaginal syndrome with major public health implications worldwide [6] and is characterized by a depletion of vaginal LB. Recurrence of BV is seen in approximately 30% of affected females and LB-containing probiotic preparations have been evaluated for treatment and prevention. A means of quantifying the growth and acidification potential of LB over a range of pH values would be useful for intra-and inter-species comparisons of LB considered as potential probiotic candidates. To address this, we studied three vaginal LB under anaerobic conditions in liquid medium over a range of pHs.A defined liquid medium rendered anaerobic with 'Oxyrase for Broth' (Oxyrase Inc, Ohio, USA) was prepared and used as described previously [11] at initial pHs 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0. In brief, media with and without buffer -and for each of these, with and without 1.25g/L human hemoglobin (Hb) -were adjusted to the required initial pH using 1M HCl before filter sterilizing through a 0.22μm filter. The buffers used were 30mM MES for pH 5.5 -6.5 and 30mM MOPS for pH 7.0 -8.0. Media were pre-reduced in 7mL screwtop plastic bijoux (Bibby Sterilin) by incubation at 37 o C for at least 2h before inoculation [11]. Suspensions of Lactobacillus crispatus NCTC 4505 (LC), Lactobacillus gasseri ATCC 9857 (LG), Lactobacillus jensenii ATCC 25258 (LJ) and Fusobacterium nucleatum ATCC 25586 (FN) in phosphate-buffered saline (PBS) were prepared such that 100 -200L added to 7mL of medium gave 10 4 cfu/mL. Controls for each medium at each initial pH included: uninoculated media to measure the pH effect of Oxyrase; rezasurin sodium at 0.002g/L as a reduction indicator; and FN to demonstrate ability to support anaerobic growth. Also, each strain was inoculated in to MES-and MOPS-containing media adjusted to pH 6.6 -which is at the overlap of the buffers' ranges -to identify any potential inhibition by the buffers. To provide growth curves for each strain in unbuffered medium, each was inoculated in to medium without buffer adjusted to pH 6.6. Bijoux were incubated statically at 37 o C and counts done after vortexing for 5s on an undiluted aliquot and serial 10 -1 dilutions in PBS on blood agar (Oxoid, UK) using a spiral plater (Don Whitley, Shipley, UK) at inoculation and at 25h, at which time pH was measured. For growth curves, additional samples were taken at 15h and 20h. Plates were incubated in 5% CO 2 for 24 -48h for LB and in anaerobic jars for 48h for FN. All chemicals were from Sigma, Poole, UK and all experiments done at least in triplicate. The log 10 cfu/mL change in colony counts between time 0 and 25h were calculated for all medium-bacterium combinations and the pH change over 25h calculated using the uninoculated Oxyrase-containing control as baseline. For each strain the Wilcoxon Rank-sum test wa...

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