Characterization of the Intestinal Lactobacilli Community following Galactooligosaccharides and Polydextrose Supplementation in the Neonatal Piglet

Abstract

Recently, prebiotic supplementation of infant formula has become common practice; however the impact on the intestinal microbiota has not been completely elucidated. In this study, neonatal piglets were randomized to: formula (FORM, n = 8), formula supplemented with 2 g/L each galactooligosaccharides (GOS) and polydextrose (PDX, F+GP, n = 9) or a sow-reared (SOW, n = 12) reference group for 19 days. The ileal (IL) and ascending colon (AC) microbiota were characterized using culture-dependent and -independent methods. 16S amplicon sequencing identified no differences at the genera level in the IL. Interestingly, six genera in the AC were significantly different between FORM and F+GP (P<0.05): Lactobacillus, Ruminococcus, Parabacteroides, Oscillospira, Hydrogenoanaerobacterium and Catabacter. In particular, the relative abundance of AC Lactobacillus was higher (P = 0.04) in F+GP as compared to FORM. Culture-dependent analysis of the IL and AC lactobacilli communities of FORM and F+GP revealed a Lactobacillus spp. composition similar to 16S amplicon sequencing. Additional analysis demonstrated individual Lactobacillus isolates were unable to ferment PDX. Conversely, a majority of lactobacilli isolates could ferment GOS, regardless of piglet diet. In addition, the ability of lactobacilli isolates to ferment the longer chain GOS fragments (DP 3 or greater), which are expected to be present in the distal intestine, was not different between FORM and F+GP. In conclusion, prebiotic supplementation of formula impacted the AC microbiota; however, direct utilization of GOS or PDX does not lead to an increase in Lactobacillus spp.

Fig 1. Lactobacilli population is dominated by few members.

Lactobacilli communities isolated from the ileum (IL) and ascending colon (AC) of 21 d old piglets fed either a bovine milk-based formula (FORM), formula supplemented with GOS and PDX (F+GP) or sow-reared (SOW). Stacked bar chart represents the percent relative abundance of each lactobacilli detected in the community. Beta diversity was calculated using the Bray-Curtis Dissimilarity Index and is visualized by a dendrogram.

Fig 2. Several isolated lactobacilli identified as GOS utilizers.

Carbohydrate utilization of lactobacilli communities isolated from the ileum (IL) and ascending colon (AC) of 21 d old piglets fed either a bovine milk-based formula (FORM), formula supplemented with GOS and PDX (F+GP). (A) Percent relative utilization (OD>0.7) of glucose, Purimune galactooligosaccharide (GOSP) or PDX. To account for isolates not capable of in vitro fermentation, utilization was normalized to maximum number of isolates capable of utilizing glucose as a sole carbon source. (B) Maximum optical density (595 nm) of Lactobacilli sp. when GOSP is supplemented as a sole carbon source. Colored bar indicates the mean maximum optical density. Not all Lactobacillus sp. were detected in each treatment or location.

Fig 3. Bacterial communities vary greatly by location, but less by diet.

Ileal (IL) and ascending colon (AC) microbiome of 21 d old piglets fed either a bovine milk-based formula (FORM), formula supplemented with GOS and PDX (F+GP) or sow-reared (SOW) illustrated by Principle Coordinate Analysis (PCoA). PCoA was constructed from the unweighted Unifrac distance measure which only considers the change in composition not abundance. PCoA of the 16S amplicon sequencing data set: (A) all three treatments; (B) formula-fed piglets only.

Fig 4. Culture-independent analysis reveals a more diverse lactobacilli community.

Lactobacilli communities from the ileum (IL) and ascending colon (AC) of 21 d old piglets fed either a bovine milk-based formula (FORM), formula supplemented with GOS and PDX (F+GP) or sow-reared (SOW) as determined by 16S amplicon sequencing. Stacked bar chart represents the percent relative abundance of each lactobacilli detected in the community. Beta diversity was calculated using the Bray-Curtis Dissimilarity Index and is visualized by a dendrogram. Lactobacilli represented by the label Lactobacillus sp. were unable to be assigned a species. Minor Lactobacillus sp. is an aggregate of lactobacilli present in > 1% percent relative abundance (S3 Table).

Fig 5. Identification of the predominant Lactobacillus spp. in each group.

Indicator species analysis (ISA) of the lactobacilli community of 21 d old piglets fed either a bovine milk-based formula (FORM), formula supplemented with GOS and PDX (F+GP) or sow-reared (SOW, included for reference). Color change demonstrates the more a species is indicative of a diet. The higher the indicator score (i.e. darker color), the more that species is indicative of that group. ISA scores for the genera Lactobacillus detected in the (A) IL; (B) AC.

Fig 6. Prebiotic supplementation leads to minimal changes in static volatile fatty acid concentration.

Volatile fatty acids detected in ileal (IL) and ascending colon (AC) contents of 21 d old piglets fed either a bovine milk-based formula (FORM), formula supplemented with GOS and PDX (F+GP) or sow-reared (SOW). VFAs detected in IL: (A) Short-chain fatty acids; (B) Branch-chain fatty acids. VFAs detected in the AC: (C) Short-chain fatty acids; (D) Branch-chain fatty acids. Values with different superscripts are significantly different (p<0.05).