Investigation of the gut microbiome, bile acid composition and host immunoinflammatory response in a model of azoxymethane-induced colon cancer at discrete timepoints.

BACKGROUND: Distinct sets of microbes contribute to colorectal cancer (CRC) initiation and progression. Some occur due to the evolving intestinal environment but may not contribute to disease. In contrast, others may play an important role at particular times during the tumorigenic process. Here, we describe changes in the microbiota and host over the course of azoxymethane (AOM)-induced tumorigenesis. METHODS: Mice were administered AOM or PBS and were euthanised 8, 12, 24 and 48 weeks later. Samples were analysed using 16S rRNA gene sequencing, UPLC-MS and qRT-PCR. RESULTS: The microbiota and bile acid profile showed distinct changes at each timepoint. The inflammatory response became apparent at weeks 12 and 24. Moreover, significant correlations between individual taxa, cytokines and bile acids were detected. One co-abundance group (CAG) differed significantly between PBS- and AOM-treated mice at week 24. Correlation analysis also revealed significant associations between CAGs, bile acids and the bile acid transporter, ASBT. Aberrant crypt foci and adenomas were first detectable at weeks 24 and 48, respectively. CONCLUSION: The observed changes precede host hyperplastic transformation and may represent early therapeutic targets for the prevention or management of CRC at specific timepoints in the tumorigenic process.

Associations between the postpartum uterine and vaginal microbiota and the subsequent development of purulent vaginal discharge vary with dairy cow breed and parity.

The objective of this study was to characterize the species composition and functional potential of the vaginal and uterine microbiota at 1 wk postpartum in dairy cows diagnosed with or without purulent vaginal discharge (PVD) at 3 wk postpartum. The hypothesis was that differences in the vaginal and uterine microbiota between cows diagnosed with (PVD+) or without (PVD-) PVD were dependent on parity and breed. Cytobrush samples of the vagina and uterus were collected at 1 wk postpartum from 36 Holstein-Friesian (7 primiparous and 29 multiparous) and 29 Jersey (10 primiparous and 19 multiparous) cows. Microbial DNA was isolated from each sample and processed for shotgun metagenomic sequencing. The odds of multiparous cows being diagnosed as PVD+ was less compared with primiparous cows (OR = 0.21). Neither the α-diversity nor ß-diversity of the uterine and vaginal microbiota were associated with PVD but the ß-diversity was different between breeds and between parities. In the vagina of primiparous cows, differences in the microbiota of PVD- and PVD+ cows were minor, but the microbiota of multiparous PVD+ cows had greater relative abundance of Fusobacterium necrophorum, Trueperella pyogenes, Porphyromonas levii, and greater functional potential for amino acid and protein synthesis, energy metabolism, and growth compared with PVD- cows. The uterus of primiparous PVD+ cows had lesser relative abundance of Bacteroides heparinolyticus compared with PVD- cows. In the uterine microbiota, differences included greater functional potential for cellulose biosynthesis and fucose catabolism in multiparous PVD+ cows compared with PVD- cows. In the uterine microbiota of primiparous PVD+ cows, the functional potential for gram-negative cell wall synthesis and for negative regulation of tumor necrosis factor signaling was lesser compared with multiparous PVD+ cows. In the vagina of Holstein-Friesian PVD+ cows, the relative abundance of Caviibacter abscessus was greater whereas in the vagina of Jersey PVD+ cows the relative abundance of Catenibacterium mitsuokai, Finegoldia magna, Klebsiella variicola, and Streptococcus anginosus was greater compared with PVD- cows. In the uterine microbiota of Holstein-Friesian cows, the functional potential for spermidine biosynthesis was reduced compared with PVD- cows. In summary, differences in the species composition and functional potential of the vaginal and uterine microbiota between PVD- and PVD+ cows were dependent on parity and breed. The findings suggest that alternative strategies may be required to treat PVD for different parities and breeds of dairy cow.

The intestinal protist Blastocystis is not a common member of the healthy infant gut microbiota in a Westernized country (Ireland).

Research into the gut microbiota of human infants is necessary in order to better understand how inter-species interactions and ecological succession shape the diversity of the gut microbiota, and in turn, how the specific composition of the gut microbiota impacts on host health both during infancy and in later years. Blastocystis is a ubiquitous intestinal protist that has been linked to a number of intestinal and extra-intestinal diseases. However, emerging data show that asymptomatic carriage is common and that Blastocystis is prevalent in the healthy adult gut microbiota. Nonetheless, little is known about the prevalence and diversity of this microorganism in the healthy infant gut, including when and how individuals become colonized by Blastocystis. Here, we surveyed the prevalence and diversity of Blastocystis in an infant population (n = 59) from an industrialized country (Ireland) using Blastocystis-specific primers at three or more time-points up to 24 months old. Only three infants were positive for Blastocystis (prevalence = 5%) and this was only noted for samples collected at month 24. This rate is comparatively low relative to previously reported prevalence rates in the contemporaneous adult population. These data suggest that infants in Westernized countries that are successfully colonized by Blastocystis most likely acquire this microorganism via horizontal transfer.

The influence of rosuvastatin on the gastrointestinal microbiota and host gene expression profiles.

Statins are the most widely prescribed medications worldwide for the treatment of hypercholesterolemia. They inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R), an enzyme involved in cholesterol synthesis in higher organisms and in isoprenoid biosynthesis in some bacteria. We hypothesized that statins may influence the microbial community in the gut through either direct inhibition or indirect mechanisms involving alterations to host responses. We therefore examined the impact of rosuvastatin (RSV) on the community structure of the murine gastrointestinal microbiota. RSV was orally administered to mice and the effects on the gut microbiota, host bile acid profiles, and markers of inflammation were analyzed. RSV significantly influenced the microbial community in both the cecum and feces, causing a significant decrease in α-diversity in the cecum and resulting in a reduction of several physiologically relevant bacterial groups. RSV treatment of mice significantly affected bile acid metabolism and impacted expression of inflammatory markers known to influence microbial community structure (including RegIIIγ and Camp) in the gut. This study suggests that a commonly used statin (RSV) leads to an altered gut microbial composition in normal mice with attendant impacts on local gene expression profiles, a finding that should prompt further studies to investigate the implications of statins for gut microbiota stability and health in humans.NEW & NOTEWORTHY This work demonstrates that rosuvastatin administration in mice affects the gastrointestinal microbiota, influences bile acid metabolism, and alters transcription of genes encoding factors involved in gut homeostasis and immunity in the gastrointestinal tract.

The altered gut microbiota in adults with cystic fibrosis.

BACKGROUND: Cystic Fibrosis (CF) is an autosomal recessive disease that affects the function of a number of organs, principally the lungs, but also the gastrointestinal tract. The manifestations of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the gastrointestinal tract, as well as frequent antibiotic exposure, undoubtedly disrupts the gut microbiota. To analyse the effects of CF and its management on the microbiome, we compared the gut microbiota of 43 individuals with CF during a period of stability, to that of 69 non-CF controls using 454-pyrosequencing of the 16S rRNA gene. The impact of clinical parameters, including antibiotic therapy, on the results was also assessed. RESULTS: The CF-associated microbiome had reduced microbial diversity, an increase in Firmicutes and a reduction in Bacteroidetes compared to the non-CF controls. While the greatest number of differences in taxonomic abundances of the intestinal microbiota was observed between individuals with CF and the healthy controls, gut microbiota differences were also reported between people with CF when grouped by clinical parameters including % predicted FEV1 (measure of lung dysfunction) and the number of intravenous (IV) antibiotic courses in the previous 12 months. Notably, CF individuals presenting with severe lung dysfunction (% predicted FEV1 ≤ 40%) had significantly (p < 0.05) reduced gut microbiota diversity relative to those presenting with mild or moderate dysfunction. A significant negative correlation (-0.383, Simpson's Diversity Index) was also observed between the number of IV antibiotic courses and gut microbiota diversity. CONCLUSIONS: This is one of the largest single-centre studies on gut microbiota in stable adults with CF and demonstrates the significantly altered gut microbiota, including reduced microbial diversity seen in CF patients compared to healthy controls. The data show the impact that CF and it's management have on gut microbiota, presenting the opportunity to develop CF specific probiotics to minimise microbiota alterations.

Spatial variation of the colonic microbiota in patients with ulcerative colitis and control volunteers.

OBJECTIVES: The relevance of spatial composition in the microbial changes associated with UC is unclear. We coupled luminal brush samples, mucosal biopsies and laser capture microdissection with deep sequencing of the gut microbiota to develop an integrated spatial assessment of the microbial community in controls and UC. DESIGN: A total of 98 samples were sequenced to a mean depth of 31,642 reads from nine individuals, four control volunteers undergoing routine colonoscopy and five patients undergoing surgical colectomy for medically-refractory UC. Samples were retrieved at four colorectal locations, incorporating the luminal microbiota, mucus gel layer and whole mucosal biopsies. RESULTS: Interpersonal variability accounted for approximately half of the total variance. Surprisingly, within individuals, asymmetric Eigenvector map analysis demonstrated differentiation between the luminal and mucus gel microbiota, in both controls and UC, with no differentiation between colorectal regions. At a taxonomic level, differentiation was evident between both cohorts, as well as between the luminal and mucosal compartments, with a small group of taxa uniquely discriminating the luminal and mucosal microbiota in colitis. There was no correlation between regional inflammation and a breakdown in this spatial differentiation or bacterial diversity. CONCLUSIONS: Our study demonstrates a conserved spatial structure to the colonic microbiota, differentiating the luminal and mucosal communities, within the context of marked interpersonal variability. While elements of this structure overlap between UC and control volunteers, there are differences between the two groups, both in terms of the overall taxonomic composition and how spatial structure is ascribable to distinct taxa.

A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese.

AIMS: In this study, we compare seven different methods which have been designed or modified to extract total DNA from raw milk and raw milk cheese with a view to its subsequent use for the PCR of bacterial DNA. MATERIALS AND RESULTS: Seven extraction methods were employed to extract total DNA from these foods, and their relative success with respect to the yield and purity of the DNA isolated, and its quality as a template for downstream PCR, was compared. Although all of the methods were successful with respect to the extraction of DNA naturally present in cheese, they varied in their relative ability to extract DNA from milk. However, when milk was spiked with a representative Gram-positive (Listeria monocytogenes EGDe) or Gram-negative (Salmonella enterica serovar Typhimurium LT2) bacterium, it was established that all methods successfully extracted DNA which was suitable for subsequent detection by PCR. CONCLUSIONS: Of the seven approaches, the PowerFood™ Microbial DNA Isolation kit (MoBio Laboratories Inc.) was found to most consistently extract highly concentrated and pure DNA with a view to its subsequent use for PCR-based amplification and also facilitated accurate detection by real-time quantitative PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurately assessing the bacterial composition of milk and cheese is of great importance to the dairy industry. Increasingly, DNA-based technologies are being employed to provide an accurate assessment of this microbiota. However, these approaches are dependent on our ability to extract DNA of sufficient yield and purity. This study compares a number of different options and highlights the relative success of these approaches. We also highlight the success of one method to extract DNA from different microbial populations as well as DNA which is suitable for real-time PCR of microbes of interest, a challenge often encountered by the food industry.

The impact of nisin on sensitive and resistant mutants of Listeria monocytogenes in cottage cheese.

AIMS: Listeria monocytogenes ΔgadD1 and ΔlisK mutants display enhanced and reduced sensitivity, respectively, to the food preservative nisin in laboratory media. However, the behaviour of these strains in a nisin-containing food has not been assessed. Here we use cottage cheese as a model food to address this issue. MATERIALS AND RESULTS: Antibiotic-resistant forms of the wild-type and mutant strains were employed to investigate the behaviour of multiple strains in a single food sample, thereby eliminating the problem of intersample variation. Using this approach, it was established that percentage survival of the ΔlisK mutant was greater than the parent strain in the absence of nisin and that this relative difference became even more dramatic in cottage cheese supplemented with nisin. The numbers of the ΔgadD1 mutant decreased more rapidly than the parent in cottage cheese without nisin, but surprisingly this trend was reversed in nisin-supplemented cheese. Upon the addition of 10 mmol l(-1) monosodium glutamate, a substrate for the glutamate decarboxylase (GAD) system, the wild-type LO28 strain regained its relative advantage over ΔgadD1. CONCLUSIONS: Care needs to be taken when predicting the behaviour of mutants of L. monocytogenes with altered resistance to nisin in food as experiments in laboratory media are not always a good indicator of how the strains will behave in such food environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study further emphasizes the importance of utilizing food matrices to confirm observations made using laboratory media.

Impact of the broad-spectrum antimicrobial peptide, lacticin 3147, on Streptococcus mutans growing in a biofilm and in human saliva.

AIMS: To evaluate the ability of the broad-spectrum lantibiotic, lacticin 3147, to prevent Streptococcus mutans biofilm formation and disrupt existing biofilms. METHODS AND RESULTS: Minimum inhibitory concentrations (MIC) and minimum biofilm inhibitory concentrations of purified lacticin 3147 were determined using a microdilution method. Lacticin 3147 effectively inhibited planktonic Strep. mutans, with MIC of 1.9-3.8 µmol l(-1). Time-kill kinetic studies confirmed that lacticin 3147 exhibited bactericidal activity against Strep. mutans at 38 µmol l(-1) (or 10× MIC). The effect of lacticin 3147 on biofilm formation and reduction was also determined. Exposure to 6.3-µmol l(-1) lacticin 3147 (2× MIC) resulted in substantial reductions in Strep. mutans biofilm formation while lacticin 3147 was less effective against 1-day-old biofilms. Culture-based analyses revealed that lacticin 3147 (50 µmol l(-1)) significantly inhibited Streptococcus spp. present in human saliva (P < 0.05) with an approximate 4-log reduction in viability compared with the control. CONCLUSIONS: These results indicate that lacticin 3147 may be an effective therapy against Strep. mutans and was shown to substantially attenuate its ability to form a biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: Lacticin 3147 has the potential to be a useful adjunct to traditional oral therapeutic approaches in addition to its use as a bioactive ingredient for food applications.

Composition and energy harvesting capacity of the gut microbiota: relationship to diet, obesity and time in mouse models.

BACKGROUND AND AIMS: Increased efficiency of energy harvest, due to alterations in the gut microbiota (increased Firmicutes and decreased Bacteroidetes), has been implicated in obesity in mice and humans. However, a causal relationship is unproven and contributory variables include diet, genetics and age. Therefore, we explored the effect of a high-fat (HF) diet and genetically determined obesity (ob/ob) for changes in microbiota and energy harvesting capacity over time. METHODS: Seven-week-old male ob/ob mice were fed a low-fat diet and wild-type mice were fed either a low-fat diet or a HF-diet for 8 weeks (n=8/group). They were assessed at 7, 11 and 15 weeks of age for: fat and lean body mass (by NMR); faecal and caecal short-chain fatty acids (SCFA, by gas chromatography); faecal energy content (by bomb calorimetry) and microbial composition (by metagenomic pyrosequencing). RESULTS: A progressive increase in Firmicutes was confirmed in both HF-fed and ob/ob mice reaching statistical significance in the former, but this phylum was unchanged over time in the lean controls. Reductions in Bacteroidetes were also found in ob/ob mice. However, changes in the microbiota were dissociated from markers of energy harvest. Thus, although the faecal energy in the ob/ob mice was significantly decreased at 7 weeks, and caecal SCFA increased, these did not persist and faecal acetate diminished over time in both ob/ob and HF-fed mice, but not in lean controls. Furthermore, the proportion of the major phyla did not correlate with energy harvest markers. CONCLUSION: The relationship between the microbial composition and energy harvesting capacity is more complex than previously considered. While compositional changes in the faecal microbiota were confirmed, this was primarily a feature of high-fat feeding rather than genetically induced obesity. In addition, changes in the proportions of the major phyla were unrelated to markers of energy harvest which changed over time. The possibility of microbial adaptation to diet and time should be considered in future studies.

Investigating the Role of Diet and Exercise in Gut Microbe-Host Cometabolism.

We investigated the individual and combined effects of diet and physical exercise on metabolism and the gut microbiome to establish how these lifestyle factors influence host-microbiome cometabolism. Urinary and fecal samples were collected from athletes and less active controls. Individuals were further classified according to an objective dietary assessment score of adherence to healthy dietary habits according to WHO guidelines, calculated from their proton nuclear magnetic resonance (1H-NMR) urinary profiles. Subsequent models were generated comparing extremes of dietary habits, exercise, and the combined effect of both. Differences in metabolic phenotypes and gut microbiome profiles between the two groups were assessed. Each of the models pertaining to diet healthiness, physical exercise, or a combination of both displayed a metabolic and functional microbial signature, with a significant proportion of the metabolites identified as discriminating between the various pairwise comparisons resulting from gut microbe-host cometabolism. Microbial diversity was associated with a combination of high adherence to healthy dietary habits and exercise and was correlated with a distinct array of microbially derived metabolites, including markers of proteolytic activity. Improved control of dietary confounders, through the use of an objective dietary assessment score, has uncovered further insights into the complex, multifactorial relationship between diet, exercise, the gut microbiome, and metabolism. Furthermore, the observation of higher proteolytic activity associated with higher microbial diversity indicates that increased microbial diversity may confer deleterious as well as beneficial effects on the host.IMPORTANCE Improved control of dietary confounders, through the use of an objective dietary assessment score, has uncovered further insights into the complex, multifactorial relationship between diet, exercise, the gut microbiome, and metabolism. Each of the models pertaining to diet healthiness, physical exercise, or a combination of both, displayed a distinct metabolic and functional microbial signature. A significant proportion of the metabolites identified as discriminating between the various pairwise comparisons result from gut microbe-host cometabolism, and the identified interactions have expanded current knowledge in this area. Furthermore, although increased microbial diversity has previously been linked with health, our observation of higher microbial diversity being associated with increased proteolytic activity indicates that it may confer deleterious as well as beneficial effects on the host.

CDO: an oncogene-, serum-, and anchorage-regulated member of the Ig/fibronectin type III repeat family.

Cell adhesion molecules of the Ig superfamily are implicated in a wide variety of biological processes, including cell migration, axon guidance and fasciculation, and growth control and tumorigenesis. Expression of these proteins can be highly dynamic and cell type specific, but little is known of the signals that regulate such specificity. Reported here is the molecular cloning and characterization of rat CDO, a novel cell surface glycoprotein of the Ig superfamily that contains five Ig-like repeats, followed by three fibronectin type III-like repeats in its extracellular region, and a 256-amino acid intracellular region that does not resemble other known proteins. In rat embryo fibroblasts, cdo mRNA expression is maximal in confluent, quiescent cells. It is rapidly and transiently down-regulated by serum stimulation of such cells, and is constitutively down-regulated in oncogene-transformed derivatives of these cells. CDO protein levels are also dramatically regulated by cell-substratum adhesion, via a mechanism that is independent of cdo mRNA expression. The amount of CDO produced at the surface of a cell may therefore be governed by a complex balance of signals, including mitogenic stimuli that regulate cdo mRNA levels, and substratum-derived signals that regulate CDO protein production. cdo mRNA is expressed at low levels in most adult rat tissues. A closely related human gene maps to chromosome 11q23-24, a region that displays frequent loss of heterozygosity in human lung, breast, and ovarian tumors. Taken together, these data suggest that loss of CDO function could play a role in oncogenesis.

Controlling Listeria monocytogenes in Cottage cheese through heterologous production of enterocin A by Lactococcus lactis.

AIMS: Enterocin A is an example of a class IIa bacteriocin with potent anti-listerial activity. This study was initiated with a view to harnessing this activity, through heterologous production by a lactococcal starter strain, to limit levels of Listeria monocytogenes in a food (Cottage cheese). METHODS AND RESULTS: Plasmid pEnt02 (containing entA, I, T and D genes under the control of a constitutive promoter) was introduced into a Lactococcus lactis strain capable of fermenting lactose. When this bacteriocin-producing starter was used in combination with a non-enterocin A producer, thereby compensating for an associated reduction in acid production, during a Cottage cheese fermentation, a decrease in L. monocytogenes (tagged with lux genes for convenience) levels was evident. CONCLUSIONS: Enterocin A, heterologously produced by a food grade lactic acid bacteria (LAB), was therefore shown to have potential for use as a biocontrol agent in food. SIGNIFICANCE AND IMPACT OF THE STUDY: Many of the most active anti-listerial compounds identified to date are enterocins. However, because of Enterococcus-associated concerns, the use of these antimicrobials in a food setting has been curtailed. Although enterocins have been heterologously produced in LAB to overcome this problem, this study represents the first occasion upon which the benefits of such heterologous production have been demonstrated in a food context.

Omics-Based Insights into Flavor Development and Microbial Succession within Surface-Ripened Cheese.

In this study, a young Cheddar curd was used to produce two types of surface-ripened cheese, using two commercial smear-culture mixes of yeasts and bacteria. Whole-metagenome shotgun sequencing was used to screen the microbial population within the smear-culture mixes and on the cheese surface, with comparisons of microorganisms at both the species and the strain level. The use of two smear mixes resulted in the development of distinct microbiotas on the surfaces of the two test cheeses. In one case, most of the species inoculated on the cheese established themselves successfully on the surface during ripening, while in the other, some of the species inoculated were not detected during ripening and the most dominant bacterial species, Glutamicibacter arilaitensis, was not a constituent of the culture mix. Generally, yeast species, such as Debaryomyces hansenii and Geotrichum candidum, were dominant during the first stage of ripening but were overtaken by bacterial species, such as Brevibacterium linens and G. arilaitensis, in the later stages. Using correlation analysis, it was possible to associate individual microorganisms with volatile compounds detected by gas chromatography-mass spectrometry in the cheese surface. Specifically, D. hansenii correlated with the production of alcohols and carboxylic acids, G. arilaitensis with alcohols, carboxylic acids and ketones, and B. linens and G. candidum with sulfur compounds. In addition, metagenomic sequencing was used to analyze the metabolic potential of the microbial populations on the surfaces of the test cheeses, revealing a high relative abundance of metagenomic clusters associated with the modification of color, variation of pH, and flavor development. IMPORTANCE Fermented foods, in particular, surface-ripened cheese, represent a model to explain the metabolic interactions which regulate microbial succession in complex environments. This study explains the role of individual species in a heterogeneous microbial environment, i.e., the exterior of surface-ripened cheese. Through whole-metagenome shotgun sequencing, it was possible to investigate the metabolic potential of the resident microorganisms and show how variations in the microbial populations influence important aspects of cheese ripening, especially flavor development. Overall, in addition to providing fundamental insights, this research has considerable industrial relevance relating to the production of fermented food with specific qualities.

Late-onset X-linked sideroblastic anemia. Missense mutations in the erythroid delta-aminolevulinate synthase (ALAS2) gene in two pyridoxine-responsive patients initially diagnosed with acquired refractory anemia and ringed sideroblasts.

X-linked sideroblastic anemia (XLSA) is caused by mutations of the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) resulting in deficient heme synthesis. The characteristic hypochromic, microcytic anemia typically becomes manifest in the first three decades of life. Hematologic response to pyridoxine is variable and rarely complete. We report two unrelated cases of highly pyridoxine-responsive XLSA in geriatric patients previously diagnosed with refractory anemia and ringed sideroblasts. A previously unaffected 77-yr-old male and an 81-yr-old female were each found to have developed severe hypochromic, microcytic anemia with ringed sideroblasts in the bone marrow, which responded dramatically to pyridoxine with normalization of hemoglobin values. Sequence analysis identified an A to C transversion in exon 7 (K299Q) of the ALAS2 gene in the male proband and his daughter. In the female proband a G to A transition was identified in exon 5 (A172T). This mutation resulted in decreased in vitro stability of bone marrow delta-aminolevulinate synthase activity. Each patient's recombinant mutant ALAS2 enzyme had marked thermolability. Addition of pyridoxal 5'-phosphate in vitro stabilized the mutant enzymes, consistent with the observed dramatic response to pyridoxine in vivo. This late-onset form of XLSA can be distinguished from refractory anemia and ringed sideroblasts by microcytosis, pyridoxine-responsiveness, and ALAS2 mutations. These findings emphasize the need to consider all elderly patients with microcytic sideroblastic anemia as candidates for XLSA, especially if pyridoxine responsiveness is demonstrated.

Lack of Heterogeneity in Bacteriocin Production Across a Selection of Commercial Probiotic Products.

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. Bacteriocin production has often been mooted as a desirable probiotic trait and, in specific cases, has been shown to promote probiotic survival within the gastrointestinal tract, contribute to the control of pathogens and even influence host gene expression in the gut. However, it is not clear what proportion of probiotic strains routinely found in commercial products produces bacteriocins, and additionally, it is not known which bacteriocins are produced most frequently. To address this, we conducted a culture-based assessment of the bacteriocinogenic ability of bacterial strains found in a variety of commercially available probiotic products. We detected eight bacteriocin-producing isolates from 16 tested products. Interestingly, in all cases, the isolates were Lactobacillus acidophilus, and the bacteriocin produced was identified as the narrow spectrum class II bacteriocin, lactacin B. The apparent absence of other bacteriocin-producing strains from across these products suggests a lack of heterogeneity in bacteriocin production within probiotic products and suggests that bacteriocin production is not being optimally harnessed as a probiotic trait.

Influence of GABA and GABA-producing Lactobacillus brevis DPC 6108 on the development of diabetes in a streptozotocin rat model.

The aim of this study was to investigate if dietary administration of γ-aminobutyric acid (GABA)-producing Lactobacillus brevis DPC 6108 and pure GABA exert protective effects against the development of diabetes in streptozotocin (STZ)-induced diabetic Sprague Dawley rats. In a first experiment, healthy rats were divided in 3 groups (n=10/group) receiving placebo, 2.6 mg/kg body weight (bw) pure GABA or L. brevis DPC 6108 (~10(9)microorganisms). In a second experiment, rats (n=15/group) were randomised to five groups and four of these received an injection of STZ to induce type 1 diabetes. Diabetic and non-diabetic controls received placebo [4% (w/v) yeast extract in dH2O], while the other three diabetic groups received one of the following dietary supplements: 2.6 mg/kg bw GABA (low GABA), 200 mg/kg bw GABA (high GABA) or ~10(9) L. brevis DPC 6108. L. brevis DPC 6108 supplementation was associated with increased serum insulin levels (P<0.05), but did not alter other metabolic markers in healthy rats. Diabetes induced by STZ injection decreased body weight (P<0.05), increased intestinal length (P<0.05) and stimulated water and food intake. Insulin was decreased (P<0.05), whereas glucose was increased (P<0.001) in all diabetic groups, compared with non-diabetic controls. A decrease (P<0.01) in glucose levels was observed in diabetic rats receiving L. brevis DPC 6108, compared with diabetic-controls. Both the composition and diversity of the intestinal microbiota were affected by diabetes. Microbial diversity in diabetic rats supplemented with low GABA was not reduced (P>0.05), compared with non-diabetic controls while all other diabetic groups displayed reduced diversity (P<0.05). L. brevis DPC 6108 attenuated hyperglycaemia induced by diabetes but additional studies are needed to understand the mechanisms involved in this reduction.

Epigenetic detection of human chromosome 14 uniparental disomy.

The recent demonstration of genomic imprinting of DLK1 and MEG3 on human chromosome 14q32 indicates that these genes might contribute to the discordant phenotypes associated with uniparental disomy (UPD) of chromosome 14. Regulation of imprinted expression of DLK1 and MEG3 involves a differentially methylated region (DMR) that encompasses the MEG3 promoter. We exploited the normal differential methylation of the DLK1/MEG3 region to develop a rapid diagnostic PCR assay based upon an individual's epigenetic profile. We used methylation-specific multiplex PCR in a retrospective analysis to amplify divergent lengths of the methylated and unmethylated MEG3 DMR in a single reaction and accurately identified normal, maternal UPD14, and paternal UPD14 in bisulfite converted DNA samples. This approach, which is based solely on differential epigenetic profiles, may be generally applicable for rapidly and economically screening for other imprinting defects associated with uniparental disomy, determining loss of heterozygosity of imprinted tumor suppressor genes, and identifying gene-specific hypermethylation events associated with neoplastic progression.

Characterization of a long-term survivor with Stüve-Wiedemann syndrome and mosaicism of a supernumerary marker chromosome.

Stüve-Wiedemann syndrome (SWS) is typically lethal in the neonatal period; only two patients have been reported with a longer survival. We report a new patient with SWS, who at 9 years of age is one of the longest survivors with this disorder. In addition to the characteristic features of SWS, she has a number of unique clinical signs, including lack of corneal and patellar reflexes, a smooth tongue with no fungiform papillae, chronic gingival abscesses, mottled, poor dentition, blotchy pigmentation of the skin, unusual infections, multiple fractures, and progressive scoliosis. Cytogenetic analysis identified mosaicism for a supernumerary marker chromosome (SMC), seen in the majority of amniocytes, blood, and skin fibroblasts. The SMC was shown to be derived from chromosome 5 and contains euchromatin. The significance of the SMC to the etiology of SWS is unknown. This patient further demonstrates that SWS is not universally lethal.