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1.  Microbiota That Affect Risk for Shigellosis in Children in Low-Income Countries 
Emerging Infectious Diseases  2015;21(2):242-250.
Co-infection with Shigella spp. and other microbes modifies diarrhea risk.
Pathogens in the gastrointestinal tract exist within a vast population of microbes. We examined associations between pathogens and composition of gut microbiota as they relate to Shigella spp./enteroinvasive Escherichia coli infection. We analyzed 3,035 stool specimens (1,735 nondiarrheal and 1,300 moderate-to-severe diarrheal) from the Global Enteric Multicenter Study for 9 enteropathogens. Diarrheal specimens had a higher number of enteropathogens (diarrheal mean 1.4, nondiarrheal mean 0.95; p<0.0001). Rotavirus showed a negative association with Shigella spp. in cases of diarrhea (odds ratio 0.31, 95% CI 0.17–0.55) and had a large combined effect on moderate-to-severe diarrhea (odds ratio 29, 95% CI 3.8–220). In 4 Lactobacillus taxa identified by 16S rRNA gene sequencing, the association between pathogen and disease was decreased, which is consistent with the possibility that Lactobacillus spp. are protective against Shigella spp.–induced diarrhea. Bacterial diversity of gut microbiota was associated with diarrhea status, not high levels of the Shigella spp. ipaH gene.
doi:10.3201/eid2101.140795
PMCID: PMC4313639  PMID: 25625766
shigellosis; Shigella; bacteria; polymicrobial infection; Escherichia coli; enteroinvasive E. coli; EIEC; Lactobacillus; rotavirus; viruses; co-occurring pathogens; enteropathogens; microbiota; ipaH gene; diarrhea; children; low-income countries
2.  Reagent and laboratory contamination can critically impact sequence-based microbiome analyses 
BMC Biology  2014;12(1):87.
Background
The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents.
Results
In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination.
Conclusions
These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-014-0087-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12915-014-0087-z
PMCID: PMC4228153  PMID: 25387460
Contamination; Microbiome; Microbiota; Metagenomics; 16S rRNA
3.  Time between Collection and Storage Significantly Influences Bacterial Sequence Composition in Sputum Samples from Cystic Fibrosis Respiratory Infections 
Journal of Clinical Microbiology  2014;52(8):3011-3016.
Spontaneously expectorated sputum is traditionally used as the sampling method for the investigation of lower airway infections. While guidelines exist for the handling of these samples for culture-based diagnostic microbiology, there is no comparable consensus on their handling prior to culture-independent analysis. The increasing incorporation of culture-independent approaches in diagnostic microbiology means that it is of critical importance to assess potential biases. The aim of this study was to assess the impact of delayed freezing on culture-independent microbiological analyses and to identify acceptable parameters for sample handling. Sputum samples from eight adult cystic fibrosis (CF) patients were collected and aliquoted into sterile Bijou bottles. Aliquots were stored at room temperature before being frozen at −80°C for increasing intervals, up to a 72-h period. Samples were treated with propidium monoazide to distinguish live from dead cells prior to DNA extraction, and 16S rRNA gene pyrosequencing was used to characterize their bacterial compositions. Substantial variation was observed in samples with high-diversity bacterial communities over time, whereas little variation was observed in low-diversity communities dominated by recognized CF pathogens, regardless of time to freezing. Partitioning into common and rare species demonstrated that the rare species drove changes in similarity. The percentage abundance of anaerobes over the study significantly decreased after 12 h at room temperature (P = 0.008). Failure to stabilize samples at −80°C within 12 h of collection results in significant changes in the detected community composition.
doi:10.1128/JCM.00764-14
PMCID: PMC4136140  PMID: 24920767
4.  Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition 
Genome Biology  2014;15(6):R76.
Background
Diarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease.
Results
We use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age.
Conclusions
Our findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.
doi:10.1186/gb-2014-15-6-r76
PMCID: PMC4072981  PMID: 24995464
5.  Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions 
Nature genetics  2013;45(4):450-455e1.
The importance of commensal microbes for human health is increasingly recognized1-5, yet the impacts of evolutionary changes in human diet and culture on commensal microbiota remain almost unknown. Two of the greatest dietary shifts in human evolution involved the adoption of carbohydrate-rich Neolithic (farming) diets6,7 (beginning ~10,000 years BP6,8), and the more recent advent of industrially processed flour and sugar (~1850)9. Here, we show that calcified dental plaque (dental calculus) on ancient teeth preserves a detailed genetic record throughout this period. Data from 34 early European skeletons indicate that the transition from hunter-gatherer to farming shifted the oral microbial community to a disease-associated configuration. The composition of oral microbiota remained surprisingly constant between Neolithic and Medieval times, after which (the now ubiquitous) cariogenic bacteria became dominant, apparently during the Industrial Revolution. Modern oral microbiota are markedly less diverse than historic populations, which might be contributing to chronic oral (and other) disease in post-industrial lifestyles.
doi:10.1038/ng.2536
PMCID: PMC3996550  PMID: 23416520
6.  Survey of Culture, GoldenGate Assay, Universal Biosensor Assay, and 16S rRNA Gene Sequencing as Alternative Methods of Bacterial Pathogen Detection 
Journal of Clinical Microbiology  2013;51(10):3263-3269.
Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.
doi:10.1128/JCM.01342-13
PMCID: PMC3811648  PMID: 23884998
7.  The Impact of Different DNA Extraction Kits and Laboratories upon the Assessment of Human Gut Microbiota Composition by 16S rRNA Gene Sequencing 
PLoS ONE  2014;9(2):e88982.
Introduction
Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories.
Methods
Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient.
Results
DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05).
Conclusion
This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples in a study are prepared with the same method, and the need for caution when cross-comparing studies that use different methods.
doi:10.1371/journal.pone.0088982
PMCID: PMC3933346  PMID: 24586470
8.  Patent Human Infections with the Whipworm, Trichuris trichiura, Are Not Associated with Alterations in the Faecal Microbiota 
PLoS ONE  2013;8(10):e76573.
Background
The soil-transmitted helminth (STH), Trichuris trichiura colonises the human large intestine where it may modify inflammatory responses, an effect possibly mediated through alterations in the intestinal microbiota. We hypothesised that patent T. trichiura infections would be associated with altered faecal microbiota and that anthelmintic treatment would induce a microbiota resembling more closely that observed in uninfected individuals.
Materials and Methods
School children in Ecuador were screened for STH infections and allocated to 3 groups: uninfected, T. trichiura only, and mixed infections with T. trichiura and Ascaris lumbricoides. A sample of uninfected children and those with T. trichiura infections only were given anthelmintic treatment. Bacterial community profiles in faecal samples were studied by 454 pyrosequencing of 16 S rRNA genes.
Results
Microbiota analyses of faeces were done for 97 children: 30 were uninfected, 17 were infected with T. trichiura, and 50 with T. trichiura and A. lumbricoides. Post-treatment samples were analyzed for 14 children initially infected with T. trichiura alone and for 21 uninfected children. Treatment resulted in 100% cure of STH infections. Comparisons of the microbiota at different taxonomic levels showed no statistically significant differences in composition between uninfected children and those with T. trichiura infections. We observed a decreased proportional abundance of a few bacterial genera from the Clostridia class of Firmicutes and a reduced bacterial diversity among children with mixed infections compared to the other two groups, indicating a possible specific effect of A. lumbricoides infection. Anthelmintic treatment of children with T. trichiura did not alter faecal microbiota composition.
Discussion
Our data indicate that patent human infections with T. trichiura may have no effect on faecal microbiota but that A. lumbricoides colonisation might be associated with a disturbed microbiota. Our results also catalogue the microbiota of rural Ecuadorians and indicate differences with individuals from more urban industrialised societies.
doi:10.1371/journal.pone.0076573
PMCID: PMC3790696  PMID: 24124574
9.  Evolution of locomotion in Anthropoidea: the semicircular canal evidence 
Our understanding of locomotor evolution in anthropoid primates has been limited to those taxa for which good postcranial fossil material and appropriate modern analogues are available. We report the results of an analysis of semicircular canal size variation in 16 fossil anthropoid species dating from the Late Eocene to the Late Miocene, and use these data to reconstruct evolutionary changes in locomotor adaptations in anthropoid primates over the last 35 Ma. Phylogenetically informed regression analyses of semicircular canal size reveal three important aspects of anthropoid locomotor evolution: (i) the earliest anthropoid primates engaged in relatively slow locomotor behaviours, suggesting that this was the basal anthropoid pattern; (ii) platyrrhines from the Miocene of South America were relatively agile compared with earlier anthropoids; and (iii) while the last common ancestor of cercopithecoids and hominoids likely was relatively slow like earlier stem catarrhines, the results suggest that the basal crown catarrhine may have been a relatively agile animal. The latter scenario would indicate that hominoids of the later Miocene secondarily derived their relatively slow locomotor repertoires.
doi:10.1098/rspb.2012.0939
PMCID: PMC3396915  PMID: 22696520
vestibular system; generalized least-squares analysis; primates
10.  Enhanced Susceptibility to Citrobacter rodentium Infection in MicroRNA-155-Deficient Mice 
Infection and Immunity  2013;81(3):723-732.
MicroRNAs (miRNAs) are small noncoding molecules that control gene expression posttranscriptionally, with microRNA-155 (miR-155) one of the first to be implicated in immune regulation. Here, we show that miR-155-deficient mice are less able to eradicate a mucosal Citrobacter rodentium infection than wild-type C57BL/6 mice. miR-155-deficient mice exhibited prolonged colonization associated with a higher C. rodentium burden in gastrointestinal tissue and spread into systemic tissues. Germinal center formation and humoral immune responses against C. rodentium were severely impaired in infected miR-155-deficient mice. A similarly susceptible phenotype was observed in μMT mice reconstituted with miR-155-deficient B cells, indicating that miR-155 is required intrinsically for mediating protection against this predominantly luminal bacterial pathogen.
doi:10.1128/IAI.00969-12
PMCID: PMC3584869  PMID: 23264052
11.  Active Aging: A Global Goal 
doi:10.1155/2013/298012
PMCID: PMC3586450  PMID: 23476642
12.  Targeted Restoration of the Intestinal Microbiota with a Simple, Defined Bacteriotherapy Resolves Relapsing Clostridium difficile Disease in Mice 
PLoS Pathogens  2012;8(10):e1002995.
Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis.
Author Summary
Pathological imbalances within the intestinal microbiota, termed dysbiosis, are often associated with chronic Clostridium difficile infections in humans. We show that infection of mice with the healthcare pathogen C. difficile leads to persistent intestinal dysbiosis that is associated with chronic disease and a highly contagious state. Using this model we rationally designed a simple mixture of phylogenetically diverse intestinal bacteria that can disrupt intestinal dysbiosis and as a result resolve disease and contagiousness. Our results validate the microbiota as a viable therapeutic target and open the way to rationally design bacteriotherapy to treat chronic C. difficile infections and potentially other forms of persistent dysbiosis.
doi:10.1371/journal.ppat.1002995
PMCID: PMC3486913  PMID: 23133377
13.  The Transcription Factor T-bet Regulates Intestinal Inflammation Mediated by Interleukin-7 Receptor+ Innate Lymphoid Cells 
Immunity  2012;37(4):674-684.
Summary
Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα+ innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21−/−Rag2−/− ulcerative colitis (TRUC) mice. TNF-α produced by CD103−CD11b+ dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.
Highlights
► Chronic colitis in TRUC mice was mediated by IL-17-producing innate lymphoid cells ► TNF-α synergized with IL-23 to induce innate IL-17 production ► Helicobacter typhlonius triggered intestinal pathology in TRUC mice ► T-bet regulated IL-7R transcription, a key checkpoint in intestinal ILC homeostasis
doi:10.1016/j.immuni.2012.09.008
PMCID: PMC3540260  PMID: 23063332
14.  The bacteriology of pouchitis: a molecular phylogenetic analysis using 16S rRNA gene cloning and sequencing 
Annals of surgery  2010;252(1):90-98.
Objective
To identify, compare and contrast the microbiota in patients with and without pouchitis after RPC for UC and FAP.
Summary Background Data
Pouchitis is the most common complication following restorative proctocolectomy (RPC). An abnormal host-microbial interaction has been implicated. We investigated the pouch microbiota in patients with and without pouchitis undergoing restorative proctocolectomy for UC and familial adenomatous polyposis (FAP).
Methods
Mucosal pouch biopsies, taken from 16 UC (pouchitis 8) and 8 FAP (pouchitis 3) patients were analysed to the species (or phylotype) level by cloning and sequencing of 3,184 full-length bacterial 16S rRNA genes.
Results
There was a significant increase in Proteobacteria (p= 0.019) and a significant decrease in Bacteroidetes (p= 0.001) and Faecalibacterium prausnitzii (p=0.029) in the total UC compared to the total FAP cohort, but only limited differences were found between the UC non-pouchitis and pouchitis groups and the FAP pouchitis and non-pouchitis groups. Bacterial diversity in the FAP non-pouchitis group was significantly greater than in UC non-pouchitis (p= 0.019) and significantly greater in UC non-pouchitis compared to UC pouchitis (p= 0.009). No individual species or phylotype specifically associated with either UC or FAP pouchitis was found.
Conclusions
UC pouch patients have a different, less diverse, gut microbiota than FAP patients. A further reduction in bacterial diversity but no significant dysbiosis occurs in those with pouchitis. The study suggests that a dysbiosis occurs in the ileal pouch of UC RPC patients which predisposes to, but may not directly cause, pouchitis.
doi:10.1097/SLA.0b013e3181e3dc8b
PMCID: PMC3292798  PMID: 20562611
15.  Bayesian estimation of bacterial community composition from 454 sequencing data 
Nucleic Acids Research  2012;40(12):5240-5249.
Estimating bacterial community composition from a mixed sample in different applied contexts is an important task for many microbiologists. The bacterial community composition is commonly estimated by clustering polymerase chain reaction amplified 16S rRNA gene sequences. Current taxonomy-independent clustering methods for analyzing these sequences, such as UCLUST, ESPRIT-Tree and CROP, have two limitations: (i) expert knowledge is needed, i.e. a difference cutoff between species needs to be specified; (ii) closely related species cannot be separated. The first limitation imposes a burden on the user, since considerable effort is needed to select appropriate parameters, whereas the second limitation leads to an inaccurate description of the underlying bacterial community composition. We propose a probabilistic model-based method to estimate bacterial community composition which tackles these limitations. Our method requires very little expert knowledge, where only the possible maximum number of clusters needs to be specified. Also our method demonstrates its ability to separate closely related species in two experiments, in spite of sequencing errors and individual variations.
doi:10.1093/nar/gks227
PMCID: PMC3384343  PMID: 22406836
16.  Dominant and diet-responsive groups of bacteria within the human colonic microbiota 
The ISME journal  2010;5(2):220-230.
The populations of dominant species within the human colonic microbiota can potentially be modified by dietary intake with consequences for health. Here we examined the influence of precisely controlled diets in 14 overweight men. Volunteers were provided successively with a control diet, diets high in resistant starch (RS) or non-starch polysaccharides (NSPs) and a reduced carbohydrate weight loss (WL) diet, over 10 weeks. Analysis of 16S rRNA sequences in stool samples of six volunteers detected 320 phylotypes (defined at >98% identity) of which 26, including 19 cultured species, each accounted for >1% of sequences. Although samples clustered more strongly by individual than by diet, time courses obtained by targeted qPCR revealed that ‘blooms' in specific bacterial groups occurred rapidly after a dietary change. These were rapidly reversed by the subsequent diet. Relatives of Ruminococcus bromii (R-ruminococci) increased in most volunteers on the RS diet, accounting for a mean of 17% of total bacteria compared with 3.8% on the NSP diet, whereas the uncultured Oscillibacter group increased on the RS and WL diets. Relatives of Eubacterium rectale increased on RS (to mean 10.1%) but decreased, along with Collinsella aerofaciens, on WL. Inter-individual variation was marked, however, with >60% of RS remaining unfermented in two volunteers on the RS diet, compared to <4% in the other 12 volunteers; these two individuals also showed low numbers of R-ruminococci (<1%). Dietary non-digestible carbohydrate can produce marked changes in the gut microbiota, but these depend on the initial composition of an individual's gut microbiota.
doi:10.1038/ismej.2010.118
PMCID: PMC3105703  PMID: 20686513
human colon; resistant starch; 16S rRNA; phylotypes; Ruminococcus; temporal change
17.  The first four million years of human evolution 
doi:10.1098/rstb.2010.0179
PMCID: PMC2981965  PMID: 20855300
18.  Dominant and diet-responsive groups of bacteria within the human colonic microbiota 
The ISME journal  2010;5(2):220-230.
The populations of dominant species within the human colonic microbiota can potentially be modified by dietary intake with consequences for health. Here we examined the influence of precisely controlled diets in 14 overweight males. Volunteers were provided successively with a control diet, diets high in resistant starch (RS) or non-starch polysaccharides (NSP), and a reduced carbohydrate weight loss (WL) diet, over 10 weeks. Analysis of 16S rRNA sequences in stool samples of six volunteers detected 320 phylotypes (defined at >98% identity) of which 26, including 19 cultured species, each accounted for >1% of sequences. Although samples clustered more strongly by individual than by diet, time courses obtained by targeted qPCR revealed that “blooms” in specific bacterial groups occurred rapidly following a dietary change. These were rapidly reversed by the subsequent diet. Relatives of Ruminococcus bromii (“R-ruminococci”) increased in most volunteers on the RS diet, accounting for a mean of 17% of total bacteria compared with 3.8% on the NSP diet, while the uncultured Oscillibacter group increased on the RS and WL diets. Relatives of Eubacterium rectale increased on RS (to mean 10.1%) but decreased, along with Collinsella aerofaciens, on WL. Inter-individual variation was marked, however, with >60% of resistant starch remaining unfermented in two volunteers on the RS diet, compared to <4% in the other 12 volunteers; these two individuals also showed low numbers of R-ruminococci (<1%). Dietary non-digestible carbohydrate can produce marked changes in the gut microbiota, but these depend on the initial composition of an individual’s gut microbiota.
doi:10.1038/ismej.2010.118
PMCID: PMC3105703  PMID: 20686513
19.  Partitioning core and satellite taxa from within cystic fibrosis lung bacterial communities 
The ISME Journal  2010;5(5):780-791.
Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections that lead to death in the majority of cases. The need to maintain lung function in these patients means that characterising these infections is vital. Increasingly, culture-independent analyses are expanding the number of bacterial species associated with CF respiratory samples; however, the potential significance of these species is not known. Here, we applied ecological statistical tools to such culture-independent data, in a novel manner, to partition taxa within the metacommunity into core and satellite species. Sputa and clinical data were obtained from 14 clinically stable adult CF patients. Fourteen rRNA gene libraries were constructed with 35 genera and 82 taxa, identified in 2139 bacterial clones. Shannon–Wiener and taxa-richness analyses confirmed no undersampling of bacterial diversity. By decomposing the distribution using the ratio of variance to the mean taxon abundance, we partitioned objectively the species abundance distribution into core and satellite species. The satellite group comprised 67 bacterial taxa from 33 genera and the core group, 15 taxa from 7 genera (including Pseudomonas (1 taxon), Streptococcus (2), Neisseria (2), Catonella (1), Porphyromonas (1), Prevotella (5) and Veillonella (3)], the last four being anaerobes). The core group was dominated by Pseudomonas aeruginosa. Other recognised CF pathogens were rare. Mantel and partial Mantel tests assessed which clinical factors influenced the composition observed. CF transmembrane conductance regulator genotype and antibiotic treatment correlated with all core taxa. Lung function correlated with richness. The clinical significance of these core and satellite species findings in the CF lung is discussed.
doi:10.1038/ismej.2010.175
PMCID: PMC3105771  PMID: 21151003
commonness and rarity; oral microbiota; polymicrobial infections; pulmonary disease; species abundance distributions
20.  High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease 
BMC Microbiology  2011;11:7.
Background
The gut microbiota is thought to play a key role in the development of the inflammatory bowel diseases Crohn's disease (CD) and ulcerative colitis (UC). Shifts in the composition of resident bacteria have been postulated to drive the chronic inflammation seen in both diseases (the "dysbiosis" hypothesis). We therefore specifically sought to compare the mucosa-associated microbiota from both inflamed and non-inflamed sites of the colon in CD and UC patients to that from non-IBD controls and to detect disease-specific profiles.
Results
Paired mucosal biopsies of inflamed and non-inflamed intestinal tissue from 6 CD (n = 12) and 6 UC (n = 12) patients were compared to biopsies from 5 healthy controls (n = 5) by in-depth sequencing of over 10,000 near full-length bacterial 16S rRNA genes. The results indicate that mucosal microbial diversity is reduced in IBD, particularly in CD, and that the species composition is disturbed. Firmicutes were reduced in IBD samples and there were concurrent increases in Bacteroidetes, and in CD only, Enterobacteriaceae. There were also significant differences in microbial community structure between inflamed and non-inflamed mucosal sites. However, these differences varied greatly between individuals, meaning there was no obvious bacterial signature that was positively associated with the inflamed gut.
Conclusions
These results may support the hypothesis that the overall dysbiosis observed in inflammatory bowel disease patients relative to non-IBD controls might to some extent be a result of the disturbed gut environment rather than the direct cause of disease. Nonetheless, the observed shifts in microbiota composition may be important factors in disease maintenance and severity.
doi:10.1186/1471-2180-11-7
PMCID: PMC3032643  PMID: 21219646
21.  The translation research in a dental setting (TRiaDS) programme protocol 
Background
It is well documented that the translation of knowledge into clinical practice is a slow and haphazard process. This is no less true for dental healthcare than other types of healthcare. One common policy strategy to help promote knowledge translation is the production of clinical guidance, but it has been demonstrated that the simple publication of guidance is unlikely to optimise practice. Additional knowledge translation interventions have been shown to be effective, but effectiveness varies and much of this variation is unexplained. The need for researchers to move beyond single studies to develop a generalisable, theory based, knowledge translation framework has been identified.
For dentistry in Scotland, the production of clinical guidance is the responsibility of the Scottish Dental Clinical Effectiveness Programme (SDCEP). TRiaDS (Translation Research in a Dental Setting) is a multidisciplinary research collaboration, embedded within the SDCEP guidance development process, which aims to establish a practical evaluative framework for the translation of guidance and to conduct and evaluate a programme of integrated, multi-disciplinary research to enhance the science of knowledge translation.
Methods
Set in General Dental Practice the TRiaDS programmatic evaluation employs a standardised process using optimal methods and theory. For each SDCEP guidance document a diagnostic analysis is undertaken alongside the guidance development process. Information is gathered about current dental care activities. Key recommendations and their required behaviours are identified and prioritised. Stakeholder questionnaires and interviews are used to identify and elicit salient beliefs regarding potential barriers and enablers towards the key recommendations and behaviours. Where possible routinely collected data are used to measure compliance with the guidance and to inform decisions about whether a knowledge translation intervention is required. Interventions are theory based and informed by evidence gathered during the diagnostic phase and by prior published evidence. They are evaluated using a range of experimental and quasi-experimental study designs, and data collection continues beyond the end of the intervention to investigate the sustainability of an intervention effect.
Discussion
The TRiaDS programmatic approach is a significant step forward towards the development of a practical, generalisable framework for knowledge translation research. The multidisciplinary composition of the TRiaDS team enables consideration of the individual, organisational and system determinants of professional behaviour change. In addition the embedding of TRiaDS within a national programme of guidance development offers a unique opportunity to inform and influence the guidance development process, and enables TRiaDS to inform dental services practitioners, policy makers and patients on how best to translate national recommendations into routine clinical activities.
doi:10.1186/1748-5908-5-57
PMCID: PMC2920875  PMID: 20646275
22.  Antibiotic Treatment of Clostridium difficile Carrier Mice Triggers a Supershedder State, Spore-Mediated Transmission, and Severe Disease in Immunocompromised Hosts ▿ †  
Infection and Immunity  2009;77(9):3661-3669.
Clostridium difficile persists in hospitals by exploiting an infection cycle that is dependent on humans shedding highly resistant and infectious spores. Here we show that human virulent C. difficile can asymptomatically colonize the intestines of immunocompetent mice, establishing a carrier state that persists for many months. C. difficile carrier mice consistently shed low levels of spores but, surprisingly, do not transmit infection to cohabiting mice. However, antibiotic treatment of carriers triggers a highly contagious supershedder state, characterized by a dramatic reduction in the intestinal microbiota species diversity, C. difficile overgrowth, and excretion of high levels of spores. Stopping antibiotic treatment normally leads to recovery of the intestinal microbiota species diversity and suppresses C. difficile levels, although some mice persist in the supershedding state for extended periods. Spore-mediated transmission to immunocompetent mice treated with antibiotics results in self-limiting mucosal inflammation of the large intestine. In contrast, transmission to mice whose innate immune responses are compromised (Myd88−/−) leads to a severe intestinal disease that is often fatal. Thus, mice can be used to investigate distinct stages of the C. difficile infection cycle and can serve as a valuable surrogate for studying the spore-mediated transmission and interactions between C. difficile and the host and its microbiota, and the results obtained should guide infection control measures.
doi:10.1128/IAI.00558-09
PMCID: PMC2737984  PMID: 19564382
23.  Comparing the microbiota of the cystic fibrosis lung and human gut 
Gut Microbes  2010;1(2):85-93.
doi:10.4161/gmic.1.2.11350
PMCID: PMC3023585  PMID: 21326915
cystic fibrosis; gut microbiota; bacterial commuity profiling

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