A Chinese rhesus macaque (Macaca mulatta) model for vaginal Lactobacillus colonization and live microbicide development

Abstract

Background: We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates.

Methods: Vaginal and rectal microflora in Chinese rhesus macaques (Macaca mulatta) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization.

Results: Chinese rhesus macaques harbored abundant vaginal Lactobacillus, with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals.

Conclusions: The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus-based topical microbicides.

Fig. 1

Rep-PCR DNA fingerprint patterns of vaginal and rectal Lactobacillus strains isolated from Chinese rhesus macaques housed at (A) ABL and (B) Southern Research Institute. (A) Lanes 1 and 19, a 1-kb plus DNA ladder; lane 2, L. johnsonii ABL-V1; lane 3, L. johnsonii ABLV2; lane 4, L. johnsonii ABL-V3; lane 5, L. johnsonii ABL-V4; lane 6, L. johnsonii ABL-V6A; lane 7, L. johnsonii ABL-R6A; lane 8, L. johnsonii ABL-R6B; lane 9, L. murinus ABL-V6B; lane 10, L. murinus ABL-R6C; lane 11, L. johnsonii ABL-V7A; lane 12, L. johnsonii ABL-R7A; lane 13, L. acidophilus ABL-V7B; lane 14, L. acidophilus ABL-R7B; lane 15, L. amylovorous ABL-V7C; lane 16, L. johnsonii ABL-V8A; lane 17, L. johnsonii ABL-R8A; lane 18, L. amylovorous ABL-V8B. (B) lanes 1 and 12, a 1-kb plus DNA ladder; lanes 2–11, all L. johnsonii isolates: lane 2, SRI-V1; lane 3, SRI-R1; lane 4, SRI-V2; lane 5, SRI-V3A; lane 6, SRI-V3B; lane 7, SRI-R3; lane 8, SRI-V4; lane 9, SRI-R4, lane 10, SRI-V5; lane 11, SRI-R5. bp: base pair of DNA fragments.

Fig. 2

Phylogenetic tree showing the relationship based on the analysis of a 1537-nucleotide region of 16S rRNA genes of vaginal Lactobacillus johnsonii from Chinese rhesus macaques to some representative human vaginal Lactobacillus species. The tree was constructed using the Clustalw program. Ljo_135-1 is a human vaginal isolate of L. johnsonii.

Fig. 3

Dynamic nature of endogenous vaginal Lactobacillus johnsonii in Chinese rhesus macaques housed at Southern Research Institute. Vaginal swabs were collected at multiple time points over one menstrual cycle, and the level of endogenous L. johnsonii recovered from each animal was studied. Levels of endogenous lactobacilli varied from 10³ to 10⁷ per swab during the cycle. Arrows indicated sample collection immediately (one day) after menses (animal SRI-1) or during menses (animals SRI-3 and SRI-4). Macaques were not synchronized for their menstrual cycles.

Fig. 4

Vaginal colonization of Lactobacillus in Chinese rhesus macaques. (A) 7-day inoculation of a human vaginal isolate of L. jensenii 1153 in three Chinese rhesus macaques (ABL-1, ABL-2 and ABL-4). Level of Lactobacillus jensenii recovered was analyzed for vaginal swabs collected at 1 and 5 days after the final bacterial inoculation. Lj 1153, L. jensenii 1153; 7 inoculations, 7-day inoculation of L. jensenii 1153. (B) Clearance of vaginal Lactobacillus by a 5-day administration of vaginal suppository of azithromycin, followed by restoration of L. johnsonii in two Chinese rhesus macaques (ABL-1 and ABL-3). Baseline level of vaginal Lactobacillus was analyzed before azithromycin administration. Level of Lactobacillus was monitored for 36 days after the final azithromycin administration. Lactobacillus johnsonii previously isolated from individual macaques was reintroduced intravaginally every other day for three times into each of the two macaques. Level of L. johnsonii was analyzed for vaginal swabs collected at 5 and 12 days after the final bacterial inoculation. AZI, vaginal suppository of azithromycin; 3 inoculations, 3 inoculations (every other day) of L. johnsonii.