A metagenomic catalog of the early-life human gut virome.

Early-life human gut microbiome is a pivotal driver of gut homeostasis and infant health. However, the viral component (known as "virome") remains mostly unexplored. Here, we establish the Early-Life Gut Virome (ELGV), a catalog of 160,478 non-redundant DNA and RNA viral sequences from 8130 gut virus-like particles (VLPs) enriched or bulk metagenomes in the first three years of life. By clustering, 82,141 viral species are identified, 68.3% of which are absent in existing databases built mainly from adults, and 64 and 8 viral species based on VLPs-enriched and bulk metagenomes, respectively, exhibit potentials as biomarkers to distinguish infants from adults. With the largest longitudinal population of infants profiled by either VLPs-enriched or bulk metagenomic sequencing, we track the inherent instability and temporal development of the early-life human gut virome, and identify differential viruses associated with multiple clinical factors. The mother-infant shared virome and interactions between gut virome and bacteriome early in life are further expanded. Together, the ELGV catalog provides the most comprehensive and complete metagenomic blueprint of the early-life human gut virome, facilitating the discovery of pediatric disease-virome associations in future.

Anionic liposome formulation for oral delivery of thuricin CD, a potential antimicrobial peptide therapeutic.

Thuricin CD is a two-peptide antimicrobial produced by Bacillus thuringiensis. Unlike previous antibiotics, it has shown narrow spectrum activity against Clostridioides difficile, a bacterium capable of causing infectious disease in the colon. However, peptide antibiotics have stability, solubility, and permeability problems that can affect their performance in vivo. This work focuses on the bioactivity and bioavailability of thuricin CD with a view to developing a formulation for delivery of active thuricin CD peptides through the gastrointestinal tract (GIT) for local delivery in the colon. The results indicate that thuricin CD is active at low concentrations only when both peptides are present. While thuricin CD was degraded by proteases and was unstable and poorly soluble in gastric fluid, it showed increased solubility in intestinal fluid, probably due to micelle encapsulation. Based on this, thuricin CD was encapsulated in anionic liposomes, which showed increased activity compared to the free peptide, maintained activity after exposure to pepsin in gastric fluid and intestinal fluid, was stable in suspension for over 21 days at room temperature and for 60 days at 4 °C, and exhibited no toxicity to epithelial intestinal cells. These findings suggest that an anionic lipid-based nano formulation may be a promising approach for local oral delivery of thuricin CD.

Sudanese Toombak smokeless tobacco users harbour significantly altered long-term cortisol body production.

The Sudanese, in particular its male population, are known to utilise a smokeless tobacco product (Toombak) which is placed in the oral cavity and can be replaced several times a day. Toombak has been shown to harm human health and is highly addictive. The effect on body cortisol response over a retrospective period in users of this product has not been previously explored. In addition, psycho-dependency scores of Toombak users have not been analysed. In this study, 37 male subjects, age 18-45 years were recruited, of which 18 were non-users of Toombak and 19 were Toombak users. One hair sample was collected from each user and non-user of Toombak. Each hair sample (n=37) was placed in a pre-prepared long piece of foil with two labels on either side marked: 'scalp-side' and 'distant-side'. Cortisol was extracted by mincing 10 mg of 'scalp-side' hair, not exceeding 3 cm, with methanol addition, incubation, and sonication. Cortisol was measured using the enzyme-linked immunosorbent assay kit (Enzo Life Sciences, UK). The amount of hair cortisol in the samples was determined using spectrophotometry at wavelength 405 nm measured in pg/ml and visualised with a four parametric logistic curve. Toombak users were further asked to complete the Fagerstrom Test for Nicotine Dependence-Smokeless Tobacco questionnaire (FTND-ST) comprising of six questions. Scores of > 5 indicated a significant dependence, while a score of < 4 marked low to moderate dependence. The mean concentration of hair cortisol in Toombak users (9.7 pg/ml) was significantly lower (p=0.023) compared to non-users (19.4 pg/ml), with total concentrations ranging from 2.1 to 55.6 pg/ml. FTND-ST scores ranged from 4 to 9, with high levels of psycho-dependency (score > 5) and nicotine tolerance found in 85 % of Toombak users. Cortisol body release in Sudanese smokeless tobacco users was found to be significantly altered. While low cortisol levels do lead to anxiolytic effects, in the long-term, this can allow for increased susceptibility to low cortisol-associated diseases.

A compendium of 32,277 metagenome-assembled genomes and over 80 million genes from the early-life human gut microbiome.

Age-specific reference genomes of the human gut microbiome can provide higher resolution for metagenomic analyses including taxonomic classification, strain-level genomic investigation and functional characterization. We present the Early-Life Gut Genomes (ELGG) catalog with 32,277 genomes representing 2172 species from 6122 fecal metagenomes collected from children under 3 years old spanning delivery mode, gestational age, feeding pattern, and geography. The ELGG substantially expanded the phylogenetic diversity by 38% over the isolate microbial genomes, and the genomic landscape of the early-life microbiome by increasing recruitment of metagenomic reads to 82.8%. More than 60% of the ELGG species lack an isolate representative. The conspecific genomes of the most abundant species from children differed in gene diversity and functions compared to adults. The ELGG genomes encode over 80 million protein sequences, forming the Early-Life Gut Proteins (ELGP) catalog with over four million protein clusters, 29.5% of which lacked functional annotations. The ELGG and ELGP references provided new insights into the early-life human gut microbiome and will facilitate studies to understand the development and mechanisms of disturbances of the human gut microbiome in early life.

Viral biogeography of the mammalian gut and parenchymal organs.

The mammalian virome has been linked to health and disease but our understanding of how it is structured along the longitudinal axis of the mammalian gastrointestinal tract (GIT) and other organs is limited. Here, we report a metagenomic analysis of the prokaryotic and eukaryotic virome occupying luminal and mucosa-associated habitats along the GIT, as well as parenchymal organs (liver, lung and spleen), in two representative mammalian species, the domestic pig and rhesus macaque (six animals per species). Luminal samples from the large intestine of both mammals harboured the highest loads and diversity of bacteriophages (class Caudoviricetes, family Microviridae and others). Mucosal samples contained much lower viral loads but a higher proportion of eukaryotic viruses (families Astroviridae, Caliciviridae, Parvoviridae). Parenchymal organs contained bacteriophages of gut origin, in addition to some eukaryotic viruses. Overall, GIT virome composition was specific to anatomical region and host species. Upper GIT and mucosa-specific viruses were greatly under-represented in distal colon samples (a proxy for faeces). Nonetheless, certain viral and phage species were ubiquitous in all samples from the oral cavity to the distal colon. The dataset and its accompanying methodology may provide an important resource for future work investigating the biogeography of the mammalian gut virome.

Alleviation effects of Bifidobacterium breve on DSS-induced colitis depends on intestinal tract barrier maintenance and gut microbiota modulation.

PURPOSE: The study aimed to investigate the discrepancy and potential mechanisms of different CLA-producing B. breve on dextran sulphate sodium (DSS)-induced colitis. METHODS: Colitis was induced in C57BL/6 J mice using DSS. Disease activity index (DAI), histopathological changes, epithelial barrier integrity and epithelial apoptosis were determined. Gut microbiota were gauged to evaluate the systemic effects of CLA-producing B. breve. RESULTS: Oral administration of different B. breve showed different effects, in which B. breve M1 and B. breve M2 alleviated the inflammation induced by DSS as well as significantly increased the concentration of mucin2 (MUC2) and goblet cells, but neither B. breve M3 nor B. breve M4 had those protective effects. Meanwhile, B. breve M1 and B. breve M2 treatments significantly up-regulated the tight junction (TJ) proteins and ameliorated the epithelial apoptosis lead by DSS challenge. Moreover, inflammatory cytokines (TNF-α, IL-6) were modulated by B. breve M1 and B. breve M2, neither B. breve M3 nor B. breve M4. Furthermore, B. breve M1 and B. breve M2 reduced the abundance of Bacteroides and increased the abundance of Odoribacter, then rebalanced the damaged gut microbiota. Colonic CLA concentrations in mice fed with B. breve M1, B. breve M2, B. breve M3 and B. breve M4 decreased successively, which showed significant positive correlation with the effectiveness of relieving colitis. CONCLUSIONS: Bifidobacterium breve M1 and B. breve M2 alleviated DSS-induced colitis by producing CLA, inhibiting the inflammatory cytokines, maintaining of the intestinal epithelial barrier and regulating the gut microbiota.

Divergent role of abiotic factors in shaping microbial community assembly of paocai brine during aging process.

Microbial communities in fermented food are shaped by a myriad of abiotic factors. The respective roles of abiotic factors in shaping the dynamic bacterial community of paocai during aging remain unclear. In the present study, 100 paocai samples (pH: 2.95-5.23; NaCl content: 0.13-15.41%; total acid: 6.61-18.33 mg/mL; total sugars: 7.96-487.90 µg/mL; total viable count: 3.55-8.99 LogCFU/mL; aging time: 5 day-15 year) were analyzed through high-throughput sequencing and the results revealed five dominant bacterial genera across different samples, including Lactobacillus, Pediococcus, Leuconostoc, Lactococcus and unclassified genera. Both NaCl and total acid (TA) were the major factors regulating bacterial community divergence in paocai. Based on these results, the microbial communities were reconstructed by manipulating the TA and NaCl contents in vitro to validate the effectiveness of these factors in shaping microbial communities during paocai fermentation. Results showed that roles of abiotic factors differentiated during fermentation. At the early stage, salt was the first abiotic filter, mainly working through promoting the abundance of Lactococcus and Leuconostoc. As the TA content increased, the selective role of salt weakened while acid became the dominant at the later stage, as evidenced by the increased abundance of Lactobacillus and Pediococcus following the increase of TA content.

Bioengineering nisin to overcome the nisin resistance protein.

The emergence and dissemination of antibiotic resistant bacteria is a major medical challenge. Lantibiotics are highly modified bacterially produced antimicrobial peptides that have attracted considerable interest as alternatives or adjuncts to existing antibiotics. Nisin, the most widely studied and commercially exploited lantibiotic, exhibits high efficacy against many pathogens. However, some clinically relevant bacteria express highly specific membrane-associated nisin resistance proteins. One notable example is the nisin resistance protein that acts by cleaving the peptide bond between ring E and the adjacent serine 29, resulting in a truncated peptide with significantly less activity. We utilised a complete bank of bioengineered nisin (nisin A) producers in which the serine 29 residue has been replaced with every alternative amino acid. The nisin A S29P derivative was found to be as active as nisin A against a variety of bacterial targets but, crucially, exhibited a 20-fold increase in specific activity against a strain expressing the nisin resistance protein. Another derivative, nisin PV, exhibited similar properties but was much less prone to oxidation. This version of nisin with enhanced resistance to specific resistance mechanisms could prove useful in the fight against antibiotic resistant pathogens.

Comparative genomics of Cp8viruses with special reference to Campylobacter phage vB_CjeM_los1, isolated from a slaughterhouse in Ireland.

Campylobacter phage vB_CjeM_Los1 was recently isolated from a slaughterhouse in the Republic of Ireland using the host Campylobacter jejuni subsp. jejuni PT14, and full-genome sequencing and annotation were performed. The genome was found to be 134,073 bp in length and to contain 169 predicted open reading frames. Transmission electron microscopy images of vB_CjeM_Los1 revealed that it belongs to the family Myoviridae, with tail fibres observed in both extended and folded conformations, as seen in T4. The genome size and morphology of vB_CjeM_Los1 suggest that it belongs to the genus Cp8virus, and seven other Campylobacter phages with similar size characteristics have also been fully sequenced. In this work, comparative studies were performed in relation to genomic rearrangements and conservation within each of the eight genomes. None of the eight genomes were found to have undergone internal rearrangements, and their sequences retained more than 98% identity with one another despite the widespread geographical distribution of each phage. Whole-genome phylogenetics were also performed, and clades were shown to be representative of the differing number of tRNAs present in each phage. This may be an indication of lineages within the genus, despite their striking homology.

The viability of probiotics in water, breast milk, and infant formula.

The aim of this study was to determine bacteriological stability of a probiotic mixture dispersed in various diluents. The commercially available probiotic (Infloran®), containing Bifidobacterium bifidum (109 CFU/250 mg tablet) and Lactobacillus acidophilus (109 CFU/250 mg tablet), was dispersed within expressed breast milk, sterile water, and infant formula and examined at temperatures of 4 and 21 °C. When stored at 4 °C, significant decreases (P < 0.05) in the level of L. acidophilus and B. bifidum were observed in expressed breast milk and sterile water after a 6-h period. However, when stored in infant formula, both strains remained stable over a 12-h period. When stored at 21 °C, a significant decrease (P < 0.05) was observed in the level of L. acidophilus in sterile water, expressed breast milk and infant formula throughout a 12-h period. However, no significant decrease was observed overtime in B. bifidum in all three diluents at this temperature. CONCLUSION: Our findings suggest that, when stored at 4 °C, this probiotic product can remain at a stable condition for 6 h in sterile water and infant formula; however, the viability of the probiotic decreases significantly after this period of time. Administration of this probiotic in sterile water can be an acceptable alternative to dispersion and administration in expressed breast milk. What is Known: • Administration of probiotics containing lactobacilli and bifidobacteria has become more widespread in neonatology, mainly as prophylaxis for the prevention of necrotising entercolitis in preterm infants. • Probiotic reconstitution, from its powder base, is not standardized and various diluents, including sterile water, breast milk, and infant formula, have been used. What is New: • When stored at 4 °C, a probiotic containing lactobacilli and bifidobacteria remains at a stable microbological condition for up to 6 h in sterile water. • Administration of this probiotic dispersed in sterile water, followed by an EBM feed, can be an acceptable alternative to dispersion and administration in EBM.

Omega-3 polyunsaturated fatty acids critically regulate behaviour and gut microbiota development in adolescence and adulthood.

BACKGROUND: Neurodevelopment is strongly influenced by maternal and early-postnatal diet. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are vital structural and functional components of the developing brain. The gut microbiota is also influenced by n-3 PUFA status, however, little is known about the role of maternal and early-life n-3 PUFA intake on offspring gut microbiota development and subsequent interactions with central nervous system functioning and behavioural outcomes. METHODS: Pregnant female C57BL/6 mice and their male offspring were fed a control (CON), omega-3 deficient (O3-) or omega-3 supplemented (O3+) diet. Cognitive, depressive and social behaviours were assessed through a battery of behaviour tests in the male offspring at both adolescence (week 4-5) and adulthood (week 11-13). Hypothalamic-pituitary-adrenal axis (HPA) activation was assessed by analysis of stress-induced corticosterone production. Fecal microbiota composition was analysed by 16S sequencing at both adolescent and adulthood. In addition, stimulated spleen cytokine levels were assessed. RESULTS: n-3 PUFA interventions induced subtle changes in offspring early-life and adolescent behaviours, which were further evident in adulthood, such that O3- animals displayed impaired communication, social and depression-related behaviours and O3+ animals displayed enhanced cognition. O3- mice displayed an elevated Firmicutes:Bacteroidetes ratio and blunted systemic LPS responsiveness. Contrastingly, O3+ mice displayed greater fecal Bifidobacterium and Lactobacillus abundance and dampened HPA-axis activity. CONCLUSIONS: Neurobehavioural development related to cognitive, anxiety and social behaviours, is highly dependent upon in utero and lifelong n-3 PUFA availability. In addition, neurobehavioural changes induced by altering n-3 PUFA status are closely associated with comprehensive alterations in gut microbiota composition, HPA-axis activity and inflammation.

Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera.

Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.

Carbohydrate catabolic diversity of bifidobacteria and lactobacilli of human origin.

Because increased proportions of particular commensal bacteria such as bifidobacteria and lactobacilli have been linked to human health through a variety of mechanisms, there is corresponding interest in identifying carbohydrates that promote growth and metabolic activity of these bacteria. We evaluated the ability of 20 carbohydrates, including several commercially available carbohydrates that are sold as prebiotic ingredients, to support growth of 32 human-derived isolates belonging to the genera Bifidobacterium and Lactobacillus, including those isolated from healthy elderly subjects. In general, bifidobacterial strains were shown to display more diverse carbohydrate utilization profiles compared to the tested Lactobacillus species, with several bifidobacterial strains capable of metabolizing xylo-oligosaccharide (XOS), arabinoxylan, maltodextrin, galactan and carbohydrates containing fructo-oligosaccharide (FOS) components. In contrast, maltodextrin, galactan, arabinogalactan and galactomannan did not support robust growth (≥0.8 OD600 nm) of any of the Lactobacillus strains assessed. Carbohydrate fermentation was variable among strains tested of the same species for both genera. This study advances our knowledge of polysaccharide utilization by human gut commensals, and provides information for the rational design of selective prebiotic food ingredients.

Transcription of two adjacent carbohydrate utilization gene clusters in Bifidobacterium breve UCC2003 is controlled by LacI- and repressor open reading frame kinase (ROK)-type regulators.

Members of the genus Bifidobacterium are commonly found in the gastrointestinal tracts of mammals, including humans, where their growth is presumed to be dependent on various diet- and/or host-derived carbohydrates. To understand transcriptional control of bifidobacterial carbohydrate metabolism, we investigated two genetic carbohydrate utilization clusters dedicated to the metabolism of raffinose-type sugars and melezitose. Transcriptomic and gene inactivation approaches revealed that the raffinose utilization system is positively regulated by an activator protein, designated RafR. The gene cluster associated with melezitose metabolism was shown to be subject to direct negative control by a LacI-type transcriptional regulator, designated MelR1, in addition to apparent indirect negative control by means of a second LacI-type regulator, MelR2. In silico analysis, DNA-protein interaction, and primer extension studies revealed the MelR1 and MelR2 operator sequences, each of which is positioned just upstream of or overlapping the correspondingly regulated promoter sequences. Similar analyses identified the RafR binding operator sequence located upstream of the rafB promoter. This study indicates that transcriptional control of gene clusters involved in carbohydrate metabolism in bifidobacteria is subject to conserved regulatory systems, representing either positive or negative control.

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 production of conjugated α-linolenic, γ-linolenic and stearidonic acids by strains of bifidobacteria and propionibacteria.

Conjugated fatty acids are regularly found in nature and have a history of biogenic activity in animals and humans. A number of these conjugated fatty acids are microbially produced and have been associated with potent anti-carcinogenic, anti-adipogenic, anti-atherosclerotic and anti-diabetogenic activities. Therefore, the identification of novel conjugated fatty acids is highly desirable. In this study, strains of bifidobacteria and propionibacteria previously shown by us and others to display linoleic acid isomerase activity were assessed for their ability to conjugate a range of other unsaturated fatty acids during fermentation. Only four, linoleic, α-linolenic, γ-linolenic and stearidonic acids, were converted to their respective conjugated isomers, conjugated linoleic acid (CLA), conjugated α-linolenic acid (CLNA), conjugated γ-linolenic acid (CGLA) and conjugated stearidonic acid (CSA), each of which contained a conjugated double bond at the 9,11 position. Of the strains assayed, Bifidobacterium breve DPC6330 proved the most effective conjugated fatty acid producer, bio-converting 70% of the linoleic acid to CLA, 90% of the α-linolenic acid to CLNA, 17% of the γ-linolenic acid to CGLA, and 28% of the stearidonic acid to CSA at a substrate concentration of 0.3 mg mL⁻¹. In conclusion, strains of bifidobacteria and propionibacteria can bio-convert linoleic, α-linolenic, γ-linolenic and stearidonic acids to their conjugated isomers via the activity of the enzyme linoleic acid isomerase. These conjugated fatty acids may offer the combined health promoting properties of conjugated fatty acids such as CLA and CLNA, along with those of the unsaturated fatty acids from which they are formed.

A study of the prevalence of methicillin-resistant Staphylococcus aureus in pigs and in personnel involved in the pig industry in Ireland.

To evaluate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the pig population in Ireland, nasal swabbing was employed in three abattoirs to screen 440 pigs from 41 geographically distributed farms. One hundred individuals involved in the pig industry were also nasally screened. No MRSA isolates were recovered from the pigs and only two of the humans tested were identified as MRSA carriers. Importantly, MRSA was not obtained from pig producers, veterinarians or abattoir employees, but was isolated from individuals working in the wider pig industry. Multi-locus sequence typing revealed that these isolates belonged to sequence types (ST) ST22 and ST1307; the latter is a previously unreported single locus variant of ST5. Five dust samples from each of the three slaughterhouses were culture-negative for MRSA. These results indicate that porcine colonisation by MRSA, and in particular the animal-related strain MRSA-ST398, was not common in Ireland during the period of study.

Efficacy of a teat dip containing the bacteriocin lacticin 3147 to eliminate Gram-positive pathogens associated with bovine mastitis.

On most dairy farms teat dips are applied to the teats of cows either before or after milking in order to prevent pathogens from gaining access to the mammary gland via the teat canal. In the present experiments, a natural teat dip was developed using a fermentate containing the live bacterium Lactococcus lactis DPC 3251. This bacterium produces lacticin 3147, a two-component lantibiotic which was previously shown to effectively kill Gram-positive mastitis pathogens. Lacticin 3147 activity in the fermentate was retained at 53% of its original level following storage for 3 weeks at 4 degrees C. In the initial experiments in vitro, 105 colony-forming units/ml (cfu/ml) of either Staphylococcus aureus, Streptococcus dysgalactiae or Streptococcus uberis were introduced into the lacticin-containing fermentate. Neither Staph. aureus nor Str. dysgalactiae could be detected after 30 min or 15 min, respectively, while Str. uberis was reduced approximately 100-fold after 15 min. Following these trials, preliminary experiments were performed in vivo on teats of lactating dairy cows. In these experiments, teats were coated with each of the challenge organisms and then dipped with the lacticin-containing fermented teat dip. Following a dip contact time of 10 min, staphylococci were reduced by 80% when compared with the undipped control teat. Streptococcal challenges were reduced by 97% for Str. dysgalactiae and by 90% for Str. uberis. These trials showed that the teat dip is able to reduce mastitis pathogens on the teats of lactating cows.

Asymptomatic carriage of Clostridium difficile in an Irish continuing care institution for the elderly: prevalence and characteristics.

INTRODUCTION: Clostridium difficile is an increasing cause of nosocomial diarrhoea and colitis. The aim of this study was to identify the prevalence and characteristics of asymptomatic C. difficile carriage in a continuing care institution for the elderly. METHODS: Stool samples were collected from 100 asymptomatic patients, whose median age was 83 years. Samples were tested for C. difficile using traditional culturing methods, 16s rDNA and 16s-23s intergenic spacer (IGS) rDNA sequencing, and analysed for toxin production and toxin genes. RESULTS: The prevalence of C. difficile carriage was 10/100 (10%) following culture and 16s and IGS sequencing. An additional seven isolates, initially identified as C. difficile, were subsequently identified by IGS rDNA sequencing as C. sordellii of the 10% that tested positive for C. difficile, seven tested positive for toxin A and B. A significant number of C. difficile carriers had recent antibiotic exposure compared with non-carriers, P = 0.046. CONCLUSION: The prevalence of asymptomatic C. difficile carriage in this institution was 10%, 7% of which were toxin positive. This study underscores the importance of increased vigilance for C. difficile using microbial and molecular methodology and identifies patients at increased risk following antibiotic administration.

Inhibitory effect of conjugated alpha-linolenic acid from bifidobacteria of intestinal origin on SW480 cancer cells.

In this study, we assessed the ability of six strains of bifidobacteria (previously shown by us to possess the ability to convert linoleic acid to c9, t11-conjugated linoleic acid (CLA) to grow in the presence of alpha-linolenic acid and to generate conjugated isomers of the fatty acid substrate during fermentation for 42 h. The six strains of bifidobacteria were grown in modified MRS (mMRS) containing alpha-linolenic acid for 42 h at 37 degrees C, after which the fatty acid composition of the growth medium was assessed by gas liquid chromatography (GLC). Indeed, following fermentation of one of the strains, namely Bifidobacterium breve NCIMB 702258, in the presence of 0.41 mg/ml alpha-linolenic acid, 79.1% was converted to the conjugated isomer, C18:3 c9, t11, c15 conjugated alpha-linolenic acid (CALA). To examine the inhibitory effect of the fermented oils produced, SW480 colon cancer cells were cultured in the presence of the extracted fermented oil (10-50 microg/ml) for 5 days. The data indicate an inhibitory effect on cell growth (p