Changes in the Vaginal Microbiota between the First and Final Visits

Final Visits

The vaginal microbiota at the first and final visits were compared. At the first visit, the vaginal samples were dominated by Lactobacillus, which is indicative of vaginal health

during pregnancy141. Samples not dominated by Lactobacillus, were dominated by

organisms associated with BV which was in agreement with the Nugent scores (Table 20 and Figure 20)242. In the only other study that looked at the vaginal microbiota in

pregnancy, a reduction in diversity was observed as compared to non-pregnant individuals, with an increase in Lactobacillus species iners,crispatus, jensenii and

johnsonii141. However, in the present study, sequencing of the V4 region did not allow for

identification of taxa at the species level. Thus, although 29 out of 51 samples having greater than 80% abundance of Lactobacillus were observed, it was not possible to

determine the species composition or to make conclusions about diversity within these samples, as samples were not collected from a non-pregnant population and comparisons between different studies cannot be done due to different methodologies used. Future work could involve examining and comparing the diversity within samples

longitudinally, as vaginal samples were collected every month during gestation.

Furthermore, it would be beneficial to collect age-matched vaginal samples from a non- pregnant subset for comparison in a larger scale study to determine the changes in the vaginal microbiome during pregnancy in a native African population.

The vaginal microbiota significantly changed after giving birth. Alpha diversity

comparisons using the Shannon index revealed that the vaginal microbiota of the women significantly increased in diversity from the first visit to the final visit. Furthermore,

Sneathia and Prevotella dominated the vaginal microbiota at the final visit, which are BV

associated organisms and Prevotella species have also been previously observed as a

dominant BV associated organism in Tanzanian populations88,242. Nugent scoring of the final visit samples did not match the microbiota profiles. In a study conducted out of Bugando Medical Centre in Mwanza, Tanzania, Nugent scoring was performed at birth

and BV was diagnosed in 28.5% of the participants150. Based on the Nugent scores obtained in the current study, 41% of participants had a positive Nugent score at the final visit. A potential reason for the increased prevalence of a positive Nugent score, could be due to the fact a majority of the women gave birth at home, thus the birthing conditions may not have been as sanitary as in a hospital. Based on dominance of Lactobacillus, only two samples were BV negative (Figure 22). OTUs for Lactobacillus were present in every sample at the final visit except for one, and the average abundance at the final visit was 12.7% compared to 70% at the first visit. No other studies have examined the vaginal microbiota of women at birth, thus the shifts observed in the current study may not

necessarily be indicative of disease but may be a normal disruption due to the birthing process. The final visit samples were collected between 8 and 30 days after birth;

however, vaginal samples were collected and sequenced from 3 days after birth. Thus, it would be extremely interesting to examine these samples and compare them to the final visit samples to determine if this increased diversity and altered OTU dominance was also observed at this time point. Furthermore, in order to gain greater understanding on the shifts of the vaginal microbiota due to the birthing process, future studies should collect vaginal samples up to six months or a year after birth to determine when the microbiota shifts back to the pre-pregnancy profile, and the factors that might influence this process.

4.2.5

Supplementation with Moringa Enriched Probiotic Yogurt

May Modulate the Infant Gut Microbiota

Infant fecal samples collected at the final visit had the bacterial DNA extracted and the V4 region of the 16S rRNA gene amplified and sequenced. Studies have shown that the infant gut microbiota during the first 3 years of life contains high interindividual

variability and does not follow a defined developmental pattern due to significant changes caused by life events such as type of feeding, introduction of solid food and antibiotic exposure. However, it is generally accepted that Bifidobacterium becomes the

dominant bacterial genus starting approximately three days after birth128,243,244. The age of the infants analyzed at the final visit were between 10 and 20 days and as seen in Figure

Bifidobacterium appeared to be more abundant in the UNP and NP groups then in any

other group and members of the family Enterobacteriaceae appeared to be more

abundant in the UN and N groups (Figure 23). Studies have shown that members of the family Enterobacteriaceae are normally the dominant organisms at birth in infants born

vaginally and numbers normally decline once Bifidobacterium begins to

dominate96,128,244. ALDEx2 analysis determined that the difference observed at the genus level between the relative abundance of Bifidobacterium and the unknown

Enterobacteriaceae was not significant, most likely due to the low sample numbers.

However, when the actual proportion of sequences aligned to Bifidobacterium and the unknown Enterobacteriaceae were compared between infant fecal samples from mothers who received probiotic yogurt (UNP and NP groups) and infant fecal samples from mothers who did not receive probiotic yogurt (UN and N groups), a significant difference was observed (Figure 27). Specifically, Bifidobacterium was three times more abundant

in the infant fecal samples from mothers who received probiotic yogurt and the unknown

Enterobacteriaceae was thirteen times more abundant in infants from mothers who did

not receive probiotic yogurt. Further ALDEx2 analysis on the phyla level revealed that Actinobacteria was significantly increased in infants from mothers who received probiotic yogurt as compared to infants from mothers who did not. If the UN and UNP groups were compared alone, Proteobacteria was also significantly increased in the UN group as compared to the UNP group (Figures 28 and 29). Enterobacteriaceae is a

member of Proteobacteria and evidence has suggested that an increased abundance of members of Enterobacteriaceae in the infant gut is associated with the development of

atopic disease245-247. Furthermore, members of both Proteobacteria and

Enterobacteriaceae include a number of organisms that have the potential to be

pathogenic and an increase of representative members of this taxa coinciding with a decrease of members of Bifidobacterium (a genus belonging to the phyla Actinobacteria) is not normally associated with health. An increase of Enterobacteriaceae and a decrease

in Bifidobacterium, has also been observed in infants receiving antibiotic treatment and

this imbalance was seen to persist even after treatment commenced248. The exact consequences of persistence of this imbalance is not completely understood, nor has it been investigated, however, a recent study has shown that a decrease in Bifidobacterium

and an increase in Enterobacteriaceae is associated with Crohn’s disease in children249.

Furthermore animal models and in vitro studies have shown that members of

Bifidobacterium promote specific immune functions, which could translate to an

importance of increased Bifidobacterium diversity in infant guts for proper immune

development250-252. In addition, secreted factors from Bifidobacterium infantis (a prominent member of the infant gut microbiota comprising up to 90% of the total

Bifidobacterium), have been shown to reduce colonic permeability and attenuate

inflammation in mouse models173. Thus, supplementation of Moringa enriched probiotic yogurt to mothers during pregnancy, may be priming their infant’s gut microbiota for healthy compositional and immune development, as well as protection against infection. It could be extremely valuable and informative to conduct a large-scale, longitudinal study on probiotic supplementation from pregnancy to three years post-partum to fully understand the potential of this proposed modulation.