Association of Gut Microbiome and Vitamin D Deficiency in Knee Osteoarthritis Patients: A Pilot Study.

BACKGROUND: Few preclinical studies have shown that Knee osteoarthritis (KOA) is linked to gut microbiome dysbiosis and chronic inflammation. This pilot study was designed to look at the gut microbiome composition in KOA patients and normal individuals with or without vitamin D deficiency (VDD, serum vitamin D <30 ng/mL). METHODS: This pilot study was conducted prospectively in 24 participants. The faecal samples of all the participants were taken for DNA extraction. The V3-V4 region of 16s rRNA was amplified, and the library was prepared and sequenced on the Illumina Miseq platform. RESULTS: The mean (±SD) age was 45.5 (±10.2) years with no defined comorbidities. Of 447 total Operational Taxonomic Units (OTUs), a differential abundance of 16 nominally significant OTUs between the groups was observed. Linear discriminate analysis (LEfSe) revealed a significant difference in bacteria among the study groups. Pseudobutyrivibrio and Odoribacter were specific for VDD, while Parabacteroides, Butyricimonas and Gordonibacter were abundant in the KOA_VDD group, and Peptococcus, Intestimonas, Delftia and Oribacterium were abundant in the KOA group. About 80% of bacterial species were common among different groups and hence labelled as core bacterial species. However, the core microbiome of KOA and VDD groups were not seen in the KOA_VDD group, suggesting that these bacterial groups were affected by the interaction of the KOA and VDD factors. CONCLUSION: Parabacteroides, Butyricimonas, Pseudobutyrivibrio, Odoribacter and Gordonibacter are the predominant bacteria in vitamin D deficient patients with or without KOA. Together these results indicate an association between the gut microbiome, vitamin D and knee osteoarthritis.

The Firmicutes/Bacteroidetes Ratio: A Relevant Marker of Gut Dysbiosis in Obese Patients?

The gut microbiota is emerging as a promising target for the management or prevention of inflammatory and metabolic disorders in humans. Many of the current research efforts are focused on the identification of specific microbial signatures, more particularly for those associated with obesity, type 2 diabetes, and cardiovascular diseases. Some studies have described that the gut microbiota of obese animals and humans exhibits a higher Firmicutes/Bacteroidetes ratio compared with normal-weight individuals, proposing this ratio as an eventual biomarker. Accordingly, the Firmicutes/Bacteroidetes ratio is frequently cited in the scientific literature as a hallmark of obesity. The aim of the present review was to discuss the validity of this potential marker, based on the great amount of contradictory results reported in the literature. Such discrepancies might be explained by the existence of interpretative bias generated by methodological differences in sample processing and DNA sequence analysis, or by the generally poor characterization of the recruited subjects and, more particularly, the lack of consideration of lifestyle-associated factors known to affect microbiota composition and/or diversity. For these reasons, it is currently difficult to associate the Firmicutes/Bacteroidetes ratio with a determined health status and more specifically to consider it as a hallmark of obesity.

Effect of Native and Acetylated Dietary Resistant Starches on Intestinal Fermentative Capacity of Normal and Stunted Children in Southern India.

The health benefits of dietary amylase resistant starch (RS) arise from intestinal microbial fermentation and generation of short chain fatty acids (SCFA). We compared the intestinal fermentative capability of stunted and nonstunted ('healthy') children in southern India using two types of RS: high amylose maize starch (HAMS) and acetylated HAMS (HAMSA). Twenty children (10 stunted and 10 healthy) aged 2 to 5 years were fed biscuits containing HAMS (10 g/day) for two weeks followed by a 2-week washout and then HAMSA biscuits (10 g/day) for 2 weeks. Fecal samples were collected at 3-4 day intervals and pH and SCFA analyzed. At entry, stunted children had lower SCFA concentrations compared to healthy children. Both types of RS led to a significant decrease in fecal pH and increase in fecal acetate and propionate in both healthy and stunted children. However, while HAMS increased fecal butyrate in both groups of children, HAMSA increased butyrate in healthy but not stunted children. Furthermore, healthy children showed a significantly greater increase than stunted children in both acetate and butyrate when fed either RS. No adverse effects were reported with either RS. Stunted children have impaired capacity to ferment certain types of RS which has implications for choice of RS in formulations aimed at improving microbial function in stunted children.

Frequency of HLA celiac disease risk alleles and haplotypes in healthy adults in Tamil Nadu.

Celiac disease (CeD) occurs only in individuals who are able to express human leukocyte antigens (HLA) DQ2 or DQ8, and these are expressed in nearly a third of healthy people in the West. As the disease is very uncommon in Tamil Nadu, we evaluated the possibility that the relevant genes are infrequent in this population. Four hundred healthy adults without any gastrointestinal abnormalities were recruited from Vellore district of Tamil Nadu. Genomic DNA was extracted from venous blood and amplified by PCR using the allele-specific primers for the following alleles-DQA1*0201, 0301, and 0501 and DQB1*02, 0201, and 0302, which determine the CeD risk haplotypes. Among the 400 healthy adults, the presence of DQ2.5 (DQB1*0201-DQA1*0501) and DQ2.2 (DQB1*02-DQA1*0201) haplotypes was found in 8.25% and 14.25%, respectively. DQ8 (DQB1*0302-DQA1*0301) haplotype was identified in only 3% of healthy individuals. Overall, approximately a quarter of healthy adults in Tamil Nadu had the potential CeD risk haplotypes of HLA DQ2.5, DQ2.2, and DQ8.

Sequential testing with different tissue transglutaminase antibodies, a new approach for diagnosis of celiac disease.

BACKGROUND: The diagnosis of celiac disease (CeD) in clinical practice relies on serological testing for IgA antibodies to human tissue transglutaminase (anti-tTG) which diagnose CeD autoimmunity. We compared three kits for their performance in diagnosis of the disease and evaluated the point prevalence of CeD autoimmunity in a South Indian urban population. METHODS: In the first part of the study, sera from 90 patients with documented CeD and 92 healthy controls were tested for anti-tTG using three different kits. One thousand nine hundred and seventeen healthy adults residing in urban areas of Vellore and Kancheepuram districts were tested for CeD autoimmunity using a sequential two-test strategy. RESULTS: The sensitivity, specificity, false positivity, false negativity, positive predictive value, and negative predictive value for the three assays respectively were as follows: 95.5%, 82.6%, 17.3%, 4.4%, 84.3%, and 95% for the Aeskulisa New Generation Assay; 85.5%, 100%, 0%, 14.4%, 100%, and 87.6% for Quanta Lite; and 71.1%, 100%, 0%, 28.8%, 100%, and 71% for Celiac Microlisa. The ROC curves showed good discrimination for all three ELISAs with an AUC of 0.947, 0.950, and 0.886 for the Aeskulisa, Quanta Lite, and Celiac Microlisa, respectively. Of 1917 (males 908, females 1009) healthy adults, 113 (5.89%) were seropositive for IgA anti-htTG in the Aeskulisa test. Two of the latter tested positive in the Quanta Lite assay and/or the Celiac Microlisa assay. The CeD autoimmunity prevalence in this urban population was 1.0 per thousand (95% confidence interval 0.3 to 3.7 per thousand). CONCLUSION: Sequential testing for anti-tTG using first a highly sensitive assay followed by a very specific assay is a new strategy for screening for CeD in clinical practice.

Molecular analysis of the human faecal archaea in a southern Indian population.

Archaea are an important constituent of the human gut microbiota, but there is no information on human gut archaea in an Indian population. In this study, faecal samples were obtained from different age groups (neonatal babies, preschool children, school-going children, adolescents, adults and elderly) of a southern Indian population, and from a tribal population also resident in southern India). 16S rRNA gene sequences specific to Archaea were amplified from pooled faecal DNA in each group, sequenced, and aligned against the NCBI database. Of the 806 adequate sequences in the study, most aligned with 22 known sequences. There were 9 novel sequences in the present study. All sequences were deposited in the GenBank nucleotide sequence database with the following accession numbers: KF607113 - KF607918. Methanobrevibacter was the most prevalent genus among all the age groups accounting for 98% in neonates, 96% in post-weaning, and 100% each in preschool, school and adult population. In the elderly, Methanobrevibacter accounted for 96% and in tribal adults, 99% of the clones belonged to Methanobrevibacter genus. Other genera detected included Caldisphaera, Halobaculum, Methanosphaeraand Thermogymnomonas. Methanobrevibacter smithii predominated in all age groups, accounting for 749 (92.9%) of the 806 sequences. Archaea can be found in the faeces of southern Indian residents immediately after birth. Methanobrevibacter smithii was the dominant faecal archeon in all age groups, with other genera being found at the extremes of age.

Prevalence of Adult Celiac Disease in India: Regional Variations and Associations.

OBJECTIVES: Although celiac disease (CeD) affects 1% of people in the northern part of India, it is believed to be uncommon in the southern and northeastern parts because of significant differences in dietary pattern and ethnicity. We estimated the prevalence of CeD in these three populations. In a subset, we also investigated differences in the prevalence of HLA-DQ 2/8 allelotype and dietary grain consumption. METHODS: A total of 23,331 healthy adults were sampled from three regions of India-northern (n=6207), northeastern (n=8149), and southern (n=8973)-and screened for CeD using IgA anti-tissue transglutaminase antibody. Positive tests were reconfirmed using a second ELISA. CeD was diagnosed if the second test was positive and these participants were further investigated. A subsample of participants was tested for HLA-DQ2/-DQ8 and underwent detailed dietary evaluation. RESULTS: Age-adjusted prevalence of celiac autoantibodies was 1.23% in northern, 0.87% in northeastern, and 0.10% in southern India (P<0.0001). Prevalence of CeD and latent CeD, respectively, was 8.53/1,000 and 3.70/1,000 in northern, 4.66/1,000 and 3.92/1,000 in northeastern, and 0.11/1,000 and 1.22/1,000 in the southern part. The population prevalence of genes determining HLA-DQ2 and/or -DQ8 expression was 38.1% in northern, 31.4% in northeastern, and 36.4% in southern India. Mean daily wheat intake was highest in northern (455 g) compared with northeastern (37 g) or southern part (25 g), whereas daily rice intake showed an inverse pattern. CONCLUSIONS: CeD and latent CeD were most prevalent in northern India and were the least in southern India. The prevalence correlated with wheat intake and did not reflect differences in the genetic background.

Probiotic potential of lactic acid bacteria present in home made curd in southern India.

BACKGROUND & OBJECTIVES: The human gut microbiota play a significant role in nutritional processes. The concept of probiotics has led to widespread consumption of food preparations containing probiotic microbes such as curd and yogurt. Curd prepared at home is consumed every day in most homes in southern India. In this study the home-made curd was evaluated for lactic acid bacteria (LAB) with probiotic potential. METHODS: Fifteen LAB (12 lactobacilli, 1 l0 actococcus , 2 Leuconostoc) and one yeast isolated from home-made curd were evaluated for resistance to acid, pepsin, pancreatin and bile salts; antimicrobial resistance; intrinsic antimicrobial activity; adherence to Caco-2 epithelial cells; ability to block pathogen adherence to Caco-2 cells; ability to inhibit interleukin (IL)-8 secretion from HT-29 epithelial cells in response to Vibrio cholerae; and ability to induce anti-inflammatory cytokine expression in THP-1 monocyte cells. RESULTS: Lactobacillus abundance in fermenting curd peaked sharply at 12 h. Nine of the strains survived exposure to acid (pH 3.0) for at least one hour, and all strains survived in the presence of pancreatin or bile salts for 3 h. None showed haemolytic activity. All were resistant to most antimicrobials tested, but were sensitive to imipenem. Most strains inhibited the growth of Salmonella Typhimurium while five inhibited growth of V. cholerae O139. Seven strains showed adherence to Caco-2 cells ranging from 20-104 per cent of adherence of an adherent strain of Escherichia coli, but all inhibited V. cholerae adherence to Caco-2 cells by 20-100 per cent. They inhibited interleukin-8 secretion from HT-29 cells, in response to V. cholerae, by 50-80 per cent. Two strains induced IL-10 and IL-12 messenger ribonucleic acid (mRNA) expression in THP-1 cells. INTERPRETATION & CONCLUSIONS: LAB in curd had properties consistent with probiotic potential, but these were not consistent across species. LAB abundance in curd increased rapidly at 12 h of fermentation at room temperature and declined thereafter.

Development of the gut microbiota in southern Indian infants from birth to 6 months: a molecular analysis.

Acquisition of the gastrointestinal microbiota at birth may have long-term health impacts. We longitudinally characterised major microbial communities in the faeces of a cohort of infants using molecular methods. Faecal samples were prospectively obtained at several time points after birth from eighty-three infants. Real-time PCR using SYBR green and primers targeted at 16S rRNA gene sequences were used to quantify Bifidobacterium, Lactobacillus acidophilus group, Bacteroides-Prevotella group, Enterobacteriaceae, Enterococcus, Clostridium coccoides-Eubacterium rectale group, Clostridium leptum group and Staphylococcus. Microbial community abundance was expressed relative to amplification of sequences conserved universally for domain bacteria. Faecal copy number of 16S rRNA genes increased non-significantly from a mean of 4·1 × 10(9)/g on day 1 to 1·1 × 10(10)/g on day 4. All microbial communities were detected from day 1 after birth. Enterobacteriaceae and lactobacilli predominated on day 1, while bifidobacteria and staphylocci increased on day 4. Bacteroides-Prevotella and C. coccoides-E. rectale increased by day 180. C. leptum was detected in half of the cohort at birth and in a slightly larger percentage by 6 months. Caesarean section was associated with delayed colonisation by several bacterial communities. Higher socio-economic status was associated with more abundant lactobacilli and Bacteroides-Prevotella at 90 and 180 d. Supplemental feeding was associated with a reduction in Enterobacteriaceae. Microbial colonisation of the gut was well established on the first day of birth, and relative abundance of microbial communities was influenced by mode of delivery, socio-economic status and supplemental feeding. These findings may have relevance to infant nutrition and growth.

Metronidazole effects on microbiota and mucus layer thickness in the rat gut.

Both mucus and mucosa-associated bacteria form a specific environment in the gut; their disruption may play a crucial role in the development of intestinal bowel disease (IBD). Metronidazole, an antibiotic used in the treatment of IBD, alters gut microbiota and reduces basal oxidative stress to proteins in colonic tissue of healthy rats. The aim of this study was to evaluate the impact of the altered microbiota due to the metronidazole on the thickness of the mucus layer. This study was performed in healthy untreated rats (control group) or rats treated by metronidazole (metronidazole-treated rats, 1 mg mL(-1) in drinking water for 7 days). Both PCR-temporal temperature gradient gel electrophoresis and quantitative PCR (qPCR) revealed an altered microbiota with an increase in bifidobacteria and enterobacteria in metronidazole-treated rats compared with control rats. Moreover, a dominant bifidobacterial species, Bifidobacterium pseudolongum, was detected. Using qPCR and FISH, we showed that bifidobacteria were also increased in the microbiota-associated mucosa. At the same time, the mucus layer thickness was increased approximately twofold. These results could explain the benefits of metronidazole treatment and warrant further investigations to define the role of bifidobacteria in the colonic mucosa.

Low levels of faecal lactobacilli in women with iron-deficiency anaemia in south India.

Fe deficiency in women contributes significantly to maternal and child morbidity in India. The intestinal bacterial flora may facilitate absorption of Fe from the caecum and proximal colon. The present study investigated the possibility that intestinal microbiota of anaemic young women may differ from that of women with normal Hb levels. The microbiota was quantified by real-time PCR in faeces of eight anaemic (Hb ≤ 100 g/l) and twenty-six normohaemic (Hb ≥ 120 g/l) women aged 18-25 years. Sequences of 16S ribosomal DNA (rDNA) specific to Bifidobacterium genus, Lactobacillus acidophilus group, Bacteroides-Prevotella-Porphyromonas group, Clostridium leptum group and Eubacterium rectale were amplified and expressed (as relative difference) relative to the universally conserved bacterial 16S rDNA sequences. Dietary intakes of energy, carbohydrate, fibre and Fe were ascertained by maintenance of a diet diary for a week. Faecal lactobacilli were significantly lower in anaemic women (median 6.6 × 10(-8), relative difference compared with total bacteria) than in the reference group (2.9 × 10(-6); P = 0.001, unpaired t test with logarithmic transformation). There was no difference between the two groups with respect to any of the other bacteria that were examined. Intakes of energy, carbohydrate, fibre, Fe and milk were similar in both the groups. Fe deficiency in young women in south India was associated with low levels of lactobacilli in the faeces. The relationship between lactobacilli and Fe deficiency needs to be explored further.

Faecal bifidobacteria in Indian neonates & the effect of asymptomatic rotavirus infection during the first month of life.

BACKGROUND AND OBJECTIVES: Bifidobacteria colonize the gut after the first week of life and remain an important component of the gut microbiota in infancy. This study was carried out to characterize the diversity and number of bifidobacteria colonizing the gut in Indian neonates and to investigate whether asymptomatic infection with rotavirus in the first month of life affected gut colonization by bifidobacteria. METHODS: DNA was isolated from faeces of 14 term-born neonates who were under surveillance for rotavirus infection. Bacterial and bifidobacterial diversity was evaluated by temporal temperature gradient electrophoresis (TTGE) of 16S rDNA amplified using total bacteria and bifidobacteria-specific primers. Real time PCR, targeting 16S rDNA, was used to quantitate faecal bifidobacteria and enterobacteria. RESULTS: TTGE of conserved bacterial 16S rDNA showed 3 dominant bands of which Escherichia coli (family Enterobacteriaceae) and Bifidobacterium (family Bifidobacteriaceae) were constant. TTGE of Bifidobacterium genus-specific DNA showed a single band in all neonates identified by sequencing as Bifidobacterium longum subsp. infantis. Faecal bifidobacterial counts (log 10 cfu/g faeces) ranged from 6.1 to 9.3 and enterobacterial counts from 6.3 to 9.5. Neonates without and with rotavirus infection in the first week of life did not show significant differences in the median count of bifidobacteria (log 10 count 7.48 vs. 7.41) or enterobacteria (log 10 count 8.79 vs. 7.92). INTERPRETATION AND CONCLUSIONS: B. longum subsp. infantis was the sole bifidobacterial species colonizing the gut of Indian neonates. Asymptomatic rotavirus infection in the first month of life was not associated with alteration in faecal bifidobacteria or enterobacteria.

Quantitative differences in intestinal Faecalibacterium prausnitzii in obese Indian children.

Gut bacteria contribute to energy conservation in man through their ability to ferment unabsorbed carbohydrate. The present study examined the composition of predominant faecal microbiota in obese and non-obese children. The participants (n 28) aged 11-14 years provided fresh faecal samples and completed a dietary survey consisting of 24 h diet recall and a FFQ of commonly used foods taken over the previous 3 months. Faecal bacteria were quantitated by real-time PCR using primers targeted at 16S rDNA. Of the participants, fifteen (seven female) were obese, with median BMI-for-age at the 99th percentile (range 97 to>99) while thirteen participants (seven female) were normal weight, with median BMI-for age being at the 50th percentile (range 1-85). Consumption of energy, carbohydrates, fat and protein was not significantly different between the obese and non-obese participants. There was no significant difference between the two groups in faecal levels of Bacteroides-Prevotella, Bifidobacterium species, Lactobacillus acidophilus group or Eubacterium rectale. Levels of Faecalibacterium prausnitzii were significantly higher in obese children than in non-obese participants (P = 0.0253). We concluded that the finding of increased numbers of F. prausnitzii in the faeces of obese children in south India adds to the growing information on alterations in faecal microbiota in obesity.

Common NOD2 mutations are absent in patients with Crohn's disease in India.

BACKGROUND: Crohn's disease is being increasingly diagnosed in the Indian subcontinent. Three apparently common mutations in the NOD2 gene are found in up to 30% of sporadic patients with Crohn's disease in western countries. We examined whether such mutations are also found in Indian patients with Crohn's disease. METHODS: Venous blood was collected from 82 patients (age range: 7-65 years, 53 men) with Crohn's disease and 149 control subjects; DNA was extracted and subjected to polymerase chain reaction using specific primers. The amplified fragments of size 185, 163 and 151 bp for R702W, G908R and 1007fs, respectively, were digested with MspI, HhaI and ApaI, and the restriction pattern noted after electrophoresis. RESULTS: Twenty-eight patients had ileocolonic disease, 26 ileal disease, 20 colonic disease and 8 had disease limited to proximal small bowel or stomach. None of the 82 patients showed any of the three NOD2 mutations. The control subjects (93 men) had a variety of chronic gastrointestinal disorders (ulcerative colitis 52, irritable bowel syndrome 30, intestinal tuberculosis 20, colon cancer 7, miscellaneous 37). None of the control subjects showed a mutation in any of the three NOD2 mutation analyses. CONCLUSION: The three NOD2 gene mutations described above are uncommon in Indian patients with Crohn's disease. This study complements information provided by recent studies on NOD2 mutations in Indians.

Bacterial succession in the colon during childhood and adolescence: molecular studies in a southern Indian village.

BACKGROUND: The colonic bacterial flora, largely anaerobic, is believed to establish and stabilize in the first 2 y of life. OBJECTIVE: This study was undertaken to determine whether the bacterial flora of the colon undergoes further changes (succession) during childhood and adolescence. DESIGN: This cross-sectional study examined fecal samples from 130 healthy children and adolescents in the age group 2-17 y and from 30 healthy adults (median age: 42 y) residing in a single village in southern India. DNA was extracted and subjected to 16S rDNA-targeted real-time polymerase chain reaction to determine the relative predominance of Bifidobacterium genus, Bacteroides-Prevotella-Porphyromonas group, Lactobacillus acidophilus group, Eubacterium rectale, and Faecalibacterium prausnitzii. RESULTS: Bifidobacterium species and Bacteroides-Prevotella group were dominant fecal bacteria overall. E. rectale and Lactobacillus species were considerably less abundant. Clear age-related differences emerged, with a steep decline in Bifidobacterium species in adults (P < 0.0001), a steep decline of Lactobacillus species >5 y of age (P < 0.0001), an increase in Bacteroides during late adolescence and in adults (P = 0.0040), an increase in E. rectale during childhood and adolescence followed by a steep decline in adults (P < 0.0001), and a late childhood peak of F. prausnitzii with decline in adolescents and adults (P < 0.0001). CONCLUSIONS: Changes in the bacterial flora occur during childhood and adolescence characterized by reduction in Lactobacillus and Bifidobacterium species and an increase in Bacteroides, E rectale, and F. prausnitzii peaked during late childhood in this population.

Estimation of faecal carriage of Clostridium difficile in patients with ulcerative colitis using real time polymerase chain reaction.

BACKGROUND & OBJECTIVE: Ulcerative colitis (UC) is a disease of unknown aetiology in which exacerbations are sometimes linked to intestinal colonization by toxin-producing Clostridium difficile. We undertook this study to detect and quantitatively assess C. difficile in the stool of patients with UC using real time polymerase chain reaction (RT-PCR), and to compare it with healthy individuals. METHODS: A total of 37 consecutive patients with UC (26 male, mean age 41.3 yr) and 36 healthy adult volunteers (20 male, mean age 36.4), none of whom had received antibiotics within two months prior to faecal collection, were included in the study. Faecal DNA was extracted, quantitative PCR (qPCR) carried out using primers to amplify species-specific segments of 16S rDNA of C. difficile, and expressed as relative fold difference against amplification of highly conserved (universal) segments. Toxins A and B were assayed by ELISA. RESULTS: Quantitative PCR detected C. difficile sensitively, and spiking with increasing numbers of the organism resulted in linear increase in amplification (R(2)=0.974). C. difficile was detected by qPCR in faeces of 20 of 36 healthy volunteers and 34 of 37 patients with UC. Relatively greater amplification of C. difficile (fold difference) was noted in UC compared to controls (P<0.0001). There was no significant difference in C. difficile amplification between patients with proctitis, left sided colitis and pancolitis, or between active and quiescent colitis. Toxin was detected in the faeces of 8 of 37 patients with UC compared to 2 of 36 healthy volunteers. INTERPRETATION & CONCLUSION: Findings of this study showed overgrowth of C. difficile in the stool of Indian patients with UC. However, its relevance to disease pathogenesis and severity in a tropical country like India needs to be investigated further.

Real-time polymerase chain reaction quantification of specific butyrate-producing bacteria, Desulfovibrio and Enterococcus faecalis in the feces of patients with colorectal cancer.

BACKGROUND AND AIM: Bacterial metabolites produced in the bowel are potentially related to the genesis of colorectal cancer. Butyrate is protective against cancer, whereas hydrogen sulfide and oxygen free radicals can be toxic to the epithelium. The present study was designed to quantitate Eubacterium rectale, Faecalibacterium prausnitzii (both butyrate-producing bacteria), Desulfovibrio (sulfate-reducing bacteria), and Enterococcus faecalis (that produces extracellular superoxide) in the feces of patients with colorectal cancer. METHODS: DNA was extracted from feces of 20 patients with colorectal cancer, nine patients with upper gastrointestinal cancer and 17 healthy volunteers. Real-time polymerase chain reaction using primers aimed at 16S rDNA was used to quantitate the above bacterial species or genus, and this was expressed relative to amplification of universal sequences conserved among all bacteria. RESULTS: Levels of E. rectale and F. prausnitzii were decreased approximately fourfold (P = 0.0088 and 0.0028, respectively) in colorectal cancer patients compared to healthy control volunteers. Levels of Desulfovibrio were not significantly different between the three groups. E. faecalis populations were significantly higher in colorectal cancer patients compared to healthy volunteers (P = 0.0294). CONCLUSIONS: Butyrate producers were decreased and E. faecalis increased in the feces of colon cancer patients. These shifts in the colonic bacterial population could potentially lead to epithelial cell damage and increased turnover and may be a factor leading to colon cancer.

Molecular studies of fecal anaerobic commensal bacteria in acute diarrhea in children.

BACKGROUND AND OBJECTIVE: The commensal bacterial flora of the colon may undergo changes during diarrhea, owing to colonization of the intestine by pathogens and to rapid intestinal transit. This study used molecular methods to determine changes in the composition of selected commensal anaerobic bacteria during and after acute diarrhea in children. MATERIALS AND METHODS: Fecal samples were obtained from 46 children with acute diarrhea in a rural community during an episode of acute diarrhea, immediately after recovery from diarrhea, and 3 months after recovery. DNA was extracted and quantitative polymerase chain reaction using SYBR green and genus- and species-specific primers targeting 16S rDNA were undertaken to quantitate the following groups of bacteria: Bifidobacterium spp., Bifidobacterium longum group, Bacteroides-Prevotella group, Bacteroides fragilis, Lactobacillus acidophilus group, Faecalibacterium prauznitzii, and Eubacterium rectale, relative to amplification of universal bacterial domain 16S rDNA. RESULTS: Bacteria belonging to the Bacteroides-Prevotella-Porphyromonas group, E rectale, L acidophilus, and F prauznitzii groups were low during acute diarrhea compared with their levels after recovery from diarrhea. The pattern was similar in rotavirus diarrhea and nonrotavirus diarrhea. Administration of amylase-resistant maize starch as adjuvant therapy was associated with lower levels of F prauznitzii at the time of recovery but did not lead to other changes in the floral pattern. CONCLUSIONS: Specific classes of fecal bacteria are lower during episodes of acute diarrhea in children than during periods of normal gastrointestinal health, suggesting specific alterations in the flora during diarrhea.

Probiotic administration alters the gut flora and attenuates colitis in mice administered dextran sodium sulfate.

BACKGROUND: Probiotics are used in the therapy of inflammatory bowel disease. This study aimed to determine whether prior administration of probiotic lactobacilli and bifidobacteria would prevent disease and change gut flora in an animal model of colitis. METHODS: Swiss albino mice received a probiotic mixture (four Lactobacillus and four Bifidobacterium species) or medium (control) for a week prior to induction of colitis by oral 4% dextran sodium sulfate (DSS) for seven days. Appropriate non-colitis controls were used. Histological damage was assessed (n = 5 per group), as was expression of mRNA for tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta1 and SOCS-1 in the colonic mucosa (n = 6 per group). Secretion of TNF-alpha was measured in distal colon organ culture (n = 5-6 per group). Levels of Bacteroides, Bifidobacterium, and Lactobacillus acidophilus in feces were quantified by real time polymerase chain reaction (PCR) targeting 16S rDNA. RESULTS: Compared to untreated DSS colitis, probiotic treatment significantly reduced weight loss (P < 0.05), shifted histological damage to lesser grades of severity (P < 0.001), reduced mRNA expression of TNF-alpha and TGF-beta1 (P < 0.05), and down-regulated production of TNF-alpha from distal colon explants (P < 0.05). Colitis induced a significant reduction in the relative proportions of Bifidobacterium, Bacteroides and Lactobacillus acidophilus group bacteria in feces, and these levels were significantly increased in probiotic-treated mice compared to DSS mice (P < 0.001). CONCLUSION: Prior administration of probiotic bacteria reduced mucosal inflammation and damage in DSS-induced colitis. DSS colitis was associated with significant changes in the fecal anaerobic bacterial flora and these changes were modulated by administration of probiotic bacteria.