Antibiotic treatment of pregnant non-obese diabetic mice leads to altered gut microbiota and intestinal immunological changes in the offspring.

The intestinal microbiota is important for tolerance induction through mucosal immunological responses. The composition of the gut microbiota of an infant is affected by environmental factors such as diet, disease and antibiotic treatment. However, already in utero, these environmental factors can affect the immunological development of the foetus and influence the future gut microbiota of the infant. To investigate the effects of antibiotic treatment of pregnant mothers on the offspring's gut microbiome and diabetes development, we treated non-obese diabetic (NOD) mice with a cocktail of antibiotics during gestation and the composition of the gut microbiota, diabetes incidence and major gut-related T lymphocyte populations were investigated in the offspring. We observed a persistent reduction in the general diversity of the gut microbiota in the offspring from NOD mothers treated with antibiotics during gestation compared with offspring from control mothers. In addition, by clustering the present bacterial taxa with principal component analysis, we found a differential clustering of gut microbiota in the offspring from NOD mothers treated with antibiotics during gestation compared with offspring from control mothers. Offspring from NOD mothers treated with antibiotics during gestation also showed some immunological alterations in the gut immune system, which could be related to the diversity of the gut microbiome and influence modulation of diabetes development at 20 weeks. Our data point out maternal derangement of the intestinal microbiota as a potential environmental risk factor for T1D development.

Effects of a high-fat diet during pregnancy and lactation are modulated by E. coli in rat offspring.

OBJECTIVE: Microbial manipulations in early life can affect gut development and inflammatory status of the neonate. The maternal diet during pregnancy and lactation also influences the health of the offspring, but the impact of maternal high-fat (HF) feeding along with modulations of the gut microbiota on body weight, fat deposition and gut function in the offspring has been poorly studied. METHODS: Rat dams were given access to either an HF or a standard low-fat diet during the last 2 weeks of pregnancy and during lactation and effects on body weight and gastrointestinal function were investigated in the 14-day-old offspring. To elucidate whether bacterial administration to the dam could modulate any effects of the diets in the rat pups, another group of dams were given Escherichia coli in their drinking water. RESULTS: Maternal HF feeding resulted in increased body and fat pad weights in the offspring, along with increased levels of the acute-phase protein, haptoglobin and decreased protein content and disaccharidase activities in the small intestine. The addition of E. coli further accentuated these responses in the young rats, which, in addition to higher body weights and increased fat deposition, also showed an increased intestinal permeability and elevated levels of haptoglobin. CONCLUSIONS: The present study demonstrates for the first time how bacterial administration to the maternal diet during the neonatal period can affect body weight and fat deposition in the offspring. The results point to a mechanistic link between the gut microbiota, increased intestinal permeability and metabolic endotoxemia, which appear to have led to increased adiposity in the young rats.

The live bacterial load and microbiota composition of prepacked "ready-to-eat" leafy greens during household conditions, with special reference to E. coli.

Ready-to-eat (RTE) leafy greens are popular products that unfortunately have been associated with numerous foodborne illness outbreaks. Since the influence of consumer practices is essential for their quality and safety, the objective of this study was to analyze the microbiota of RTE products throughout shelf life during simulated household conditions. Products from different companies were analyzed in terms of plate counts, and resealed and unopened packages were compared. High bacterial loads were found, up to a total plate count of 9.6 log10 CFU/g, and Enterobacteriaceae plate counts up to 6.0 CFU/g on the expiration date. The effect of consumer practice varied, thus no conclusions regarding resealed or unopened bags could be drawn. The tested products contained opportunistic pathogens, such as Enterobacter homaechei, Hafnia paralvei and Pantoea agglomerans. Amplicon sequencing revealed that the relative abundance of major taxonomic groups changed during shelf life; Pseudomonadaceae and Xanthomonadaceae decreased, while Flavobacteriaceae and Marinomonadaceae inceased. Inoculation with E. coli CCUG 29300T showed that the relative abundance of Escherichia-Shigella was lower on rocket than on other tested leafy greens. Inoculation with E. coli strain 921 indicate growth at the beginning of shelf-life time, while E. coli 731 increases at the end, seemingly able to adapt to cold storage conditions. The high levels of live microorganisms, the detection of opportunistic pathogens, and the ability of E. coli strains to grow at refrigeration temperature raise concerns and indicate that the shelf life may be shortened to achieve a safer product. Due to variations between products, further studies are needed to define how long the shelf-life of these products should be, to ensure a safe product even at the end of the shelf-life period.

Epidemiological and molecular assessment of a measles outbreak in a highly vaccinated population of northeast Italy.

Two distinct measles outbreaks, unrelated from the epidemiological point of view but caused by genetically related strains, occurred in the Friuli Venezia Giulia region of northeastern Italy. Forty-two cases were reported during the period April-May 2008. In the first outbreak the index case was a teacher who introduced the virus into the Pordenone area, involving eight adolescents and young adults. The other concomitant outbreak occurred in the city of Trieste with 33 cases. The containment of the epidemics can be explained by the high MMR vaccine coverage in an area where the first dose was delivered to 93·4% and the second dose to 88·3% of the target children. Phylogenetic analysis of 14 measles virus strains showed that they belonged to a unique D4 genotype indistinguishable from the MVs/Enfield.GBR/14.07 strain, probably introduced from areas (i.e. Piedmont and Germany) where this genotype was present or had recently caused a large epidemic.

Cervical cancer prevention and health inequalities: an ad-hoc survey in Italian women.

OBJECTIVE: To assess knowledge and attitudes towards cervical cancer prevention in a sample of 2400 Italian women. STUDY DESIGN: Cross-sectional study. METHODS: The study was conducted through a standardized questionnaire administered in the workplace. RESULTS: Regular Pap testing was reported by 65.6% of the sample, and 86.9% were aware of the human papillomavirus (HPV) vaccine. Just over half of the women (51.8%) stated that they would pay for the vaccine for themselves or family members. Significant differences in responses were associated with monthly income and educational level. CONCLUSION: Introduction of payment for the HPV vaccine may increase health inequalities significantly. For overall improvement in the quality of life, effective prevention and treatment services should be made available to all.

Anti-transglutaminase antibodies in non-coeliac children suffering from infectious diseases.

Anti-transglutaminase antibodies are the diagnostic markers of coeliac disease. A role is suggested for infectious agents in the production of anti-transglutaminase antibodies. The aim was to measure positive anti-transglutaminase antibody levels in children with infectious diseases and to compare immunological and biological characteristics of the anti-transglutaminase antibodies derived from these children with that from coeliac patients. Two hundred and twenty-two children suffering from infectious diseases were enrolled prospectively along with seven biopsy-proven coeliacs. Serum samples were tested for anti-transglutaminase antibodies and anti-endomysium antibodies; positive samples were tested for coeliac-related human leucocyte antigen (HLA)-DQ2/8 and anti-viral antibodies. Purified anti-transglutaminase antibodies from the two study groups were tested for urea-dependent avidity, and their ability to induce cytoskeletal rearrangement and to modulate cell-cycle in Caco-2 cells, using phalloidin staining and bromodeoxyuridine incorporation assays, respectively. Nine of 222 children (4%) tested positive to anti-transglutaminase, one of whom also tested positive for anti-endomysium antibodies. This patient was positive for HLA-DQ2 and was diagnosed as coeliac following intestinal biopsy. Of the eight remaining children, two were positive for HLA-DQ8. Levels of anti-transglutaminase returned to normal in all subjects, despite a gluten-containing diet. Purified anti-transglutaminase of the two study groups induced actin rearrangements and cell-cycle progression. During an infectious disease, anti-transglutaminase antibodies can be produced temporarily and independently of gluten. The infection-triggered anti-transglutaminase antibodies have the same biological properties as that of the coeliacs, with the same in-vivo potential for damage.

Blueberry husks and multi-strain probiotics affect colonic fermentation in rats.

The aim was to investigate how blueberry husks and/or mixtures of probiotic strains (Lactobacillus crispatus DSM16743, L. gasseri DSM16737 and L. plantarum DSM15313 (LABmix), or Bifidobacterium infantis DSM15159 and DSM15161 (BIFmix)) affect colonic fermentation, caecal counts of lactobacilli, bifidobacteria and Enterobacteriaceae, body weight gain, and blood concentrations of carboxylic acids (CA) and ammonia in rats. Dietary fibres in blueberry husks were fermented to 61 % in colon, and the elevated faecal excretion of fibre and protein contributed to the high faecal bulking capacity (1.3). The caecal pool of CA was higher in rats fed blueberry husks than the fibre-free control (P < 0.05), and the propionic acid proportion was higher in the distal colon than in the control group (P < 0.05). Probiotics lowered the caecal amount of CA when added to blueberry husks (P < 0.001), while the propionic acid proportion was higher with LABmix (P < 0.01) than blueberry husks only. The propionic acid and butyric acid concentrations in blood were higher in rats fed blueberry husks and probiotics than those fed blueberry husks only (P < 0.01), implying that the absorption of these acids was facilitated by the bacteria. The caecal counts of lactobacilli, bifidobacteria and Enterobacteriaceae were lower in rats fed blueberry husks than the control diet (P < 0.05). The body weight gain was partly influenced by the caecal tissue and contents weights, and BIFmix decreased the ammonia concentration in blood (P < 0.05). We conclude that colonic fermentation is differentially affected by dietary fibre and probiotics, which may be of importance when developing foods with certain health effects.

Lactobacillus fermentum and Lactobacillus plantarum increased gut microbiota diversity and functionality, and mitigated Enterobacteriaceae, in a mouse model.

Probiotics should bring 'balance' to the intestinal microbiota by stimulating beneficial bacteria, whilst mitigating adverse ones. Balance can also be interpreted as high alpha-diversity. Contrary, Escherichia coli is often regarded as an adverse component of the resident intestinal microbiota. The aim of the present study was to implement a mouse model for in vivo screening of Lactobacillus-strains for ability to increase gut-microbiota diversity and to mitigate E. coli. Mice were divided into six groups, two dietary control-groups and four groups administered strains of Lactobacillus fermentum and/or Lactobacillus plantarum. All animals were pre-treated with antibiotics, and E. coli in order to equalise the microbiota from the start. After 7 weeks of Lactobacillus administration, the animals were sacrificed: DNA was extracted from caecum tissue, and the microbiota composition was analysed with terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene sequencing. The diversity of the caecal microbiota decreased when the dietary carbohydrate source was limited to corn starch. Conversely, the diversity was restored by Lactobacillus-supplements. The tested combinations of two Lactobacillus strains exerted different influences, not only on the taxonomic level, but also on the inferred microbiome functions. The mixture of L. fermentum GOS47 and L. fermentum GOS1 showed potential for anti-inflammatory activity and short chain fatty acid production. On the other hand, co-administration of L. fermentum GOS57 and L. plantarum GOS42 significantly decreased the viable count of Enterobacteriaceae. These results warrant further investigation of the tested strains as candidates for probiotics. Furthermore, the findings demonstrated that the current experimental animal model is suitable for in vivo studies of the effect of bacterial supplements on the gut-microbiota.

Lactobacillus plantarum 299v reduces colonisation of Clostridium difficile in critically ill patients treated with antibiotics.

BACKGROUND: The incidence of Clostridium difficile-associated disease (CDAD) in hospitalised patients is increasing. Critically ill patients are often treated with antibiotics and are at a high risk of developing CDAD. Lactobacillus plantarum 299v (Lp299v) has been found to reduce recurrence of CDAD. We investigated intensive care unit (ICU) patients with respect to the impact of Lp299v on C. difficile colonisation and on gut permeability and parameters of inflammation and infection in that context. METHODS: Twenty-two ICU patients were given a fermented oatmeal gruel containing Lp299v, and 22 received an equivalent product without the bacteria. Faecal samples for analyses of C. difficile and Lp299v were taken at inclusion and then twice a week during the ICU stay. Other cultures were performed on clinical indication. Infection and inflammation parameters were analysed daily. Gut permeability was assessed using a sugar probe technique. RESULTS: Colonisation with C. difficile was detected in 19% (4/21) of controls but in none of the Lp299v-treated patients (P<0.05). CONCLUSIONS: Enteral administration of the probiotic bacterium Lp299v to critically ill patients treated with antibiotics reduced colonisation with C. difficile.

Endotoxin- and D-galactosamine-induced liver injury improved by the administration of Lactobacillus, Bifidobacterium and blueberry.

BACKGROUND: D-galactosamine together with lipopolysaccharide can lead to a pronounced secretion by Kupffer cells of pro-inflammatory mediators, which have been shown to be early and important mediators of liver injury. Probiotics and dietary supplementation with fruit or vegetable extracts with high content of antioxidants, such as blueberry, could be beneficial in protecting against hepatotoxicity. AIMS: To investigate whether blueberry and probiotics could attenuate liver injury induced by D-galactosamine and lipopolysaccharide. SUBJECTS: Sprague-Dawley rats were used. METHODS: Six experimental groups: acute liver injury control and five groups of liver injury treated by blueberry alone or by each of the probiotics strains (Lactobacillus plantarum DSM 15313 and Bifidobacterium infantis DSM 15159) with and without blueberry. Samples were collected 24 h after induction for bacterial test, liver function test, short chain fatty acids, myeloperoxidase, cytokines, malondialdehyde and glutathione. RESULTS: Alanine aminotransferase levels decreased significantly in all groups compared to liver injury control and DSM 15313 groups. Bilirubin, liver TNF-alpha, myeloperoxidase and acetic acid in cecum content decreased significantly in all groups, while liver glutathione values increased significantly in all groups compared to liver injury control. Liver IL-1beta and bacterial translocation to the liver and mesenteric lymph nodes decreased significantly in all groups except B. infantis DSM 15159 group compared to the liver injury control. Enterobacteriaceae count in cecum decreased significantly in the groups with blueberry plus probiotics compared to the other groups. CONCLUSION: Blueberry and probiotics exert protective effects on acute liver injury. They reduce the hepatocytes injury, the inflammation and the pro-inflammatory cytokines, and improve the barrier functions and antioxidant activity.

Composition of the bacterial population of refrigerated beef, identified with direct 16S rRNA gene analysis and pure culture technique.

The composition of the dominating population of freshly cut beef, and beef stored at 4 degrees C for 8 d, was studied by direct analysis of the 16S rRNA gene (PCR amplification, cloning and sequencing) and compared with pure culture technique where the isolates picked from the viable plate count were identified by sequencing of the 16S rRNA gene. The composition of the bacterial population was recorded at two different time points, at the start when the viable plate count of the meat was 4 x 10(2) colony forming unit (cfu) per cm(2) and when it was 5 x 10(7) cfu per cm(2). Direct gene analysis by PCR amplification generated 30 clones, and 79 isolates were picked from the plate count, and identified by 16S rRNA gene sequencing. At the low initial bacterial load of the beef, the two sampling strategies showed variations in the composition of species. Direct 16S rRNA gene analysis revealed a domination of Bacillus-like sequences while no such sequences were found in isolates from the viable plate count. Instead the population of the plate count was dominated by Chryseobacterium spp. In contrast, the two sampling strategies matched on the multiplying beef population, where both methods indicated Pseudomonas spp. as the dominating group (99% of the population-sequences), irrespectively of sampling strategy. Pseudomonas panacis/Pseudomons brennerii was the dominating taxon (99% similarity to type strain), but sequences with highest similarity to Pseudomonas lundensis (99%), Pseudomonas beteli (99%) and Pseudomonas koreensis (100%) were also found.

HBV, HCV, and TTV detection by in situ polymerase chain reaction could reveal occult infection in hepatocellular carcinoma: comparison with blood markers.

OBJECTIVE: To report a retrospective analysis on the presence of hepatitis B virus (HBV), hepatitis C virus (HCV), and transfusion transmitted virus (TTV) sequences in formalin fixed, paraffin embedded liver biopsies from eight patients with hepatocellular carcinoma, in comparison with blood markers. METHODS: A direct in situ polymerase chain reaction (PCR) technique was developed for the detection and localisation of genomic signals in the liver tissue. Conventional serological and molecular methods were used for blood evaluation. RESULTS: In situ PCR showed the presence of one of the three viruses (four HCV, two HBV, and one TTV) in seven of the eight patients. In addition, a co-infection with HBV and HCV was detected in one patient. HCV and HBV sequences were located in the cytoplasm and the nucleus, respectively. When compared with blood markers, these findings were compatible with one occult HBV and two occult HCV infections. CONCLUSIONS: These findings provide further evidence for occult HBV and HCV infections in cancerous tissues from patients with hepatocellular carcinomas. In situ PCR could be an additional tool for evaluating the viral aetiology of hepatocellular carcinoma alongside conventional diagnostic procedures.

A population based seroepidemiological survey of Chlamydia pneumoniae infections in schoolchildren.

AIM: A serosurvey was carried out in schoolchildren from a northeastern area of Italy to define the burden of Chlamydia pneumoniae infection. METHODS: A sample of 649 schoolchildren underwent a simplified version of the International Study of Asthma and Allergies in Childhood questionnaire and IgG and IgA antibodies were investigated using an enzyme immunoassay, followed by a microimmunofluorescence assay in reactive sera. RESULTS: Of the children examined, 29% and 19.7% had IgG and IgA antibodies, respectively. The IgG prevalence increased with age. No other sociodemographical variable was related to C pneumoniae infection. An association was established between IgA prevalence and previous otitis media. CONCLUSIONS: A mesoendemic (intermediate between high and low endemic level) pattern of C pneumoniae infection is present in schoolchildren from this area and the prevalence rate is related to age. Moreover, this is the first epidemiological evidence of the role of C pneumoniae in otitis.

Immunological alteration and changes of gut microbiota after dextran sulfate sodium (DSS) administration in mice.

Ulcerative colitis (UC) is characterized by chronic inflammation of the colonic mucosa. Administration of dextran sulfate sodium (DSS) to animals is a frequently used model to mimic human colitis. Deregulation of the immune response to the enteric microflora or pathogens as well as increased intestinal permeability have been proposed as disease-driving mechanisms. To enlarge the understanding of the pathogenesis, we have studied the effect of DSS on the immune system and gut microbiota in mice. Intestinal inflammation was verified through histological evaluation and myeloperoxidase activity. Immunological changes were assessed by flow cytometry in spleen, Peyer's patches and mesenteric lymph nodes and through multiplex cytokine profiling. In addition, quantification of the total amount of bacteria on colonic mucosa as well as the total amount of lactobacilli, Akkermansia, Desulfovibrio and Enterobacteriaceae was performed by the use of quantitative PCR. Diversity and community structure were analysed by terminal restriction fragment length polymorphism (T-RFLP) patterns, and principal component analysis was utilized on immunological and T-RFLP patterns. DSS-induced colitis show clinical and histological similarities to UC. The composition of the colonic microflora was profoundly changed and correlated with several alterations of the immune system. The results demonstrate a relationship between multiple immunological changes and alterations of the gut microbiota after DSS administration. These data highlight and improve the definition of the immunological basis of the disease and suggest a role for dysregulation of the gut microbiota in the pathogenesis of colitis.

Probiotics in foods not containing milk or milk constituents, with special reference to Lactobacillus plantarum 299v.

Lactic acid fermentation is the simplest and safest way of preserving food and has probably always been used by humans. Species such as Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus acidophilus, and Lactobacillus salivarius are common in the human mucosa, from the mouth to the rectum. In food, L. paracasei and L. rhamnosus are usually associated with dairy products whereas L. plantarum is found in fermented foods of plant origin. A probiotic food product containing no milk constituent was launched in Sweden in 1994. The product is a lactic acid fermented oatmeal gruel that is mixed in a fruit drink. It contains approximately 5 x 10(10) colony-forming units of L. plantarum 299v/L. The strain L. plantarum 299v originates from the human intestinal mucosa and has been shown in rats to decrease translocation, improve mucosal status, improve liver status, improve the immunologic status of the mucosa, and reduce mucosal inflammation. In humans, L. plantarum 299v can increase the concentration of carboxylic acids in feces and decrease abdominal bloating in patients with irritable bowel disease. It can also decrease fibrinogen concentrations in blood. Should probiotics be administrated through foods, the probiotic organism must remain vigorous in the food until consumption and the food must remain palatable, ie, the food carrier and the organism must suit each other. L. plantarum 299v not only affects the bacterial flora of the intestinal mucosa but may also regulate the host's immunologic defense. The mechanisms involved need to be clarified.

Dynamic in vitro calcification of bioprosthetic porcine valves: evidence of apatite crystallization.

OBJECTIVE: Calcification is the most important cause of structural deterioration of glutaraldehyde-fixed bioprosthetic valves. Devitalization of tissue favors calcium deposits in the shape of apatite crystals. Host factors influence the extent and progression of calcification, but the phenomenon can also occur in vitro in the absence of a viable milieu. Whether calcific deposits obtained in vitro are similar to those found in vivo is unknown. METHODS: Four porcine frame-mounted bioprostheses (St Jude Medical Bioimplant; St Jude Medical, Inc, St Paul, Minn) were tested in vitro by using a pulsatile accelerated calcification testing device at a frequency of 300 cycles per minute at 37 degrees C for 19 x 10(6) cycles with a rapid synthetic calcification solution (final product [calcium x phosphate], 130 mg/dL(2)). Three of the same type of xenografts explanted from human subjects because of calcific failure (time in place, 108 +/- 25.63 mo) served as control grafts. Each sample underwent gross and x-ray examination, histology, transmission and scanning electron microscopy, atomic absorption spectroscopy, electron microprobe analysis, and x-ray powder diffraction methods. RESULTS: All in vitro bioprostheses were heavily calcific, with intrinsic Von Kossa stain-positive deposits and a mean calcium content of 205.285 +/- 64.87 mg/g dry weight. At transmission electron microscopy, nuclei of calcification involved mostly collagen fibers and interfibrillar spaces and, more rarely, cell debris and nuclei. Electron microprobe analysis showed a Ca/P atoms ratio of 4.5:3, a value intermediate between hydroxyapatite and its precursor, octacalciumphosphate. X-ray powder diffraction showed a well-separated and sharp peak, which is typical of hydroxyapatite. Aggregates of plate-like crystals up to 8 microm in size were observed at scanning electron microscopy, with a typical tabular hexagonal shape consistent with apatite. The morphologic and chemical findings in human explants were similar. CONCLUSIONS: Intrinsic calcification of glutaraldehyde-fixed porcine valves was induced in vitro. Electron microprobe analysis and x-ray powder diffraction findings were in keeping with apatite crystallization, such as that occurring in valve xenografts implanted in vivo. The model may be of value to accelerate the screening of anticalcific agents and may reduce the need for animal experiments.

Dynamics of bacterial translocation in acute liver injury induced by D-galactosamine in rat.

Bacterial translocation may play a role in acute liver injuries as high rates of infectious and septic complications are observed in these clinical conditions. Increased passage of endotoxin and translocating bacteria not only potentiates the extent of liver injury, but may also play a determining role in its final outcome. In this paper the incidence of bacteria] translocation in acute liver injury in rats is evaluated with other important pathological changes observed at different time points after liver injury induced by intraperitoneal administration of D-galactosamine. The bacterial translocation to the blood and other extraintestinal sites starts 3 h after induction of liver injury and is not found to be related to light microscopic changes in the liver or ileal or cecal mucosa, detectable levels of endotoxin in the portal blood, or DNA changes in the small and large intestinal mucosa, but corresponds to the release of liver enzymes in the serum.

Identification of the translocating bacteria in rats with acute liver injury and their relation to the bacterial flora of the intestinal mucosa.

The bacterial flora of the intestine and the bacteria found in liver, mesenteric lymph nodes, portal and arterial blood after D-galactosamine-induced liver injury, with and without pretreatment with Lactobacillus plantarum DSM 9843, were studied in the rat. Dominating representatives were identified to species level by 16S rDNA sequencing and typed by randomly amplified polymorphic DNA (RAPD) and by restriction endonuclease analysis (REA) for strain definition. It was proven that bacterial strains from the intestine occur at extraintestinal sites after liver injury. Lactobacillus spp. dominated the intestinal flora and were also the most frequently found genus in the liver and the mesenteric lymph nodes. Some of the blood isolates, identified as Staphylococcus aureus, Proteus vulgaris and Bacteroides merdae, were not found as a dominating part of the mucosal flora. Treatment with L. plantarum before liver injury decreased translocation and made the intestinal flora increasingly dominated by lactobacilli.

Identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions.

Based on partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions of 14 enterococcal type strains, Enterococcus faecalis, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, Enterococcus raffinosus and Enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. Furthermore, two species cluster groups could be identified, including (i) Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, and (ii) Enterococcus casseliflavus/Enterococcus flavescens, Enterococcus pseudoavium, Enterococcus dispar and Enterococcus sulfureus. There were identical DNA sequences in the V6 region within each group. Temporal temperature gradient gel electrophoresis (TTGE) of the PCR products from 16 type strains, 12 enterococcal reference strains and 8 clinical isolates revealed that a single nucleotide divergence in DNA sequences was sufficient for separation, identification and division of the studied enterococcal strains into corresponding TTGE profiles. It was concluded that partial DNA sequence analysis and TTGE profiling of PCR-amplified 16S rDNA variable V6 regions provide useful tools for the identification of clinically important Enterococcus spp.

T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages.

The fecal microbiota of two healthy Swedish infants was monitored over time by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Principal component analysis (PCA) of the T-RFLP profiles revealed that the fecal flora in both infants was quite stable during breast-feeding and a major change occurred after weaning. The two infants had different sets of microbiota at all sampling time points. 16S rDNA clone libraries were constructed and the predominant terminal restriction fragments (T-RFs) were identified by comparing T-RFLP patterns in the fecal community with that of corresponding 16S rDNA clones. Sequence analysis indicated that the infants were initially colonized mostly by members of Enterobacteriaceae, Veillonella, Enterococcus, Streptococcus, Staphylococcus and Bacteroides. The members of Enterobacteriaceae and Bacteroides were predominant during breast-feeding in both infants. However, Enterobacteriaceae decreased while members of clostridia increased after weaning. T-RFLP in combination with PCA and 16S rRNA gene sequencing was shown to be an effective strategy for comparing fecal microbiota in infants and pointing out the major changes.