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Background: There is increasing interest in the pulmonary microbiome's bacterial and viral communities, particularly in the context of chronic airway infections in cystic fibrosis (CF). However, the isolation of microbial DNA from the sputum from patients with CF is technically challenging and the optimal protocols for the analysis of viral species, including bacteriophage, from clinical samples remains difficult. Materials and Methods: In this study, we evaluate a set of methods developed for processing and analyzing sputum from patients with CF with the goal of detecting Pf bacteriophage virion-derived nucleic acid. We evaluate the impact of bead beating, deoxyribonuclease digestion, and heating steps in these protocols focusing on the quantitative assessment of Pseudomonas aeruginosa and Pf bacteriophage in sputum. Results: Based on these comparative data, we describe an optimized protocol for processing sputum from patients with CF and isolating DNA for polymerase chain reaction or sequencing-based studies. Conclusion: These studies demonstrate the assessment of a specific bacteriophage and bacteria in sputum from patients with CF.

Pressures to publish, perverse incentives, financial interest and gender are amongst the most commonly discussed risk factors for scientific misconduct. However, evidence of their association with actual data fabrication and falsification is inconclusive. A recent case-controlled analysis of articles containing problematic image duplications suggested that country of affiliation of first and last authors is a significant predictor of scientific misconduct. The same analysis found null or negative associations with individual proxies of publication rate, impact and gender. The latter findings, in line with previous evidence, failed to support common hypotheses about the prevalence and causes of misconduct, but country-level effects may have confounded these results. Here we extend and complete previous results by comparing, via matched-controls analysis, articles from authors in the same country. We found that evidence for individual-level risk factors may be significant in some countries, and null or opposite in others. In particular, in countries where publications are rewarded with cash incentives, and especially China, the risk of problematic image duplication was higher for more productive, more frequently cited, earlier-career researchers working in lower-ranking institutions, in accordance with a “misaligned incentives” explanation for scientific misconduct. However, a null or opposite pattern was observed in all other countries, and especially the USA, UK and Canada, countries where concerns for misaligned incentives are commonly expressed. In line with previous results, we failed to observe a statistically significant association with industry funding and with gender. This is the first direct evidence of a link between publication performance and risk of misconduct and between university ranking and risk of misconduct. Commonly hypothesised individual risk factors for scientific misconduct, including career status and productivity, might be relevant in countries where cash-reward policies generate perverse incentives. In most scientifically active countries, however, where other incentives systems are in place, these patterns are not observed, and other risk factors might be more relevant. Policies to prevent and correct scientific misconduct may need to be tailored to a countries’ or institutions’ specific context.

There is increasing interest in the pulmonary microbiome’s bacterial and viral communities, particularly in the context of chronic airway infections in cystic fibrosis (CF). However, the isolation of microbial DNA from the sputum from patients with CF is technically challenging and the optimal protocols for the analysis of viral species, including bacteriophage, from clinical samples remains challenging. Here, we evaluate a set of methods developed for processing and analyzing sputum from patients with CF with a particular emphasis on detecting bacteriophage viron-derived nucleic acid. We evaluate the impact of bead-beating, deoxyribonuclease digestion, and heating steps in these protocols focusing on the quantitative assessment of Pseudomonas aeruginosa and Pf bacteriophage in sputum as a proof of concept. Based on these comparative data, we describe an optimized protocol for processing sputum from patients with CF and isolating DNA for PCR or sequencing-based studies. These studies will facilitate future assessments of bacteriophage and bacteria in sputum from patients with CF.Author SummaryPatients with cystic fibrosis (CF) have thickened secretions and develop chronic infections in their airways. While we culture bacterial pathogens from expectorated sputum this method favors detection of certain organisms. There is greater understanding that the microbiome with in the airway of patients with cystic fibrosis is varied and contains more than just the bacteria that we selectively culture. Processing sputum is difficult as it is very thick. There are many different ways to process sputum depending on what aspect of the sputum is to be studied. We have an interest in a specific bacteriophage, or a virus that infects bacteria. We sought to find the best method to take sputum from a CF patient and extract DNA so that we could detect both bacteria and bacteriophage DNA. We compared different methods that included different combinations of heat, mechanical homogenization, chemical lysis and DNA extraction by commercially available kits. We describe the method we found easiest to execute and produced the best yield for detection of both bacteria and bacteriophage. Our purpose of publishing this method in detail is to facilitate further study of viral and bacterial communities in the sputum of patients with CF.

It is commonly hypothesized that scientists are more likely to engage in data falsification and fabrication when they are subject to pressures to publish, when they are not restrained by forms of social control, when they work in countries lacking policies to tackle scientific misconduct, and when they are male. Evidence to test these hypotheses, however, is inconclusive due to the difficulties of obtaining unbiased data. Here we report a pre-registered test of these four hypotheses, conducted on papers that were identified in a previous study as containing problematic image duplications through a systematic screening of the journal PLoS ONE. Image duplications were classified into three categories based on their complexity, with category 1 being most likely to reflect unintentional error and category 3 being most likely to reflect intentional fabrication. We tested multiple parameters connected to the hypotheses above with a matched-control paradigm, by collecting two controls for each paper containing duplications. Category 1 duplications were mostly not associated with any of the parameters tested, as was predicted based on the assumption that these duplications were mostly not due to misconduct. Categories 2 and 3, however, exhibited numerous statistically significant associations. Results of univariable and multivariable analyses support the hypotheses that academic culture, peer control, cash-based publication incentives and national misconduct policies might affect scientific integrity. No clear support was found for the “pressures to publish” hypothesis. Female authors were found to be equally likely to publish duplicated images compared to males. Country-level parameters generally exhibited stronger effects than individual-level parameters, because developing countries were significantly more likely to produce problematic image duplications. This suggests that promoting good research practices in all countries should be a priority for the international research integrity agenda. Electronic supplementary material The online version of this article (10.1007/s11948-018-0023-7) contains supplementary material, which is available to authorized users.

The human distal gut is home to a rich and dense microbial community with representatives of all three domains of life which are intricately connected with our physiology and health. The combined genomes of these microbes, collectively called the human microbiome, vastly expand the metabolic capacities of our own genome, allowing us to break down and extract energy from dietary compounds that human enzymes cannot digest. In addition, the variable composition of these communities and their biotransformations might explain inter-individual differences in toxicities, tolerances and efficacies for certain drugs. Recent advances in sequencing technologies and bioinformatics have provided exciting new insights into the genomes of our microbial symbionts, their functional capacities and the interactions between these microbes and their human host. This review summarizes the metabolic conversions of dietary components and pharmaceuticals that take place in the human distal gut, as well as their implications for human health. LINKED ARTICLES: This article is part of a themed section on When Pharmacology Meets the Microbiome: New Targets for Therapeutics? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.24/issuetoc.

Marine mammals play crucial ecological roles in the oceans, but little is known about their microbiotas. Here we study the bacterial communities in 337 samples from 5 body sites in 48 healthy dolphins and 18 healthy sea lions, as well as those of adjacent seawater and other hosts. The bacterial taxonomic compositions are distinct from those of other mammals, dietary fish and seawater, are highly diverse and vary according to body site and host species. Dolphins harbour 30 bacterial phyla, with 25 of them in the mouth, several abundant but poorly characterized Tenericutes species in gastric fluid and a surprisingly paucity of Bacteroidetes in distal gut. About 70% of near-full length bacterial 16S ribosomal RNA sequences from dolphins are unique. Host habitat, diet and phylogeny all contribute to variation in marine mammal distal gut microbiota composition. Our findings help elucidate the factors structuring marine mammal microbiotas and may enhance monitoring of marine mammal health., Little is known about the microbiota of marine mammals, despite the crucial ecological roles played by these animals. Here, Bik et al. describe the bacterial communities associated with various body sites in dolphins and sea lions, as well as the microbiota of their dietary fish and adjacent seawater.

Have you ever been curious to find out which microbes live inside your intestinal tract (otherwise known as your gut)? Our gut is full of microbes that are intricately connected to many physiological processes, and these communities vary between persons or as a result of changes in our diet, lifestyle or health. Changes in these microbial communities are associated with certain diseases, and the analysis of the composition of our gut microbiome can be used as a clinical tool by medical professionals.

Background Hospitalization and antibiotic treatment can put patients at high risk for Clostridium difficile infection, where a disturbance of the gut microbiome allows for Clostridium difficile proliferation and associated symptoms, including mild, moderate, or severe diarrhea. Clostridium difficile infection is challenging to treat, often recurrent, and leads to almost 30,000 annual deaths in the USA alone. Here we present a case where SmartGut™, an at-home, self-administered sequencing-based clinical intestinal screening test, was used to identify the presence of Clostridium difficile in a patient with worsening diarrhea. Identification of this pathogen and subsequent treatment led to a significant improvement in symptoms. Case presentation The patient is a 29-year-old white woman with a history of severe irritable bowel syndrome with diarrhea, hemorrhoidectomy, and anal sphincterotomy complicated by a perianal fistula and perirectal abscesses that required extended courses of broad-spectrum antibiotics. In June 2016, she developed intermittent Clostridium difficile infections, which required continued antibiotic use. Months later she used an at-home, self-administered, intestinal microbial test, the first of which was negative for the presence of Clostridium difficile, but it detected the relative abundance of microbes associated with irritable bowel syndrome outside the healthy reference ranges. In the subsequent 2 months after the negative Clostridium difficile result, her gastrointestinal symptoms worsened dramatically. A second microbiome test resulted in a positive Clostridium difficile finding and continued abnormal microbial parameters, which led the treating physician to refer her to a gastroenterologist. Additional testing confirmed the presence of Clostridium difficile with a toxin-positive strain. She received treatment immediately and her gastrointestinal symptoms improved significantly over the next week. Conclusions This case report suggests that more frequent DNA testing for Clostridium difficile infections may be indicated in patients that are at high-risk for Clostridium difficile infection, especially for patients with irritable bowel syndrome, and those who undergo gastrointestinal surgery and/or an extended antibiotic treatment. This report also shows that such testing could be effectively performed using at-home, self-administered sequencing-based clinical intestinal microbial screening tests. Further research is needed to investigate whether the observations reported here extrapolate to a larger cohort of patients.

The present study analyzed 960 papers published in Molecular and Cellular Biology (MCB) from 2009-2016 and found 59 (6.1%) to contain inappropriately duplicated images. The 59 instances of inappropriate image duplication led to 42 corrections, 5 retractions and 12 instances in which no action was taken. Our experience suggests that the majority of inappropriate image duplications result from errors during figure preparation that can be remedied by correction. Nevertheless, ~10% of papers with inappropriate image duplications in MCB were retracted. If this proportion is representative, then as many as 35,000 papers in the literature are candidates for retraction due to image duplication. The resolution of inappropriate image duplication concerns after publication required an average of 6 h of journal staff time per published paper. MCB instituted a pilot program to screen images of accepted papers prior to publication that identified 12 manuscripts (14.5% out of 83) with image concerns in two months. The screening and correction of papers before publication required an average of 30 min of staff time per problematic paper. Image screening can identify papers with problematic images prior to publication, reduces post-publication problems and requires significantly less staff time than the correction of problems after publication.

Microbial community analysis can be biased by multiple technical factors, such as storage conditions, DNA extraction, or amplification conditions. In a high-throughput laboratory that relies on samples obtained from thousands of different subjects, knowledge of the extent of subject-introduced sampling and storage variation on the outcome of the inferred microbiome, as well as the effect of laboratory-introduced variation caused by reagent batches, equipment, or operator on the consistency of these processes within the laboratory is paramount. Here, we analyzed the effect of sampling from different parts of the same stool specimen or on different consecutive days, as well as short-term storage of samples at different temperatures on microbiome profiles obtained by 16S rRNA gene amplification. Each of these factors had relatively little effect on the microbial composition. In addition, replicate amplification of 44 stool samples showed reproducible results. Finally, 363 independent replicate extractions and amplifications of a single human homogenized stool (HS) specimen showed reproducible results (average Lin’s correlation = 0.95), with little variation introduced by HS batch, operator, extraction equipment, or DNA sequencer. In all cases, variations between replicates were significantly smaller than those between individual samples; subject identity always was the largest determinant. We propose that homogenized stool specimens could be used as quality control to routinely monitor the laboratory process and to validate new methods.

In most industrialized countries, screening programs for cervical cancer have shifted from cytology (Pap smear or ThinPrep) alone on clinician-obtained samples to the addition of screening for human papillomavirus (HPV), its main causative agent. For HPV testing, self-sampling instead of clinician-sampling has proven to be equally accurate, in particular for assays that use nucleic acid amplification techniques. In addition, HPV testing of self-collected samples in combination with a follow-up Pap smear in case of a positive result is more effective in detecting precancerous lesions than a Pap smear alone. Self-sampling for HPV testing has already been adopted by some countries, while others have started trials to evaluate its incorporation into national cervical cancer screening programs. Self-sampling may result in more individuals willing to participate in cervical cancer screening, because it removes many of the barriers that prevent women, especially those in low socioeconomic and minority populations, from participating in regular screening programs. Several studies have shown that the majority of women who have been underscreened but who tested HPV-positive in a self-obtained sample, will visit a clinic for follow-up diagnosis and management. Additionally, a self-collected sample can also be used for vaginal microbiome analysis, which can provide additional information about HPV infection persistence as well as vaginal health in general.

The composition of the vaginal microbiome, including both the presence of pathogens involved in sexually transmitted infections (STI) as well as commensal microbiota, has been shown to have important associations for a woman’s reproductive and general health. Currently, healthcare providers cannot offer comprehensive vaginal microbiome screening, but are limited to the detection of individual pathogens, such as high-risk human papillomavirus (hrHPV), the predominant cause of cervical cancer. There is no single test on the market that combines HPV, STI, and microbiome screening. Here, we describe a novel inclusive women’s health assay that combines self-sampling with sequencing-based HPV detection and genotyping, vaginal microbiome analysis, and STI-associated pathogen detection. The assay includes genotyping and detection of 14 hrHPV types, 5 low-risk HPV types (lrHPV), as well as the relative abundance of 32 bacterial taxa of clinical importance, includingLactobacillus,Sneathia,Gardnerella, and 4 pathogens involved in STI, with high sensitivity, specificity, and reproducibility. For each of these taxa, healthy ranges were determined in a group of 50 self-reported healthy women. The hrHPV portion of the test was evaluated against the Digene High-Risk HPV HC2 DNA test with vaginal samples obtained from 185 women. Results were concordant for 181/185 of the samples (overall agreement of 97.83%, Cohen’s kappa = 0.93), with sensitivity and specificity values of 94.74% and 98.64%, respectively. Two discrepancies were caused by the Digene assay’s known cross-reactivity with low-risk HPV types, while two additional samples were found to contain hrHPV not detected by Digene. This novel assay could be used to complement conventional cervical cancer screening, because its self-sampling format can expand access among women who would otherwise not participate, and because of its additional information about the composition of the vaginal microbiome and the presence of pathogens.

It is commonly hypothesized that scientists are more likely to engage in data falsification and fabrication when they are subject to pressures to publish, when they are not restrained by forms of social control, when they work in countries lacking policies to tackle scientific misconduct, and when they are male. Evidence to test these hypotheses, however, is inconclusive due to the difficulties of obtaining unbiased data.Here we report a pre-registered test of these four hypotheses, conducted on papers that were identified in a previous study as containing problematic image duplications through a systematic screening of the journal PLoS ONE. Image duplications were classified into three categories based on their complexity, with category 1 being most likely to reflect unintentional error and category 3 being most likely to reflect intentional fabrication. Multiple parameters connected to the hypotheses above were tested with a matched-control paradigm, by collecting two controls for each paper containing duplications.Category 1 duplications were mostly not associated with any of the parameters tested, in accordance with the assumption that these duplications were mostly not due to misconduct. Category 2 and 3, however, exhibited numerous statistically significant associations. Results of univariable and multivariable analyses support the hypotheses that academic culture, peer control, cash-based publication incentives and national misconduct policies might affect scientific integrity. Significant correlations between the risk of image duplication and individual publication rates or gender, however, were only observed in secondary and exploratory analyses.Country-level parameters generally exhibited effects of larger magnitude than individual-level parameters, because a subset of countries was significantly more likely to produce problematic image duplications. Promoting good research practices in all countries should be a priority for the international research integrity agenda.

The composition of the vaginal microbiome, including both the presence of pathogens involved in sexually transmitted infections (STI) as well as commensal microbiota, has been shown to have important associations for a woman´s reproductive and general health. Currently, healthcare providers cannot offer comprehensive vaginal microbiome screening, but are limited to the detection of individual pathogens, such as high-risk human papillomavirus (hrHPV), the predominant cause of cervical cancer. There is no single test on the market that combines HPV, STI, and microbiome screening. Here, we describe a novel inclusive vaginal health assay that combines self-sampling with sequencing-based HPV detection and genotyping, vaginal microbiome analysis, and STI-associated pathogen detection. The assay includes genotyping and detection of 14 hrHPV types, 5 low-risk HPV types (lrHPV), as well as the relative abundance of 31 bacterial taxa of clinical importance, including Lactobacillus, Sneathia, Gardnerella, and 3 pathogens involved in STI, with high sensitivity, specificity, and reproducibility. For each of these taxa, reference ranges were determined in a group of 50 self-reported healthy women. The HPV sequencing portion of the test was evaluated against the digene High-Risk HPV HC2 DNA test. For hrHPV genotyping, agreement was 95.3% with a kappa of 0.804 (601 samples); after removal of samples in which the digene hrHPV probe showed cross-reactivity with lrHPV types, the sensitivity and specificity of the hrHPV genotyping assay were 94.5% and 96.6%, respectively, with a kappa of 0.841. For lrHPV genotyping, agreement was 93.9% with a kappa of 0.788 (148 samples), while sensitivity and specificity were 100% and 92.9%, respectively. This novel assay could be used to complement conventional cervical cancer screening, because its self-sampling format can expand access among women who would otherwise not participate, and because of its additional information about the composition of the vaginal microbiome and the presence of pathogens. Fil: Bik, Elisabeth M.. Ubiome; Fil: Bird, Sara W.. Ubiome; Fil: Bustamante, Juan Pablo. Universidad Nacional de Entre Ríos. Instituto de Investigación y Desarrollo en Bioingeniería y Bioinformática - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigación y Desarrollo en Bioingeniería y Bioinformática; Argentina Fil: Leon, Luis E.. Ubiome

Aims In order to develop complementary health management strategies for marine mammals, we used culture-based and culture-independent approaches to identify gastrointestinal lactobacilli of the common bottlenose dolphin, Tursiops truncatus. Methods and Results We screened 307 bacterial isolates from oral and rectal swabs, milk and gastric fluid, collected from 38 dolphins in the U.S. Navy Marine Mammal Program, for potentially beneficial features. We focused our search on lactobacilli and evaluated their ability to modulate TNF secretion by host cells and inhibit growth of pathogens. We recovered Lactobacillus salivarius strains which secreted factors that stimulated TNF production by human monocytoid cells. These Lact. salivarius isolates inhibited growth of selected marine mammal and human bacterial pathogens. In addition, we identified a novel Lactobacillus species by culture and direct sequencing with 96·3% 16S rDNA sequence similarity to Lactobacillus ceti. Conclusions Dolphin-derived Lact. salivarius isolates possess features making them candidate probiotics for clinical studies in marine mammals. Significance and Impact of the Study This is the first study to isolate lactobacilli from dolphins, including a novel Lactobacillus species and a new strain of Lact. salivarius, with potential for veterinary probiotic applications. The isolation and identification of novel Lactobacillus spp. and other indigenous microbes from bottlenose dolphins will enable the study of the biology of symbiotic members of the dolphin microbiota and facilitate the understanding of the microbiomes of these unique animals.

Changes in the relative abundances of many intestinal microorganisms, both those that naturally occur in the human gut microbiome and those that are considered pathogens, have been associated with a range of diseases. To more accurately diagnose health conditions, medical practitioners could benefit from a molecular, culture-independent assay for the quantification of these microorganisms in the context of a healthy reference range. Here we present the targeted sequencing of the microbial 16S rRNA gene of clinically relevant gut microorganisms as a method to provide a gut screening test that could assist in the clinical diagnosis of certain health conditions. We evaluated the possibility of detecting 46 clinical prokaryotic targets in the human gut, 28 of which could be identified with high precision and sensitivity by a bioinformatics pipeline that includes sequence analysis and taxonomic annotation. These targets included 20 commensal, 3 beneficial (probiotic), and 5 pathogenic intestinal microbial taxa. Using stool microbiome samples from a cohort of 897 healthy individuals, we established a reference range defining clinically relevant relative levels for each of the 28 targets. Our assay quantifies 28 targets in the context of a healthy reference range and correctly reflected 38/38 verification samples of real and synthetic stool material containing known gut pathogens. Thus, we have established a method to determine microbiome composition with a focus on clinically relevant taxa, which has the potential to contribute to patient diagnosis, treatment, and monitoring. More broadly, our method can facilitate epidemiological studies of the microbiome as it relates to overall human health and disease.

Recent developments in sequencing methods and bioinformatics analysis tools have greatly enabled the culture-independent analysis of complex microbial communities associated with environmental samples, plants, and animals. This has led to a spectacular increase in the number of studies on both membership and functionalities of these hitherto invisible worlds, in particular those of the human microbiome. The wide variety in available microbiome tools and platforms can be overwhelming, and making sound conclusions from scientific research can be challenging. Here, I will review 1) the methodological and analytic hoops a good microbiome study has to jump through, including DNA extraction and choice of bioinformatics tools, 2) the hopes this field has generated for diseases such as autism and inflammatory bowel diseases, and 3) some of the hypes that it has created, e.g., by confusing correlation and causation, and the recent pseudoscientific commercialization of microbiome research.

Metagenomic libraries represent subsamples of the total DNA found at a study site and offer unprecedented opportunities to study ecological and functional aspects of microbial communities. To examine the depth of a community sequencing effort, rarefaction analysis of the ribosomal small subunit (SSU/16S/18S) gene in the metagenome is usually performed. The fragmentary, non-overlapping nature of SSU sequences in metagenomic libraries poses a problem for this analysis, however. We introduce a software package - Megraft - that grafts SSU fragments onto full-length SSU sequences, accounting for observed and unobserved variability, for accurate assessment of species richness and sequencing depth in metagenomics endeavors.

The vast majority of bacterial diversity lies within phylum-level lineages called "candidate phyla," which lack isolated representatives and are poorly understood. These bacteria are surprisingly abundant in the oral cavity of marine mammals. We employed a genome-resolved metagenomic approach to recover and characterize genomes and functional potential from microbes in the oral gingival sulcus of two bottlenose dolphins (Tursiops truncatus). We detected organisms from 24 known bacterial phyla and one archaeal phylum. We also recovered genomes from two deep-branching, previously uncharacterized phylum-level lineages (here named "Candidatus Delphibacteria" and "Candidatus Fertabacteria"). The Delphibacteria lineage is found in both managed and wild dolphins; its metabolic profile suggests a capacity for denitrification and a possible role in dolphin health. We uncovered a rich diversity of predicted Cas9 proteins, including the two longest predicted Cas9 proteins to date. Notably, we identified the first type II CRISPR-Cas systems encoded by members of the Candidate Phyla Radiation. Using their spacer sequences, we subsequently identified and assembled a complete Saccharibacteria phage genome. These findings underscore the immense microbial diversity and functional potential that await discovery in previously unexplored environments.

We have developed a microfluidic device that allows the isolation and genome amplification of individual microbial cells, thereby enabling organism-level genomic analysis of complex microbial ecosystems without the need for culture. This device was used to perform a directed survey of the human subgingival crevice and to isolate bacteria having rod-like morphology. Several isolated microbes had a 16S rRNA sequence that placed them in candidate phylum TM7, which has no cultivated or sequenced members. Genome amplification from individual TM7 cells allowed us to sequence and assemble >1,000 genes, providing insight into the physiology of members of this phylum. This approach enables single-cell genetic analysis of any uncultivated minority member of a microbial community.

Genetics, diet, and other environmental exposures are thought to be major factors in the development and composition of the intestinal microbiota of animals. However, the relative contributions of these factors in adult animals, as well as variation with time in a variety of important settings, are still not fully understood. We studied a population of inbred, female mice fed the same diet and housed under the same conditions. We collected fecal samples from 46 individual mice over two weeks, sampling four of these mice for periods as long as 236 days for a total of 190 samples, and determined the phylogenetic composition of their microbial communities after analyzing 1,849,990 high-quality pyrosequencing reads of the 16S rRNA gene V3 region. Even under these controlled conditions, we found significant inter-individual variation in community composition, as well as variation within an individual over time, including increases in alpha diversity during the first 2 months of co-habitation. Some variation was explained by mouse membership in different cage and vendor shipment groups. The differences among individual mice from the same shipment group and cage were still significant. Overall, we found that 23% of the variation in intestinal microbiota composition was explained by changes within the fecal microbiota of a mouse over time, 12% was explained by persistent differences among individual mice, 14% by cage, and 18% by shipment group. Our findings suggest that the microbiota of controlled populations of inbred laboratory animals may not be as uniform as previously thought, that animal rearing and handling may account for some variation, and that as yet unidentified factors may explain additional components of variation in the composition of the microbiota within populations and individuals over time. These findings have implications for the design and interpretation of experiments involving laboratory animals.

The purpose of this study was to evaluate the composition and richness of bacterial communities associated with low-birthweight (LBW) infants in relation to host body site, individual, and age. Bacterial 16S rRNA genes from saliva samples, skin swabs, and stool samples collected on postnatal days 8, 10, 12, 15, 18, and 21 from six LBW (five premature) infants were amplified, pyrosequenced, and analyzed within a comparative framework that included analogous data from normal-birthweight (NBW) infants and healthy adults. We found that body site was the primary determinant of bacterial community composition in the LBW infants. However, site specificity depended on postnatal age: saliva and stool compositions diverged over time but were not significantly different until the babies were 15 days old. This divergence was primarily driven by progressive temporal turnover in the distal gut, which proceeded at a rate similar to that of age-matched NBW infants. Neonatal skin was the most adult-like in microbiota composition, while saliva and stool remained the least so. Compositional variation among infants was marked and depended on body site and age. Only the smallest, most premature infant received antibiotics during the study period; this heralded a coexpansion of Pseudomonas aeruginosa and a novel Mycoplasma sp. in the oral cavity of this vaginally delivered, intubated patient. We conclude that concurrent molecular surveillance of multiple body sites in LBW neonates reveals a delayed compositional differentiation of the oral cavity and distal gut microbiota and, in the case of one infant, an abundant, uncultivated oral Mycoplasma sp., recently detected in human vaginal samples., IMPORTANCE Complications of premature birth are the most common cause of neonatal mortality. Colonization by the indigenous microbiota, which begins at delivery, may predispose some high-risk newborns to invasive infection or necrotizing enterocolitis (NEC), and protect others, yet neonatal microbiome dynamics are poorly understood. Here, we present the first cultivation-independent time series tracking microbiota assembly across multiple body sites in a synchronous cohort of hospitalized low-birthweight (LBW) neonates. We take advantage of archived samples and publically available sequence data and compare our LBW infant findings to those from normal-birthweight (NBW) infants and healthy adults. Our results suggest potential windows of opportunity for the dispersal of microbes within and between hosts and support recent findings of substantial baseline spatiotemporal variation in microbiota composition among high-risk newborns.

The fruit fly Drosophila is a classic model organism to study adaptation as well as the relationship between genetic variation and phenotypes. Although associated bacterial communities might be important for many aspects of Drosophila biology, knowledge about their diversity, composition, and factors shaping them is limited. We used 454-based sequencing of a variable region of the bacterial 16S ribosomal RNA gene to characterize the bacterial communities associated with wild and laboratory Drosophila isolates. In order to specifically investigate effects of food source and host species on bacterial communities, we analyzed samples from wild Drosophila melanogaster and D. simulans collected from a variety of natural substrates, as well as from adults and larvae of nine laboratory reared Drosophila species. We find no evidence for host species effects in lab reared flies, instead lab of origin and stochastic effects, which could influence studies of Drosophila phenotypes, are pronounced. In contrast, the natural Drosophila associated microbiota appears to be predominantly shaped by food substrate with an additional but smaller effect of host species identity. We identify a core member of this natural microbiota that belongs to the genus Gluconobacter and is common to all wild caught flies in this study, but absent from the laboratory. This makes it a strong candidate for being part of what could be a natural D. melanogaster and D. simulans core microbiome. Furthermore we were able to identify candidate pathogens in natural fly isolates., Focus has shifted from bacterial diversity to the factors we identified as important for Drosophila associated bacterial communities. This is reflected by a change of the title and removal of Suppl. Table 2. Figure 3B was added. Table 1 now corresponds better to the text. Table 2 has become more comprehensive. Parts of the M&M section were clarified. There are no changes to the major results

Background Our current understanding of the composition and stability of the human distal gut microbiota is based largely on studies of infants and adults living in developed countries. In contrast, little is known about the gut microbiota and its variation over time in older children and adolescents, especially in developing countries. Methodology/Principal Findings We compared the diversity, composition, and temporal stability of the fecal microbiota of healthy children, ages 9 to 14 years, living in an urban slum in Bangladesh with that of children of the same age range in an upper-middle class suburban community in the United States. We analyzed >8,000 near full-length 16S rRNA gene sequences and over 845,000 pyrosequencing reads of the 16S rRNA V1–V3 region. The distal gut of Bangladeshi children harbored significantly greater bacterial diversity than that of U.S. children, including novel lineages from several bacterial phyla. Bangladeshi and U.S. children had distinct fecal bacterial community membership and structure; the microbiota of Bangladeshi children was enriched in Prevotella, Butyrivibrio, and Oscillospira and depleted in Bacteroides relative to U.S. children (although similar to Bangladeshi adults). Furthermore, community membership and structure in Bangladeshi children was significantly less stable month-to-month than U.S. children. Conclusions/Significance Together, these results suggest that differing environmental or genetic factors may shape the microbiota of healthy children in the two countries. Further investigation is necessary to understand the mechanisms and factors that underlie these differences, and to incorporate these findings into new strategies for the prevention and treatment of childhood and adolescent diseases.

In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDVCO139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1-acceptor DNA is probably located downstream of rfbDVCO139. The DNA region between rfaDVCO139 and rfbQRSVCO139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found in V. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.

The role played by microbial invasion of the amniotic cavity (MIAC) in preterm pre-labor rupture of membranes (pPROM) is inadequately characterized, in part because of reliance on cultivation-based methods.Amniotic fluid from 204 subjects with pPROM was analyzed with both cultivation and molecular methods in a retrospective cohort study. Broad-range and group-specific polymerase chain reaction (PCR) assays targeted small subunit ribosomal DNA (rDNA), or other gene sequences, from bacteria, fungi, and archaea. Results were correlated with measurements of host inflammation, as well as pregnancy and perinatal outcomes.The prevalence of MIAC was 34% (70/204) by culture, 45% (92/204) by PCR, and 50% (101/204) by both methods combined. The number of bacterial species revealed by PCR (44 species-level phylotypes) was greater than that by culture (14 species) and included as-yet uncultivated taxa. Some taxa detected by PCR have been previously associated with the gastrointestinal tract (e.g., Coprobacillus sp.), the mouth (e.g., Rothia dentocariosa), or the vagina in the setting of bacterial vaginosis (e.g., Atopobium vaginae). The relative risk for histologic chorioamnionitis was 2.1 for a positive PCR [95% confidence interval (CI), 1.4-3.0] and 2.0 for a positive culture (95% CI, 1.4-2.7). Bacterial rDNA abundance exhibited a dose relationship with gestational age at delivery (R(2) = 0.26; P0.01). A positive PCR was associated with lower mean birthweight, and with higher rates of respiratory distress syndrome and necrotizing enterocolitis (P0.05 for each outcome).MIAC in pPROM is more common than previously recognized and is associated in some cases with uncultivated taxa, some of which are typically associated with the gastrointestinal tract. The detection of MIAC by molecular methods has clinical significance.

Vibrio cholerae CVD101 is a very effective live vaccine. Although this strain does not produce active cholera toxin because of a mutation in the gene for the cholera toxin A subunit, it still shows residual pathogenicity. To attenuate CVD101 further, we set out to isolate derivatives of CVD101 which were limited in their ability to proliferate in vivo. Two delta-aminolevulinic acid auxotrophs of CVD101, designated V286 and V287, were isolated by transposon mutagenesis and penicillin enrichment. Southern blotting revealed that the mutants differed with respect to the location of the transposon insertion. Under aerobic conditions, in the absence of delta-aminolevulinic acid, both mutants showed diminished growth compared with CVD101. The growth of V286 was most severely affected. Microaerophilic growth of both mutants was less affected. Competition experiments with a rabbit model showed that strain V286 was found in numbers 10(3)- to 10(4)-fold lower than its parental strain. This observation indicates that strain V286 is impaired in its ability to colonize the rabbit intestine. It also supports an important role for aerobic growth in the colonization of the intestine by V. cholerae. Vaccination of rabbits with a single dose of strain V286 resulted in full protection against challenge with a virulent strain. Strain V286 was not shed from rabbits in a cultivatable form. Our results suggest that delta-aminolevulinic acid auxotrophy can attenuate V. cholerae by limiting its ability to colonize without affecting its capacity to induce protective immunity. Furthermore, this type of mutation may prevent the spread of V. cholerae vaccine strains in the environment.

The human body is an ecosystem harboring complex site-specific microbial communities. The majority of these human-associated microbes are found in the intestinal tract, where they play important roles in energy uptake, vitamin synthesis, and epithelial and immunity development. Recent molecular studies have characterized the human-associated microbiotas in more detail than conventional culture-dependent techniques, showing a large degree of microbial diversity and differences between anatomical sites and individuals. Investigating the composition and function of microbial symbionts will facilitate better understanding of their roles in human health and disease.

Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like element, and these strains are highly polymorphic with regard to the site of integration in the chromosome. In contrast, Mycobacterium bovis BCG contains a single copy of the insertion element, and in all strains this copy is integrated at the same site in the chromosome. In this study, we determined the sequence of the single-copy insertion element from M. bovis BCG, IS987, and its flanking regions. The analysis of IS987 revealed that this element was virtually identical to the sequence of IS986 from M. tuberculosis. IS987 is located in a region containing direct repeats (DRs). The cloned flanking regions contained 20 virtually identical DRs of 36 bp, each separated by 35 to 41 bp of spacer DNA. Analysis of chromosomal DNA by the polymerase chain reaction revealed the presence of a cluster of 49 DRs, and IS987 is inserted in the 30th DR. Furthermore, the DR sequences were found to occur only in species of the M. tuberculosis complex and not in nine other mycobacterial species tested. Analysis of 14 M. tuberculosis strains revealed the presence of one insertion sequence element in the DR-containing region of eight strains, two insertion sequence elements were located in the DR region of five strains, and one strain did not contain an insertion sequence element in this region. Additionally, the DR-containing regions of these 14 M. tuberculosis strains were polymorphic in length and composition. We conclude that the DR cluster is a specific, hot-spot region for integration of insertion elements in the chromosome of M. tuberculosis complex strains.

Background Preterm delivery causes substantial neonatal mortality and morbidity. Unrecognized intra-amniotic infections caused by cultivation-resistant microbes may play a role. Molecular methods can detect, characterize and quantify microbes independently of traditional culture techniques. However, molecular studies that define the diversity and abundance of microbes invading the amniotic cavity, and evaluate their clinical significance within a causal framework, are lacking. Methods and Findings In parallel with culture, we used broad-range end-point and real-time PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from amniotic fluid of 166 women in preterm labor with intact membranes. We sequenced up to 24 rRNA clones per positive specimen and assigned taxonomic designations to approximately the species level. Microbial prevalence, diversity and abundance were correlated with host inflammation and with gestational and neonatal outcomes. Study subjects who delivered at term served as controls. The combined use of molecular and culture methods revealed a greater prevalence (15% of subjects) and diversity (18 taxa) of microbes in amniotic fluid than did culture alone (9.6% of subjects; 11 taxa). The taxa detected only by PCR included a related group of fastidious bacteria, comprised of Sneathia sanguinegens, Leptotrichia amnionii and an unassigned, uncultivated, and previously-uncharacterized bacterium; one or more members of this group were detected in 25% of positive specimens. A positive PCR was associated with histologic chorioamnionitis (adjusted odds ratio [OR] 20; 95% CI, 2.4 to 172), and funisitis (adjusted OR 18; 95% CI, 3.1 to 99). The positive predictive value of PCR for preterm delivery was 100 percent. A temporal association between a positive PCR and delivery was supported by a shortened amniocentesis-to-delivery interval (adjusted hazard ratio 4.6; 95% CI, 2.2 to 9.5). A dose-response association was demonstrated between bacterial rDNA abundance and gestational age at delivery (r2 = 0.42; P

Almost immediately after a human being is born, so too is a new microbial ecosystem, one that resides in that person's gastrointestinal tract. Although it is a universal and integral part of human biology, the temporal progression of this process, the sources of the microbes that make up the ecosystem, how and why it varies from one infant to another, and how the composition of this ecosystem influences human physiology, development, and disease are still poorly understood. As a step toward systematically investigating these questions, we designed a microarray to detect and quantitate the small subunit ribosomal RNA (SSU rRNA) gene sequences of most currently recognized species and taxonomic groups of bacteria. We used this microarray, along with sequencing of cloned libraries of PCR-amplified SSU rDNA, to profile the microbial communities in an average of 26 stool samples each from 14 healthy, full-term human infants, including a pair of dizygotic twins, beginning with the first stool after birth and continuing at defined intervals throughout the first year of life. To investigate possible origins of the infant microbiota, we also profiled vaginal and milk samples from most of the mothers, and stool samples from all of the mothers, most of the fathers, and two siblings. The composition and temporal patterns of the microbial communities varied widely from baby to baby. Despite considerable temporal variation, the distinct features of each baby's microbial community were recognizable for intervals of weeks to months. The strikingly parallel temporal patterns of the twins suggested that incidental environmental exposures play a major role in determining the distinctive characteristics of the microbial community in each baby. By the end of the first year of life, the idiosyncratic microbial ecosystems in each baby, although still distinct, had converged toward a profile characteristic of the adult gastrointestinal tract.

The microbiota of the human stomach and the influence of Helicobacter pylori colonization on its composition remain largely unknown. We characterized bacterial diversity within the human gastric mucosa by using a small subunit 16S rDNA clone library approach and analyzed 1,833 sequences generated by broad-range bacterial PCR from 23 gastric endoscopic biopsy samples. A diverse community of 128 phylotypes was identified, featuring diversity at this site greater than previously described. The majority of sequences were assigned to the Proteobacteria , Firmicutes , Actinobacteria , Bacteroidetes , and Fusobacteria phyla. Ten percent of the phylotypes were previously uncharacterized, including a Deinococcus -related organism, relatives of which have been found in extreme environments but not reported before in humans. The gastric clone libraries from 19 subjects contained H. pylori rDNA; however, only 12 of these subjects tested positive for H. pylori by conventional laboratory methods. Statistical analysis revealed a large degree of intersubject variability of the gastric ecosystem. The presence of H. pylori did not affect the composition of the gastric community. This gastric bacterial rDNA data set was significantly different from sequence collections of the human mouth and esophagus described in other studies, indicating that the human stomach may be home to a distinct microbial eco-system. The gastric microbiota may play important, as-yet-undiscovered roles in human health and disease.

Complex microbial ecosystems occupy the skin, mucosa and alimentary tract of all mammals, including humans. Recent advances have highlighted the tremendous diversity of these microbial communities and their importance to host physiology, but questions remain about the ecological processes that establish and maintain the microbiota throughout life. The prevailing view, that the gastrointestinal microbiota of adult humans is a climax community comprised of the superior competitors for a stable set of niches, does not account for all of the experimental data. We argue here that the unique history of each community and intrinsic temporal dynamics also influence the structure of human intestinal communities.

Enterobacter cloacae is becoming an increasingly important nosocomial pathogen. Outbreaks of E. cloacae in intensive care units and burns units have been described frequently. In December 1999, a neonate with line sepsis was transferred from a university hospital to the neonatal unit of the Diakonessen Hospital. Blood culture yielded E. cloacae. An outbreak of E. cloacae was occurring in the university hospital at that time. In February 2000, a second neonate in our hospital developed line sepsis caused by E. cloacae. Direct measures taken included cohorting of infected children, disinfection of incubators, thermometers and wards, and screening patients. Of nine neonates, seven were colonized with E. cloacae. Despite these measures, the outbreak continued. Forty-one patients were screened; 15 were colonized. Environmental searches yielded E. cloacae in a sink and on two thermometers. Sixteen isolates were typed by arbitrarily primed PCR using four primers. All the patient isolates and the two isolates from thermometers were identical. The strain isolated from the sink was unrelated. Amplified fragment length polymorphism typing showed that the outbreak clone was identical to that in the university hospital. After the introduction of disposable thermometer covers, E. cloacae colonization slowly decreased.

The reproducibility of the binary typing (BT) protocol developed for epidemiological typing of Staphylococcus aureus was analyzed in a biphasic multicenter study. In a Dutch multicenter pilot study, 10 genetically unique isolates of methicillin-resistant S. aureus (MRSA) were characterized by the BT assay as presented by van Leeuwen et al. [J. Clin. Microbiol. 2001 39 (1) 328]. The BT assay, including a standardized DNA extraction protocol was performed in duplicate in eleven medical microbiology laboratories. Two different hybridization detection procedures were applied and a prelabeled DNA sample as process control was included. Only three laboratories accurately identified all strains. Divergence in technical procedures resulted in misinterpretation due to an increasing number of faint or absent hybridization signals in combination with high background staining. The binary type of the process control was determined correctly by all participating laboratories. The feasibility of the BT protocol was related directly to the skill of the laboratory personnel. On the basis of the national study, we concluded that the DNA extraction protocol needed modification to improve DNA yield and purity. Subsequently, seven European laboratories participated in an international study to determine the reproducibility of the modified BT protocol. Each center was asked to analyze 10 DNA samples previously extracted from 10 MRSA strains (phase 1) and, additionally, to analyze 10 MRSA strains, using the standardized or their in-house DNA isolation protocol (phase 2). A prelabeled DNA process control sample was included again. The binary types of all DNA samples were identified correctly by all but one laboratories. This latter laboratory diverged from the protocol by adding an excess of labeled DNA to the hybridization mixture, resulting in a high background and, therefore, noninterpretable BT results. All centers produced identical BT results for the process control. Five of the seven centers correctly identified the binary types of all 10 MRSA strains in phase 2 of the international study. Three of these centers used their in-house DNA extraction protocol. Divergence from the standard BT protocol in the remaining two centers resulted in no interpretable BT data for the 10 MRSA strains. The study demonstrated that each center that followed the BT protocol to the letter could generate reproducible results, irrespective whether or not an in-house DNA isolation protocol was used. The current BT protocol thus represents a simple method generating robust, reproducible genotype data for S. aureus strains.

Therapeutic food interventions have reduced mortality in children with severe acute malnutrition (SAM), but incomplete restoration of healthy growth remains a major problem. The relationships between the type of nutritional intervention, the gut microbiota, and therapeutic responses are unclear. In the current study, bacterial species whose proportional representation define a healthy gut microbiota as it assembles during the first two postnatal years were identified by applying a machine-learning-based approach to 16S ribosomal RNA data sets generated from monthly faecal samples obtained from birth onwards in a cohort of children living in an urban slum of Dhaka, Bangladesh, who exhibited consistently healthy growth. These age-discriminatory bacterial species were incorporated into a model that computes a 'relative microbiota maturity index' and 'microbiota-for-age Z-score' that compare postnatal assembly (defined here as maturation) of a child's faecal microbiota relative to healthy children of similar chronologic age. The model was applied to twins and triplets (to test for associations of these indices with genetic and environmental factors, including diarrhoea), children with SAM enrolled in a randomized trial of two food interventions, and children with moderate acute malnutrition. Our results indicate that SAM is associated with significant relative microbiota immaturity that is only partially ameliorated following two widely used nutritional interventions. Immaturity is also evident in less severe forms of malnutrition and correlates with anthropometric measurements. Microbiota maturity indices provide a microbial measure of human postnatal development, a way of classifying malnourished states, and a parameter for judging therapeutic efficacy. More prolonged interventions with existing or new therapeutic foods and/or addition of gut microbes may be needed to achieve enduring repair of gut microbiota immaturity in childhood malnutrition and improve clinical outcomes.

The emergence of the novel Vibrio cholerae strain, 0139 Bengal, which caused a large epidemic in Southeast Asia, underlines the adaptability of pathogenic microorganisms. Recent studies reveal that horizontal transfer of cell-wall polysaccharide genes played a central role in the emergence of this strain and that its genesis may not be as unique as initially believed.

Only Vibrio cholerae strains of serotype O1 are known to cause epidemics, while non-O1 strains are associated with sporadic cases of cholera. It was therefore unexpected that the recent cholera epidemic in Asia was caused by a non-O1 strain with the serotype O139. We provide evidence that O139 arose from a strain closely related to the causative agent of the present cholera pandemic, V. cholerae O1 El Tor, by acquisition of novel DNA which was inserted into, and replaced part of, the O antigen gene cluster of the recipient strain. Part of the novel DNA was sequenced and two open reading frames (otnA and otnB) were observed, the products of which showed homology to proteins involved in capsule and O antigen synthesis, respectively. This suggests that the otnAB DNA determines the distinct antigenic properties of the O139 cell surface. The otnAB DNA was not detected in O1 strains, but was present in two non-O1 V. cholerae strains with serotypes O69 and O141. In the O69 and O139 strains the otnAB genes were located proximate to the putative insertion sequence (IS) element rfbQRS, which is associated with O antigen synthesis genes in O1 strains, and may have played a role in the insertion of the otnAB DNA in the recipient chromosome. Our results suggest that the O139 strain arose by horizontal gene transfer between a non-O1 and an O1 strain. The acquired DNA has altered the antigenic properties of the recipient O1 strain, providing a selective advantage in a region where a large part of the population is immune to O1 strains.(ABSTRACT TRUNCATED AT 250 WORDS)

During the screening of a Mycobacterium tuberculosis lambda gt-11 gene library with monoclonal antibodies, we detected a recombinant clone, lambda PH7311, which contained a mycobacterial DNA insert that hybridized specifically with DNA of M. tuberculosis complex strains. Part of this insert was sequenced and used for the development of an M. tuberculosis complex-specific polymerase chain reaction (PCR). Only strains belonging to species of the M. tuberculosis complex group contained an amplifiable fragment of 158 base pairs (bp). This fragment was absent in all strains tested belonging to 15 other mycobacterial species. After amplification by PCR and dot blot hybridization with a digoxigenin-labeled oligonucleotide, the limit of detection of purified genomic M. tuberculosis DNA amounted to a quantity corresponding to 20 bacterial cells. By this technique about 10(3) M. tuberculosis bacteria were detectable in sputum. Using PCR, we were also able to detect M. tuberculosis cells in clinical material such as pleural fluid, bronchial washings, and biopsies, and these results were comparable with those obtained by classical bacterial culture. Of 34 M. tuberculosis strains, 5 did not carry the amplifiable 158-bp fragment, which occurs usually as a single copy in the chromosome. Evidence is presented that the 158-bp fragment is located near a repeated sequence in the chromosome. We presume that strains which did not carry the 158-bp fragment have lost a chromosomal segment by a genetic rearrangement induced by the repetitive DNA element.

Diverse and complex microbial ecosystems are found in virtually every environment on earth, yet we know very little about their composition and ecology. Comprehensive identification and quantification of the constituents of these microbial communities--a 'census'--is an essential foundation for understanding their biology. To address this problem, we developed, tested and optimized a DNA oligonucleotide microarray composed of 10,462 small subunit (SSU) ribosomal DNA (rDNA) probes (7167 unique sequences) selected to provide quantitative information on the taxonomic composition of diverse microbial populations. Using our optimized experimental approach, this microarray enabled detection and quantification of individual bacterial species present at fractional abundances of