Fucosylated Human Milk Oligosaccharide Foraging within the Species Bifidobacterium pseudocatenulatum Is Driven by Glycosyl Hydrolase Content and Specificity.

Human milk enriches members of the genus Bifidobacterium in the infant gut. One species, Bifidobacterium pseudocatenulatum, is found in the gastrointestinal tracts of adults and breastfed infants. In this study, B. pseudocatenulatum strains were isolated and characterized to identify genetic adaptations to the breastfed infant gut. During growth on pooled human milk oligosaccharides (HMOs), we observed two distinct groups of B. pseudocatenulatum, isolates that readily consumed HMOs and those that did not, a difference driven by variable catabolism of fucosylated HMOs. A conserved gene cluster for fucosylated HMO utilization was identified in several sequenced B. pseudocatenulatum strains. One isolate, B. pseudocatenulatum MP80, which uniquely possessed GH95 and GH29 α-fucosidases, consumed the majority of fucosylated HMOs tested. Furthermore, B. pseudocatenulatum SC585, which possesses only a single GH95 α-fucosidase, lacked the ability to consume the complete repertoire of linkages within the fucosylated HMO pool. Analysis of the purified GH29 and GH95 fucosidase activities directly on HMOs revealed complementing enzyme specificities with the GH95 enzyme preferring 1-2 fucosyl linkages and the GH29 enzyme favoring 1-3 and 1-4 linkages. The HMO-binding specificities of the family 1 solute-binding protein component linked to the fucosylated HMO gene cluster in both SC585 and MP80 are similar, suggesting differential transport of fucosylated HMO is not a driving factor in each strain's distinct HMO consumption pattern. Taken together, these data indicate the presence or absence of specific α-fucosidases directs the strain-specific fucosylated HMO utilization pattern among bifidobacteria and likely influences competitive behavior for HMO foraging in situ. IMPORTANCE Often isolated from the human gut, microbes from the bacterial family Bifidobacteriaceae commonly possess genes enabling carbohydrate utilization. Isolates from breastfed infants often grow on and possess genes for the catabolism of human milk oligosaccharides (HMOs), glycans found in human breast milk. However, catabolism of structurally diverse HMOs differs between bifidobacterial strains. This study identifies key gene differences between Bifidobacterium pseudocatenulatum isolates that may impact whether a microbe successfully colonizes an infant gut. In this case, the presence of complementary α-fucosidases may provide an advantage to microbes seeking residence in the infant gut. Such knowledge furthers our understanding of how diet drives bacterial colonization of the infant gut.

Dietary Bovine Lactoferrin Reduces Staphylococcus aureus in the Tissues and Modulates the Immune Response in Piglets Systemically Infected with S. aureus.

BACKGROUND: Bovine lactoferrin (bLf) reduces Staphylococcus aureus infection in premature infants and promotes the growth of Bifidobacterium infantis, a predominant infant gut species. We hypothesized that bLf in combination with B. infantis would reduce the severity of systemic S. aureus infection. OBJECTIVE: The aim was to determine the effects of oral administration of bLf and B. infantis on the course of systemic S. aureus infection. METHODS: Colostrum-deprived piglets were fed formulas containing 4 g whey/L (CON group) or bLf (LF group). One-half of the piglets in each group were gavaged with B. infantis (109 colony-forming units/d), resulting in 2 additional groups (BI or COMB, respectively). On day 7, piglets were intravenously injected with S. aureus. Blood samples were collected preinfection and every 12 h postinfection for immune analyses. Tissue samples were collected on day 12 for analysis of bacterial abundance and gene expression. RESULTS: Preinfection, LF piglets had lower serum interleukin 10 (IL-10), a higher percentage of lymphocytes, and a lower percentage of neutrophils than BI or COMB piglets. After infection, dietary bLf increased piglet weight gain, reduced staphylococcal counts in the kidneys, and tended to lower staphylococcal counts in the lungs and heart. Dietary bLf also decreased kidney IL-10 and increased lung interferon γ (IFN-γ) mRNA. B. infantis increased splenic IFN-γ expression. Renal Toll-like receptor 2 was upregulated in BI piglets but not in COMB piglets. Postinfection, BI piglets had increased serum IL-10 and decreased memory T cell populations. LF and COMB piglets had fewer circulating monocytes and B cells than CON or BI piglets. CONCLUSIONS: Dietary bLf and B. infantis produced independent and tissue-specific effects. Piglets fed bLf alone or in combination with B. infantis mounted a more effective immune response and exhibited lower bacterial abundance. This study provides biological underpinnings to the clinical benefits of bLf observed in preterm infants but does not support B. infantis administration during S. aureus infection.

Human milk oligosaccharide consumption by probiotic and human-associated bifidobacteria and lactobacilli.

Human milk contains high concentrations of nondigestible complex oligosaccharides (human milk oligosaccharides; HMO) that reach the colon and are subsequently fermented by the infant gut microbiota. Using a high-throughput, low-volume growth determination, we evaluated the ability of 12 lactobacilli and 12 bifidobacteria strains, including several commercial probiotics, to ferment HMO and their constituent monomers. Of the 24 strains tested, only Bifidobacterium longum ssp. infantis ATCC 15697 and Bifidobacterium infantis M-63 were able to ferment 3'-sialyllactose, 6'-sialyllactose, 2'-fucosyllactose, and 3'-fucosyllactose. Bifidobacterium infantis M-63 degraded almost 90% of the 2'-fucosyllactose but left most of the fucose in the supernatant, as detected by HPLC. Among bifidobacteria, only the B. infantis strains and Bifidobacterium breve ATCC 15700 were able to ferment lacto-N-neotetraose (LNnT). Among lactobacilli, Lactobacillus acidophilus NCFM was found to be the most efficient at utilizing LNnT. The extracellular ß-galactosidase (lacL, LBA1467) of L. acidophilus NCFM cleaves the terminal galactose of LNnT for growth, leaving lacto-N-triose II in the media as detected by HPLC. Inactivation of lacL abolishes growth of L. acidophilus NCFM on LNnT. These results contribute to our knowledge of HMO-microbe interactions and demonstrate the potential for synbiotic combinations of pre- and probiotics.

Prebiotic Galactooligosaccharide Metabolism by Probiotic Lactobacilli and Bifidobacteria.

Galactooligosaccharides (GOS) are bifidogenic and lactogenic prebiotics; however, GOS utilization is strain-dependent. In this study, commercially available bifidobacteria and lactobacilli probiotic strains were evaluated for growth in the presence of GOS. Several bifidobacteria and lactobacilli grew on GOS; however, the specific GOS oligomers utilized for growth differed. A subset of probiotic bifidobacteria and lactobacilli revealed three different GOS utilization profiles delineated by the degrees of polymerization (DP) of GOS: (1) utilization of 2 DP GOS, (2) utilization of ≤3 DP GOS, and (3) utilization of all DP GOS. Specifically, Lactobacillus acidophilus NCFM (LA_NCFM) was found to efficiently consume all GOS oligomers. Extracellular ß-galactosidase activity in the cell-free supernatant of LA_NCFM correlated with accumulation of galactose. In a LacL-deficient LA_NCFM strain, GOS utilization was abolished. This is the first report of LacL's role in GOS metabolism in LA_NCFM. In vitro GOS utilization should be considered when GOS are delivered with probiotic bifidobacteria and lactobacilli.

Cronobacter sakazakii ATCC 29544 Autoaggregation Requires FliC Flagellation, Not Motility.

Cronobacter sakazakii is an opportunistic nosocomial and foodborne pathogen that causes severe infections with high morbidity and mortality rates in neonates, the elderly, and immunocompromised individuals. Little is known about the pathogenesis mechanism of this pathogen and if there are any consequences of C. sakazakii colonization in healthy individuals. In this study, we characterized the mechanisms of autoaggregation in C. sakazakii ATCC 29544 (CS29544). Autoaggregation in CS29544 occurred rapidly, within 30 min, and proceeded to a maximum of 70%. Frameshift mutations in two flagellum proteins (FlhA and FliG) were identified in two nonautoaggregating CS29544 clonal variant isolates. Strategic gene knockouts were generated to determine if structurally intact and functional flagella were required for autoaggregation in CS29544. All structural knockouts (ΔflhA, ΔfliG, and ΔfliC) abolished autoaggregation, whereas the functional knockout (ΔmotAB) did not prevent autoaggregation. Complementation with FliC (ΔfliC/cfliC) restored autoaggregation. Autoaggregation was also disrupted by the addition of exogenous wild-type CS29544 filaments in a dose-dependent manner. Finally, filament supercoils tethering neighboring wild-type CS29544 cells together were observed by transmission electron microscopy. In silico analyses suggest that direct interactions of neighboring CS29544 FliC filaments proceed by hydrophobic bonding between the externally exposed hypervariable regions of the CS29544 FliC flagellin protein. Further research is needed to confirm if flagella-mediated autoaggregation plays a prominent role in C. sakazakii pathogenesis.

Dietary Broccoli Alters Rat Cecal Microbiota to Improve Glucoraphanin Hydrolysis to Bioactive Isothiocyanates.

Broccoli consumption brings many health benefits, including reducing the risk of cancer and inflammatory diseases. The objectives of this study were to identify global alterations in the cecal microbiota composition using 16S rRNA sequencing analysis and glucoraphanin (GRP) hydrolysis to isothiocyanates ex vivo by the cecal microbiota, following different broccoli diets. Rats were randomized to consume AIN93G (control) or different broccoli diets; AIN93G plus cooked broccoli, a GRP-rich powder, raw broccoli, or myrosinase-treated cooked broccoli. Feeding raw or cooked broccoli for four days or longer both changed the cecal microbiota composition and caused a greater production of isothiocyanates ex vivo. A more than two-fold increase in NAD(P)H: quinone oxidoreductase 1 activity of the host colon mucosa after feeding cooked broccoli for seven days confirmed the positive health benefits. Further studies revealed that dietary GRP was specifically responsible for the increased microbial GRP hydrolysis ex vivo, whereas changes in the cecal microbial communities were attributed to other broccoli components. Interestingly, a three-day withdrawal from a raw broccoli diet reversed the increased microbial GRP hydrolysis ex vivo. Findings suggest that enhanced conversion of GRP to bioactive isothiocyanates by the cecal microbiota requires four or more days of broccoli consumption and is reversible.

Molecular weight distribution and fermentation of mechanically pre-treated konjac enzymatic hydrolysates.

There is interest in novel fibers as potential prebiotics for new and reformulated food products. Two konjac glucomannan (KGM) hydrolysates were developed by enzymatic hydrolysis with (KGMH I) or without (KGMH II) mechanical shear pre-treatment. These were characterized and evaluated as fermentation substrates using five lactobacilli and three bifidobacteria. Enzymatic treatment of native KGM reduced the average molecular weights of supernatant and pellet by ∼3-fold. Additional mechanical shear pre-treatment further reduced supernatant and pellet molecular weights by 5% and 35%, respectively. We postulated that pulverized and depolymerized short-chain KGM would better promote the growth of lactobacilli and bifidobacteria. Most lactobacilli fermented KGM hydrolysates. Lactobacillus acidophilus and Lactobacillus plantarum fermented KGMH I and II better than they fermented inulin. Overall, bifidobacteria were not strong fermenters of KGM hydrolysates. Both pulverization and enzymatic depolymerization significantly affected KGM molecular weight, suggesting that human gastrointestinal bacteria can utilize KGM hydrolysates with reduced weights.

A dynamic regression analysis tool for quantitative assessment of bacterial growth written in Python.

Herein, an open-source method to generate quantitative bacterial growth data from high-throughput microplate assays is described. The bacterial lag time, maximum specific growth rate, doubling time and delta OD are reported. Our method was validated by carbohydrate utilization of lactobacilli, and visual inspection revealed 94% of regressions were deemed excellent.

Myrosinase-dependent and -independent formation and control of isothiocyanate products of glucosinolate hydrolysis.

Brassicales contain a myrosinase enzyme that hydrolyzes glucosinolates to form toxic isothiocyanates (ITC), as a defense against bacteria, fungi, insects and herbivores including man. Low levels of ITC trigger a host defense system in mammals that protects them against chronic diseases. Because humans typically cook their brassica vegetables, destroying myrosinase, there is a great interest in determining how human microbiota can hydrolyze glucosinolates and release them, to provide the health benefits of ITC. ITC are highly reactive electrophiles, binding reversibly to thiols, but accumulating and causing damage when free thiols are not available. We found that addition of excess thiols released protein-thiol-bound ITC, but that the microbiome supports only poor hydrolysis unless exposed to dietary glucosinolates for a period of days. These findings explain why 3-5 servings a week of brassica vegetables may provide health effects, even if they are cooked.

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

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.

In vitro impact of human milk oligosaccharides on Enterobacteriaceae growth.

Human milk oligosaccharides (HMOs) function as prebiotics in the infant gut by selecting for specific species of bifidobacteria and bacteroides, but little is known about their potential utilization by Enterobacteriaceae, the relative numbers of which have been linked to the onset of necrotizing enterocolitis in preterm infants. In this study, the in vitro growth of purified HMOs and other related carbohydrates was evaluated using individual strains of Enterobacteriaceae and an Enterobacteriaceae consortia enriched from piglet feces. None of the Enterobacteriaceae strains grew on 2'-fucosyllactose, 6'-sialyllactose, or lacto-N-neotetraose (LNnT); however, several strains were capable of utilizing galactooligosaccharides, maltodextrin, and the mono- and disaccharide components of HMOs for growth. The enriched fecal consortia also did not grow on 2'-fucosyllactose or 6'-sialyllactose, but there was limited growth on LNnT. It was concluded that 2'-fucosyllactose and 6'-sialyllactose supplementation of preterm infant formula should not lead to an increase in Enterobacteriaceae; however, supplementation with LNnT may require further study.

A piglet model for studying Candida albicans colonization of the human oro-gastrointestinal tract.

Pigs from a variety of sources were surveyed for oro-gastrointestinal (oro-GIT) carriage of Candida albicans. Candida albicans-positive animals were readily located, but we also identified C. albicans-free pigs. We hypothesized that pigs could be stably colonized with a C. albicans strain of choice, simply by feeding yeast cells. Piglets were farrowed routinely and remained with the sow for 4 days to acquire a normal microbiota. Piglets were then placed in an artificial rearing environment and fed sow milk replacer. Piglets were inoculated orally with one of three different C. albicans strains. Piglets were weighed daily, and culture swabs were collected to detect C. albicans orally, rectally and in the piglet's environment. Stable C. albicans colonization over the course of the study did not affect piglet growth. Necropsy revealed mucosally associated C. albicans throughout the oro-GIT with the highest abundance in the esophagus. Uninoculated control piglets remained C. albicans-negative. These data establish the piglet as a model to study C. albicans colonization of the human oro-GIT. Similarities between oro-GIT colonization in humans and pigs, as well as the ease of working with the piglet model, suggest its adaptability for use among investigators interested in understanding C. albicans-host commensal interactions.

Consumption of different soymilk formulations differentially affects the gut microbiomes of overweight and obese men.

The effects of consuming foods on the intestinal microbiome of obese individuals remain unclear. The objective of this study was to compare the effects of consuming low glycinin soymilk (LGS, 49.5% ß-conglycinin/6% glycinin), conventional soymilk (S, 26.5% ß-conglycinin/38.7% glycinin) or bovine milk (M, 0% ß-conglycinin/0% glycinin) on the intestinal microbiome in overweight and obese men. In a randomized double-blind study, participants (64 men, BMI > 25, 20-45 y old), organized in three groups, consumed 500 mL of LGS, S or M daily for 3 mo. Three fecal samples were collected before (baseline) and after 3 mo of consumption. Dietary energy and macronutrient intake were monitored monthly and remained constant throughout the study (p > 0.05). Microbial composition was analyzed with qPCR and bTEFAP. Within groups, qPCR analysis showed that the total bacteria increased in all treatments over time (p < 0.001). Bacteroides-Prevotella (p = 0.001) and Lactobacillus (p < 0.001) increased in LGS and M, respectively. Bifidobacterium was significantly reduced in LGS (p = 0.003) and S (p < 0.001). Bacterial diversity decreased for LGS, S and M (p = 0.004, 0.005, 0.001; respectively). Unweighted UniFrac analysis revealed that the microbial communities were more similar within than between individuals. The Firmicutes to Bacteroidetes ratio decreased in both LGS and S groups and remained relatively unchanged in the M group (Time p = 0.012; Interaction p = 0.059). Indicator analysis revealed several genera that were indicative of each treatment including Lactobacillus and Prevotella. Consumption of the three beverages differentially altered the microbiota in overweight and obese men including a potentially beneficial alteration of the Firmicutes to Bacteroidetes ratio in both soymilk groups.

Identification of lactose phosphotransferase systems in Lactobacillus gasseri ATCC 33323 required for lactose utilization.

Improving the annotation of sugar catabolism-related genes requires functional characterization. Our objective was to identify the genes necessary for lactose utilization by Lactobacillus gasseri ATCC 33323 (NCK334). The mechanism of lactose transport in many lactobacilli is a lactose/galactose-specific permease, yet no orthologue was found in NCK334. Characterization of an EI knockout strain [EI (enzyme I) is required for phosphotransferase system transporter (PTS) function] demonstrated that L. gasseri requires PTS(s) to utilize lactose. In order to determine which PTS(s) were necessary for lactose utilization, we compared transcript expression profiles in response to lactose for the 15 complete PTSs identified in the NCK334 genome. PTS 6CB (LGAS_343) and PTS 8C (LGAS_497) were induced in the presence of lactose 107- and 53-fold, respectively. However, L. gasseri ATCC 33323 PTS 6CB, PTS 8C had a growth rate similar to that of the wild-type on semisynthetic deMan, Rogosa, Sharpe (MRS) medium with lactose. Expression profiles of L. gasseri ATCC 33323 PTS 6CB, PTS 8C in response to lactose identified PTS 9BC (LGAS_501) as 373-fold induced, whereas PTS 9BC was not induced in NCK334. Elimination of growth on lactose required the inactivation of both PTS 6CB and PTS 9BC. Among the six candidate phospho-ß-galactosidase genes present in the NCK334 genome, LGAS_344 was found to be induced 156-fold in the presence of lactose. In conclusion, we have determined that: (1) NCK334 uses a PTS to import lactose; (2) PTS 6CB and PTS 8C gene expression is strongly induced by lactose; and (3) elimination of PTS 6CB and PTS 9BC is required to prevent growth on lactose.