Accurate Profiling of Microbial Communities from Massively Parallel Sequencing using Convex Optimization

We describe the Microbial Community Reconstruction ({\bf MCR}) Problem, which is fundamental for microbiome analysis. In this problem, the goal is to reconstruct the identity and frequency of species comprising a microbial community, using short sequence reads from Massively Parallel Sequencing (MPS) data obtained for specified genomic regions. We formulate the problem mathematically as a convex optimization problem and provide sufficient conditions for identifiability, namely the ability to reconstruct species identity and frequency correctly when the data size (number of reads) grows to infinity. We discuss different metrics for assessing the quality of the reconstructed solution, including a novel phylogenetically-aware metric based on the Mahalanobis distance, and give upper-bounds on the reconstruction error for a finite number of reads under different metrics. We propose a scalable divide-and-conquer algorithm for the problem using convex optimization, which enables us to handle large problems (with $\sim10^6$ species). We show using numerical simulations that for realistic scenarios, where the microbial communities are sparse, our algorithm gives solutions with high accuracy, both in terms of obtaining accurate frequency, and in terms of species phylogenetic resolution.Comment: To appear in SPIRE 1

Randomized clinical trial to evaluate the effect of fecal microbiota transplant for initial Clostridium difficile infection in intestinal microbiome

Objective The aim of this study was to evaluate the impact of fecal donor-unrelated donor mix (FMT-FURM) transplantation as first-line therapy for C. difficile infection (CDI) in intestinal microbiome. Methods We designed an open, two-arm pilot study with oral vancomycin (250mg every 6 h for 10–14 days) or FMT-FURM as treatments for the first CDI episode in hospitalized adult patients in Hospital Universitario “Dr. Jose Eleuterio Gonzalez”. Patients were randomized by a closed envelope method in a 1: 1 ratio to either oral vancomycin or FMT-FURM. CDI resolution was considered when there was a reduction on the Bristol scale of at least 2 points, a reduction of at least 50% in the number of bowel movements, absence of fever, and resolution of abdominal pain (at least two criteria). From each patient, a fecal sample was obtained at days 0, 3, and 7 after treatment. Specimens were cultured to isolate C. difficile, and isolates were characterized by PCR. Susceptibility testing of isolates was performed using the agar dilution method. Fecal samples and FMT-FURM were analyzed by 16S rRNA sequencing. Results We included 19 patients; 10 in the vancomycin arm and 9 in the FMT-FURM arm. However, one of the patients in the vancomycin arm and two patients in the FMT-FURM arm were eliminated. Symptoms resolved in 8/9 patients (88.9%) in the vancomycin group, while symptoms resolved in 4/7 patients (57.1%) after the first FMT-FURM dose (P = 0.26) and in 5/7 patients (71.4%) after the second dose (P = 0.55). During the study, no adverse effects attributable to FMT-FURM were observed in patients. Twelve isolates were recovered, most isolates carried tcdB, tcdA, cdtA, and cdtB, with an 18-bp deletion in tcdC. All isolates were resistant to ciprofloxacin and moxifloxacin but susceptible to metronidazole, linezolid, fidaxomicin, and tetracycline. In the FMT-FURM group, the bacterial composition was dominated by Firmicutes, Bacteroidetes, and Proteobacteria at all-time points and the microbiota were remarkably stable over time. The vancomycin group showed a very different pattern of the microbial composition when comparing to the FMT-FURM group over time. Conclusion The results of this preliminary study showed that FMT-FURM for initial CDI is associated with specific bacterial communities that do not resemble the donors’ sample.Peer reviewedFinal Published versio

Forensic analysis of the microbiome of phones and shoes

BACKGROUND: Microbial interaction between human-associated objects and the environments we inhabit may have forensic implications, and the extent to which microbes are shared between individuals inhabiting the same space may be relevant to human health and disease transmission. In this study, two participants sampled the front and back of their cell phones, four different locations on the soles of their shoes, and the floor beneath them every waking hour over a 2-day period. A further 89 participants took individual samples of their shoes and phones at three different scientific conferences. RESULTS: Samples taken from different surface types maintained significantly different microbial community structures. The impact of the floor microbial community on that of the shoe environments was strong and immediate, as evidenced by Procrustes analysis of shoe replicates and significant correlation between shoe and floor samples taken at the same time point. Supervised learning was highly effective at determining which participant had taken a given shoe or phone sample, and a Bayesian method was able to determine which participant had taken each shoe sample based entirely on its similarity to the floor samples. Both shoe and phone samples taken by conference participants clustered into distinct groups based on location, though much more so when an unweighted distance metric was used, suggesting sharing of low-abundance microbial taxa between individuals inhabiting the same space. CONCLUSIONS: Correlations between microbial community sources and sinks allow for inference of the interactions between humans and their environment. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40168-015-0082-9) contains supplementary material, which is available to authorized users

Episodic memory and learning rates in amyotrophic lateral sclerosis without dementia

In amyotrophic lateral sclerosis (ALS), memory deficits may be primary or secondary to executive dysfunction. We assessed episodic memory and executive function of nondemented ALS patients, comparing episodic memory profiles and learning rates of ALS patients with those of mild cognitive impairment (MCI) subjects and cognitively healthy controls (HC). In a multidisciplinary tertiary centre for motor neuron disease, 72 nondemented ALS patients, 57 amnestic MCI (aMCI), 89 single non amnestic MCI with compromised executive functions (dysexecutive MCI), and 190 HC were enrolled. They were screened using the Frontal Assessment Battery and Mini Mental State Examination. Episodic memory performances and learning rates were tested using the Rey Auditory Verbal Learning Test (RAVLT). Episodic memory dysfunction (immediate recall) was found in 14 ALS patients (19.4%). The ALS group had lower performance than HC on immediate recall, without differences in learning rate, and better performance than aMCI subjects on all RAVLT measures. Compared to dysexecutive MCI subjects, ALS patients had only better verbal learning abilities. ALS patients with executive dysfunction had a lower score on immediate and delayed recalls, verbal learning, and primacy effect than ALS patients without executive dysfunction. The immediate recall among couples of diagnostic groups differed in a statistically significant way except for the ALS/dysexecutive MCI groups. In ALS patients, episodic memory performances and learning rates appeared to be better than in aMCI subjects and similar to those with dysexecutive MCI, suggesting also a secondary functional damage due to executive impairment

Interpreting 16S metagenomic data without clustering to achieve sub-OTU resolution

The standard approach to analyzing 16S tag sequence data, which relies on clustering reads by sequence similarity into Operational Taxonomic Units (OTUs), underexploits the accuracy of modern sequencing technology. We present a clustering-free approach to multi-sample Illumina datasets that can identify independent bacterial subpopulations regardless of the similarity of their 16S tag sequences. Using published data from a longitudinal time-series study of human tongue microbiota, we are able to resolve within standard 97% similarity OTUs up to 20 distinct subpopulations, all ecologically distinct but with 16S tags differing by as little as 1 nucleotide (99.2% similarity). A comparative analysis of oral communities of two cohabiting individuals reveals that most such subpopulations are shared between the two communities at 100% sequence identity, and that dynamical similarity between subpopulations in one host is strongly predictive of dynamical similarity between the same subpopulations in the other host. Our method can also be applied to samples collected in cross-sectional studies and can be used with the 454 sequencing platform. We discuss how the sub-OTU resolution of our approach can provide new insight into factors shaping community assembly.Comment: Updated to match the published version. 12 pages, 5 figures + supplement. Significantly revised for clarity, references added, results not change

Cannibalism of Live Lymphocytes by Human Metastatic but Not Primary Melanoma Cells

The phenomenon of cell cannibalism, which generally refers to the engulfment of cells within other cells, was described in malignant tumors, but its biological significance is still largely unknown. In the present study, we investigated the occurrence, the in vivo relevance, and the underlying mechanisms of cannibalism in human melanoma. As first evidence, we observed that tumor cannibalism was clearly detectable in vivo in metastatic lesions of melanoma and often involved T cells, which could be found in a degraded state within tumor cells. Then, in vitro experiments confirmed that cannibalism of T cells was a property of metastatic melanoma cells but not of primary melanoma cells. In particular, morphologic analyses, including time-lapse cinematography and electron microscopy, revealed a sequence of events, in which metastatic melanoma cells were able to engulf and digest live autologous melanoma-specific CD8+ T cells. Importantly, this cannibalistic activity significantly increased metastatic melanoma cell survival, particularly under starvation condition, supporting the evidence that tumor cells may use the eating of live lymphocytes as a way to ‘‘feed’’ in condition of low nutrient supply. The mechanism underlying cannibalism involved a complex framework, including lysosomal protease cathepsin B activity, caveolae formation, and ezrin cytoskeleton integrity and function. In conclusion, our study shows that human metastatic melanoma cells may eat live T cells, which are instead programmed to kill them, suggesting a novel mechanism of tumor immune escape. Moreover, our data suggest that cannibalism may represent a sort of ‘‘feeding’’ activity aimed at sustaining survival and progression of malignant tumor cells in an unfavorable microenvironment. (Cancer Res 2006; 66(7): 3629-38

Formyltetrahydrofolate Synthetase Gene Diversity in the Guts of Higher Termites with Different Diets and Lifestyles

In this study, we examine gene diversity for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in homoacetogenesis, recovered from the gut microbiota of six species of higher termites. The "higher" termites (family Termitidae), which represent the majority of extant termite species and genera, engage in a broader diversity of feeding and nesting styles than the "lower" termites. Previous studies of termite gut homoacetogenesis have focused on wood-feeding lower termites, from which the preponderance of FTHFS sequences recovered were related to those from acetogenic treponemes. While sequences belonging to this group were present in the guts of all six higher termites examined, treponeme-like FTHFS sequences represented the majority of recovered sequences in only two species (a wood-feeding Nasutitermes sp. and a palm-feeding Microcerotermes sp.). The remaining four termite species analyzed (a Gnathamitermes sp. and two Amitermes spp. that were recovered from subterranean nests with indeterminate feeding strategies and a litter-feeding Rhynchotermes sp.) yielded novel FTHFS clades not observed in lower termites. These termites yielded two distinct clusters of probable purinolytic Firmicutes and a large group of potential homoacetogens related to sequences previously recovered from the guts of omnivorous cockroaches. These findings suggest that the gut environments of different higher termite species may select for different groups of homoacetogens, with some species hosting treponeme-dominated homoacetogen populations similar to those of wood-feeding, lower termites while others host Firmicutes-dominated communities more similar to those of omnivorous cockroaches

The unifrac significance test is sensitive to tree topology

Long et al. (BMC Bioinformatics 2014, 15(1):278) describe a “discrepancy” in using UniFrac to assess statistical significance of community differences. Specifically, they find that weighted UniFrac results differ between input trees where (a) replicate sequences each have their own tip, or (b) all replicates are assigned to one tip with an associated count. We argue that these are two distinct cases that differ in the probability distribution on which the statistical test is based, because of the differences in tree topology. Further study is needed to understand which randomization procedure best detects different aspects of community dissimilarities

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

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