DNA fingerprinting of Lactobacillus crispatus strain CTV-05 by repetitive element sequence-based PCR analysis in a pilot study of vaginal colonization

Abstract

Lactobacillus crispatus is one of the predominant hydrogen peroxide (H(2)O(2))-producing species found in the vagina and is under development as a probiotic for the treatment of bacterial vaginosis. In this study, we assessed whether DNA fingerprinting by repetitive element sequence-based PCR (rep-PCR) can be used to distinguish the capsule strain of L. crispatus (CTV-05) from other endogenous strains as well as other species of vaginal lactobacilli. Vaginal and rectal lactobacilli were identified to the species level by using whole-chromosome probe DNA hybridization. The DNAs from L. crispatus, L. jensenii, L. gasseri, and an as-yet-unnamed H(2)O(2)-negative Lactobacillus species designated 1086V were subjected to rep-PCR. The results of gel electrophoresis and ethidium bromide staining of the DNA fingerprints obtained were compared. L. crispatus CTV-05 had a unique DNA fingerprint compared to all other lactobacilli. DNA fingerprints for 27 production lots of L. crispatus sampled from 1994 through 2001 were identical to that of the original strain isolated in 1993, suggesting strain stability. In a pilot study of nine women, this DNA fingerprinting method distinguished CTV-05 from other endogenous vaginal lactobacilli prior to and after vaginal capsule use. rep-PCR DNA fingerprinting is useful for strain typing and for evaluating longitudinal loss or acquisition of vaginal lactobacilli used as probiotics.

Figures

FIG. 1.

rep-PCR DNA fingerprints generated from Lactobacillus ATCC strains and vaginal isolates. The vaginal isolates were previously identified to the species level by using whole-chromosome probes produced from Lactobacillus ATCC strains. Each gel is representative of isolates having DNA homology to one Lactobacillus species. Clinical isolates within a group of Lactobacillus species were from different women. L. crispatus, L. jensenii, Lactobacillus sp. strain 1086V, and L. gasseri were chosen because of their vaginal prevalence. Lane 1 of each gel contains the 1-kb DNA size ladder. (A) Isolates homologous to the whole-chromosome probe of L. crispatus ATCC 33197. L. crispatus CTV-05 and ATCC 33197 are shown in lanes 2 and 3, respectively. Lanes 4 to 11 show the rep-PCR patterns for L. crispatus isolates from eight women. (B) Isolates homologous to the whole-chromosome probe of L. jensenii ATCC 25258. ATCC 25258 was run in lane 2. Lanes 3 to 12 show the rep-PCR patterns for 10 L. jensenii isolates, all from different women. (C) Isolates homologous to the Lactobacillus sp. strain 1086V whole-chromosome probe. The original Lactobacillus sp. strain 1086V is shown in lane 2. Lanes 3 to 12 show the rep-PCR patterns for Lactobacillus sp. strain 1086V-like isolates from nine women. (D) Isolates homologous to the whole-chromosome probe of L. gasseri ATCC 4963. L. gasseri ATCC 4963 and 9857 are shown in lanes 2 and 3, respectively. The rep-PCR DNA patterns for L. gasseri isolates from eight women are shown in lanes 4 to 11.

FIG. 2.

rep-PCR DNA fingerprints generated from different production lots of L. crispatus CTV-05 gelatin capsules produced from 1995 to 2001(lane 3, LB 045—January 1995; lane 4, LB 096—September 1996; lane 5, LB 112—June 1997; lane 6, LB 124—May 1998; and lane 7, LB 227—2001). The original strain CTV-05, isolated in 1993, was run in lane 2, and a 1-kb DNA size ladder was run in lane 1.

FIG. 3.

rep-PCR DNA fingerprints of lactobacilli isolated from two women in the L. crispatus CTV-05 pilot study. b, baseline visit; f1 and f2, follow-up visits 1 and 2, respectively. Each lane represents a different colony type of lactobacilli. For each gel, lane 1 is a 1-kb DNA size ladder and lane 2 is a control DNA fingerprint of L. crispatus CTV-05. (A) Patient 107. An H2O2-producing strain of L. crispatus was present at the baseline visit (lane 3) and at the first (lanes 4 to 6 and lane 8) and second (lane 9) follow-up visits. L. crispatus CTV-05 was not detected at the first follow-up visit but was detected at the third visit (lanes 11 and 12), suggesting that strain CTV-05 was present at lower levels than endogenous L. crispatus strains at the first follow-up visit. A third Lactobacillus strain having a fingerprint closely resembling the DNA fingerprints of strains homologous to L. jensenii ATCC 25258 (Fig. 1B, lane 2) was also detected at the second (lane 7) and third (lane 10) visits. (B) Patient 102. An H2O2-producing Lactobacillus strain was detected at the baseline visit (lanes 3 and 4) and at the second (lane 5) and third (lane 10) visits. Although the two H2O2-producing colony types from the baseline visit differed phenotypically, they had identical rep-PCR DNA fingerprints. Two additional strains, an H2O2-negative strain (lane 6) and L. crispatus strain CTV-05 (lane 7), were detected at the first follow-up visit. All three strains were detected at the second follow-up visit (lanes 8 to 11).