Genetically dictated change in host mucus carbohydrate landscape exerts a diet-dependent effect on the gut microbiota.

We investigate how host mucus glycan composition interacts with dietary carbohydrate content to influence the composition and expressed functions of a human gut community. The humanized gnotobiotic mice mimic humans with a nonsecretor phenotype due to knockout of their α1-2 fucosyltransferase (Fut2) gene. The fecal microbiota of Fut2(-) mice that lack fucosylated host glycans show decreased alpha diversity relative to Fut2(+) mice and exhibit significant differences in community composition. A glucose-rich plant polysaccharide-deficient (PD) diet exerted a strong effect on the microbiota membership but eliminated the effect of Fut2 genotype. Additionally fecal metabolites predicted host genotype in mice on a polysaccharide-rich standard diet but not on a PD diet. A more detailed mechanistic analysis of these interactions involved colonization of gnotobiotic Fut2(+) and Fut2(-) mice with Bacteroides thetaiotaomicron, a prominent member of the human gut microbiota known to adaptively forage host mucosal glycans when dietary polysaccharides are absent. Within Fut2(-) mice, the B. thetaiotaomicron fucose catabolic pathway was markedly down-regulated, whereas BT4241-4247, an operon responsive to terminal ß-galactose, the precursor that accumulates in the Fut2(-) mice, was significantly up-regulated. These changes in B. thetaiotaomicron gene expression were only evident in mice fed a PD diet, wherein B. thetaiotaomicron relies on host mucus consumption. Furthermore, up-regulation of the BT4241-4247 operon was also seen in humanized Fut2(-) mice. Together, these data demonstrate that differences in host genotype that affect the carbohydrate landscape of the distal gut interact with diet to alter the composition and function of resident microbes in a diet-dependent manner.

Host-centric proteomics of stool: a novel strategy focused on intestinal responses to the gut microbiota.

The diverse community of microbes that inhabits the human bowel is vitally important to human health. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interaction. Therefore, unbiased and sensitive methods focused on host proteome characterization are needed. Herein we describe a novel method for applying shotgun proteomics to the analysis of feces, focusing on the secreted host proteome. We have conducted the most complete analysis of the extracellular mouse gut proteome to date by employing a gnotobiotic mouse model. Using mice colonized with defined microbial communities of increasing complexity or a complete human microbiota ('humanized'), we show that the complexity of the host stool proteome mirrors the complexity of microbiota composition. We further show that host responses exhibit signatures specific to the different colonization states. We demonstrate feasibility of this approach in human stool samples and provide evidence for a "core" stool proteome as well as personalized host response features. Our method provides a new avenue for noninvasive monitoring of host-microbiota interaction dynamics via host-produced proteins in stool.

Complex interactions among diet, gastrointestinal transit, and gut microbiota in humanized mice.

BACKGROUND & AIMS: Diet has major effects on the intestinal microbiota, but the exact mechanisms that alter complex microbial communities have been difficult to elucidate. In addition to the direct influence that diet exerts on microbes, changes in microbiota composition and function can alter host functions such as gastrointestinal (GI) transit time, which in turn can further affect the microbiota. METHODS: We investigated the relationships among diet, GI motility, and the intestinal microbiota using mice that are germ-free (GF) or humanized (ex-GF mice colonized with human fecal microbiota). RESULTS: Analysis of gut motility revealed that humanized mice fed a standard polysaccharide-rich diet had faster GI transit and increased colonic contractility compared with GF mice. Humanized mice with faster transit due to administration of polyethylene glycol or a nonfermentable cellulose-based diet had similar changes in gut microbiota composition, indicating that diet can modify GI transit, which then affects the composition of the microbial community. However, altered transit in mice fed a diet of fermentable fructooligosaccharide indicates that diet can change gut microbial function, which can affect GI transit. CONCLUSIONS: Based on studies in humanized mice, diet can affect GI transit through microbiota-dependent or microbiota-independent pathways, depending on the type of dietary change. The effect of the microbiota on transit largely depends on the amount and type (fermentable vs nonfermentable) of polysaccharides present in the diet. These results have implications for disorders that affect GI transit and gut microbial communities, including irritable bowel syndrome and inflammatory bowel disease.

A refined palate: bacterial consumption of host glycans in the gut.

The human intestine houses a dense microbial ecosystem in which the struggle for nutrients creates a continual and dynamic selective force. Host-produced mucus glycans provide a ubiquitous source of carbon and energy for microbial species. Not surprisingly, many gut resident bacteria have become highly adapted to efficiently consume numerous distinct structures present in host glycans. We propose that sophistication in mucus consumption is a trait most likely to be found in gut residents that have co-evolved with hosts, microbes that have adapted to the complexity associated with the host glycan landscape.

An infant-associated bacterial commensal utilizes breast milk sialyloligosaccharides.

Lactating mothers secrete milk sialyloligosaccharides (MSOs) that function as anti-adhesives once provided to the neonate. Particular infant-associated commensals, such as Bifidobacterium longum subsp. infantis, consume neutral milk oligosaccharides, although their ability to utilize acidic oligosaccharides has not been assessed. Temporal glycoprofiling of acidic HMO consumed during fermentation demonstrated a single composition, with several isomers, corresponding to sialylated lacto-N-tetraose. To utilize MSO, B. longum subsp. infantis deploys a sialidase that cleaves α2-6 and α2-3 linkages. NanH2, encoded within the HMO catabolic cluster is up-regulated during HMO fermentation and is active on sialylated lacto-N-tetraose. These results demonstrate that commensal microorganisms do utilize MSO, a substrate that may be enriched in the distal gastrointestinal tract.

Tyramine and phenylethylamine biosynthesis by food bacteria.

Tyramine poisoning is caused by the ingestion of food containing high levels of tyramine, a biogenic amine. Any foods containing free tyrosine are subject to tyramine formation if poor sanitation and low quality foods are used or if the food is subject to temperature abuse or extended storage time. Tyramine is generated by decarboxylation of the tyrosine through tyrosine decarboxylase (TDC) enzymes derived from the bacteria present in the food. Bacterial TDC have been only unequivocally identified and characterized in Gram-positive bacteria, especially in lactic acid bacteria. Pyridoxal phosphate (PLP)-dependent TDC encoding genes (tyrDC) appeared flanked by a similar genetic organization in several species of lactic acid bacteria, suggesting a common origin by a single mobile genetic element. Bacterial TDC are also able to decarboxylate phenylalanine to produce phenylethylamine (PEA), another biogenic amine. The molecular knowledge of the genes involved in tyramine production has led to the development of molecular methods for the detection of bacteria able to produce tyramine and PEA. These rapid and simple methods could be used for the analysis of the ability to form tyramine by bacteria in order to evaluate the potential risk of tyramine biosynthesis in food products.

A randomized placebo-controlled comparison of 2 prebiotic/probiotic combinations in preterm infants: impact on weight gain, intestinal microbiota, and fecal short-chain fatty acids.

OBJECTIVE: To compare the effect of 2 prebiotic/probiotic products on weight gain, stool microbiota, and stool short-chain fatty acid (SCFA) content of premature infants. PATIENTS AND METHODS: This randomized, blinded, placebo-controlled trial included 90 premature infants treated with either a dietary supplement containing 2 lactobacillus species plus fructooligosaccharides (CUL, Culturelle, ConAgra, Omaha, NE), a supplement containing several species of lactobacilli and bifidobacteria plus fructooligosaccharides (PBP, ProBioPlus DDS, UAS Laboratories, Eden Prairie, MN), or placebo (a dilute preparation of Pregestamil formula) twice daily for 28 days or until discharge if earlier. The primary outcome was weight gain. Secondary outcomes were stool bacterial analysis by culture and 16S rDNA quantitative polymerase chain reaction and stool SCFA content measured by high performance liquid chromatography. RESULTS: Both prebiotic/probiotic combinations contained more bacterial species than noted on the label. No significant effect on infant growth of either prebiotic/probiotic supplement was observed. By cultures, 64% of infants receiving PBP became colonized with bifidobacteria, compared with 18% of infants receiving CUL and 27% of infants receiving placebo (chi-square, P = 0.064). No differences were noted between groups in colonization rates for lactobacilli, Gram-negative enteric bacteria, or staphylococci. By 16S rDNA polymerase chain reaction analysis, the bifidobacteria content in the stools of the infants receiving PBP was higher than in the infants receiving CUL or placebo (Kruskal-Wallis, P = 0.011). No significant differences in stool SCFA content were detected between groups. No adverse reactions were noted. CONCLUSIONS: Infants receiving PBP were more likely to become colonized with bifidobacteria. No significant differences in weight gain or stool SCFA content were detected.

Role of hypermutability in the evolution of the genus Oenococcus.

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium primarily responsible for malolactic fermentation in wine. A recent comparative genomic analysis of O. oeni PSU-1 with other sequenced lactic acid bacteria indicates that PSU-1 lacks the mismatch repair (MMR) genes mutS and mutL. Consistent with the lack of MMR, mutation rates for O. oeni PSU-1 and a second oenococcal species, O. kitaharae, were higher than those observed for neighboring taxa, Pediococcus pentosaceus and Leuconostoc mesenteroides. Sequence analysis of the rpoB mutations in rifampin-resistant strains from both oenococcal species revealed a high percentage of transition mutations, a result indicative of the lack of MMR. An analysis of common alleles in the two sequenced O. oeni strains, PSU-1 and BAA-1163, also revealed a significantly higher level of transition substitutions than were observed in other Lactobacillales species. These results suggest that the genus Oenococcus is hypermutable due to the loss of mutS and mutL, which occurred with the divergence away from the neighboring Leuconostoc branch. The hypermutable status of the genus Oenococcus explains the observed high level of allelic polymorphism among known O. oeni isolates and likely contributed to the unique adaptation of this genus to acidic and alcoholic environments.

Updated molecular knowledge about histamine biosynthesis by bacteria.

Histamine poisoning is caused by the ingestion of food containing high levels of histamine, a biogenic amine. Histamine could be expected in virtually all foods that contain proteins or free histidine and that are subject to conditions enabling microbial activity. In most histamine-containing foods the majority of the histamine is generated by decarboxylation of the histidine through histidine decarboxylase enzymes derived from the bacteria present in food. Bacterial histidine decarboxylases have been extensively studied and characterized in different organisms and two different enzymes groups have been distinguished, pyridoxal phosphate- and the pyruvoyl-dependent. Pyridoxal phosphate-dependent histidine decarboxylases are encountered in gram-negative bacteria belonging to various species. Pyruvoyl-dependent histidine decarboxylases are found in gram-positive bacteria and specially in lactic acid bacteria implicated in food fermentation or spoilage. The molecular organization of the genes involved in histamine production have been elucidated in several histamine-producer bacteria. This molecular knowledge has led to the development of molecular methods for the rapid detection of bacteria possessing the ability to produce histamine. The detection of histamine-producer bacteria is of great importance for its potential health hazard as well as from an economic point of view since products exceeding recommended limits can be refused in commercial transactions.

Rapid determination of the bacterial composition of commercial probiotic products by terminal restriction fragment length polymorphism analysis.

Label claims on probiotic products often do not represent the true constituents. With the increased use of probiotics in clinical studies, it is necessary to know the true composition of probiotic products to better interpret study outcomes. We used terminal restriction fragment length polymorphism analysis to rapidly determine the overall bacterial composition of 14 commercial probiotic products and validated the results with species-specific polymerase chain reaction. The results show that many probiotic products contain unadvertised additional lactobacilli and bifidobacteria, whereas others are missing species listed on the product label. In summary, terminal restriction fragment length polymorphism is a rapid method for profiling the microbial contents of probiotic products used in clinical studies.

Molecular methods for the detection of biogenic amine-producing bacteria on foods.

Biogenic amines are low molecular weight organic bases that can be detected in raw and processed foods. Several toxicological problems resulting from the ingestion of food containing biogenic amines have been described. Biogenic amines are mainly produced by the decarboxylation of certain amino acids by microbial action. Since the ability of microorganisms to decarboxylate amino acid is highly variable, being in most cases strain-specific, the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the risk of biogenic amine food content and to prevent biogenic amine accumulation in food products. Molecular methods for the early and rapid detection of these producer bacteria are becoming an alternative to traditional culture methods. PCR methods offer the advantages of speed, sensitivity, simplicity and specific detection of amino acid decarboxylase genes. Moreover, these molecular methods detect potential biogenic amine risk formation in food before the amine is produced. The aim of the present review is to give a complete overview of the molecular methods proposed in the literature for the detection of biogenic amine-producing bacteria. These genetic procedures allow the introduction of early control measures to avoid the development of these bacteria.

A multifactorial design for studying factors influencing growth and tyramine production of the lactic acid bacteria Lactobacillus brevis CECT 4669 and Enterococcus faecium BIFI-58.

A central composite face design was used to study growth and tyramine production of two strains of lactic acid bacteria, Lactobacillus brevis CECT 4669 and Enterococcus faecium BIFI-58. The effects of five physicochemical factors (incubation temperature and time, environmental pH, added tyrosine concentration, and pyridoxal-5-phosphate (PLP) supplementation) on cell growth and tyramine production were analyzed under aerobic and anaerobic conditions. The parameters of the quadratic model for each response variable were estimated by multiple linear regression (MLR), and statistical analysis of the results led to the elucidation of mathematical models capable of predicting the behavior of the responses as a function of the main variables involved in the process. Incubation time was found to be the most important variable influencing growth in L. brevis, while pH showed the highest contribution in E. faecium. The production of tyramine was dependent on the added tyrosine concentration and incubation time. The proposed MLR model predicted the optimum conditions that gave maximum responses for L. brevis and E. faecium growth and tyramine production. In both strains, this model predicted that the anaerobic condition at acidic pH (4.4) in the presence of a high tyrosine concentration favors tyramine production.

First genetic characterization of a bacterial beta-phenylethylamine biosynthetic enzyme in Enterococcus faecium RM58.

Enterococcus faecium RM58 produces beta-phenylethylamine and tyramine. A gene from Ent. faecium RM58 coding for a 625 amino-acid residues protein that shows 85% identity to Enterococcus faecalis tyrosine decarboxylase has been expressed in Escherichia coli, resulting in L-phenylalanine and L-tyrosine decarboxylase activities. Both activities were lost when a truncated protein lacking 84 amino acids at its C-terminus was expressed in E. coli. This study constitutes the first genetic characterization of a bacterial protein having L-phenylalanine decarboxylase activity and solves a long-standing question regarding the specificity of tyrosine decarboxylases in enterococci.

PCR detection of foodborne bacteria producing the biogenic amines histamine, tyramine, putrescine, and cadaverine.

This study describes an easy PCR method for the detection of foodborne bacteria that potentially produce histamine, tyramine, putrescine, and cadaverine. Synthetic oligonucleotide pairs for the specific detection of the gene coding for each group of bacterial histidine, tyrosine, ornithine, or lysine decarboxylases were designed. Under the conditions used in this study, the assay yielded fragments of 372 and 531 bp from histidine decarboxylase-encoding genes, a 825-bp fragment from tyrosine decarboxylases, fragments of 624 and 1,440 bp from ornithine decarboxylases, and 1,098- and 1,185-bp fragments from lysine decarboxylases. This is the first PCR method for detection of cadaverine-producing bacteria. The method was successfully applied to several biogenic amine-producing bacterial strains.

Individualized Responses of Gut Microbiota to Dietary Intervention Modeled in Humanized Mice.

Diet plays an important role in shaping the structure and function of the gut microbiota. The microbes and microbial products in turn can influence various aspects of host physiology. One promising route to affect host function and restore health is by altering the gut microbiome using dietary intervention. The individuality of the microbiome may pose a significant challenge, so we sought to determine how different microbiotas respond to the same dietary intervention in a controlled setting. We modeled gut microbiotas from three healthy donors in germfree mice and defined compositional and functional alteration following a change in dietary microbiota-accessible carbohydrates (MACs). The three gut communities exhibited responses that differed markedly in magnitude and in the composition of microbiota-derived metabolites. Adjustments in community membership did not correspond to the magnitude of changes in the microbial metabolites, highlighting potential challenges in predicting functional responses from compositional data and the need to assess multiple microbiota parameters following dietary interventions. IMPORTANCE Dietary modification has long been used empirically to modify symptoms in inflammatory bowel disease, irritable bowel syndrome, and a diverse group of diseases with gastrointestinal symptoms. There is both anecdotal and scientific evidence to suggest that individuals respond quite differently to similar dietary changes, and the highly individualized nature of the gut microbiota makes it a prime candidate for these differences. To overcome the typical confounding factors of human dietary interventions, here we employ ex-germfree mice colonized by microbiotas of three different humans to test how different microbiotas respond to a defined change in carbohydrate content of diet by measuring changes in microbiota composition and function using marker gene-based next-generation sequencing and metabolomics. Our findings suggest that the same diet has very different effects on each microbiota's membership and function, which may in turn explain interindividual differences in response to a dietary ingredient.

Modulation of a Circulating Uremic Solute via Rational Genetic Manipulation of the Gut Microbiota.

Renal disease is growing in prevalence and has striking co-morbidities with metabolic and cardiovascular disease. Indoxyl sulfate (IS) is a toxin that accumulates in plasma when kidney function declines and contributes to the progression of chronic kidney disease. IS derives exclusively from the gut microbiota. Bacterial tryptophanases convert tryptophan to indole, which is absorbed and modified by the host to produce IS. Here, we identify a widely distributed family of tryptophanases in the gut commensal Bacteroides and find that deleting this gene eliminates the production of indole in vitro. By altering the status or abundance of the Bacteroides tryptophanase, we can modulate IS levels in gnotobiotic mice and in the background of a conventional murine gut community. Our results demonstrate that it is possible to control host IS levels by targeting the microbiota and suggest a possible strategy for treating renal disease.

Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus.

Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 µg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission.

Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines.

This study describes a simple and rapid multiplex-PCR method to determine the ability to produce histamine, tyramine and putrescine by bacteria. The assay is an improved method based on an assay designed for lactic acid bacteria. This improved method includes a pair of primers based on sequences from histidine decarboxylases from Gram-negative bacteria. Under the optimised conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases of Gram-positive bacteria, 534-bp fragment from histidine decarboxylases of Gram-negative bacteria, 924-bp from bacterial tyrosine decarboxylases, and 1446-bp fragment from bacterial ornithine decarboxylases. The method was successfully applied to several biogenic amine-producing bacterial strains, even when DNAs of several target organisms were included in the same reaction. This simple method could be easily incorporated in food control laboratories to detect potentially biogenic amine-producing bacteria in foods.

Multiplex PCR method for the simultaneous detection of histamine-, tyramine-, and putrescine-producing lactic acid bacteria in foods.

In a screening of primers, we have selected three pairs of primers for a multiplex PCR assay for the simultaneous detection of lactic acid bacteria (LAB) strains, which potentially produce histamine, tyramine, and putrescine on fermented foods. These primers were based on sequences from histidine, tyrosine, and ornithine decarboxylases from LAB. Under the optimized conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases, a 924-bp fragment from tyrosine decarboxylases, and a 1,446-bp fragment from ornithine decarboxylases. When the DNAs of several target organisms were included in the same reaction, two or three corresponding amplicons of different sizes were observed. This assay was useful for the detection of amine-producing bacteria in control collection strains and in a LAB collection. No amplification was observed with DNA from nonproducing LAB strains. This article is the first describing a multiplex PCR approach for the simultaneous detection of potentially amine-producing LAB in foods. It can be easily incorporated into the routine screening for the accurate selection of starter LAB and in food control laboratories.

Metabolome progression during early gut microbial colonization of gnotobiotic mice.

The microbiome has been implicated directly in host health, especially host metabolic processes and development of immune responses. These are particularly important in infants where the gut first begins being colonized, and such processes may be modeled in mice. In this investigation we follow longitudinally the urine metabolome of ex-germ-free mice, which are colonized with two bacterial species, Bacteroides thetaiotaomicron and Bifidobacterium longum. High-throughput mass spectrometry profiling of urine samples revealed dynamic changes in the metabolome makeup, associated with the gut bacterial colonization, enabled by our adaptation of non-linear time-series analysis to urine metabolomics data. Results demonstrate both gradual and punctuated changes in metabolite production and that early colonization events profoundly impact the nature of small molecules circulating in the host. The identified small molecules are implicated in amino acid and carbohydrate metabolic processes, and offer insights into the dynamic changes occurring during the colonization process, using high-throughput longitudinal methodology.