Denoising the Denoisers: an independent evaluation of microbiome sequence error-correction approaches

High-depth sequencing of universal marker genes such as the 16S rRNA gene is a common strategy to profile microbial communities. Traditionally, sequence reads are clustered into operational taxonomic units (OTUs) at a defined identity threshold to avoid sequencing errors generating spurious taxonomic units. However, there have been numerous bioinformatic packages recently released that attempt to correct sequencing errors to determine real biological sequences at single nucleotide resolution by generating amplicon sequence variants (ASVs). As more researchers begin to use high resolution ASVs, there is a need for an in-depth and unbiased comparison of these novel “denoising” pipelines. In this study, we conduct a thorough comparison of three of the most widely-used denoising packages (DADA2, UNOISE3, and Deblur) as well as an open-reference 97% OTU clustering pipeline on mock, soil, and host-associated communities. We found from the mock community analyses that although they produced similar microbial compositions based on relative abundance, the approaches identified vastly different numbers of ASVs that significantly impact alpha diversity metrics. Our analysis on real datasets using recommended settings for each denoising pipeline also showed that the three packages were consistent in their per-sample compositions, resulting in only minor differences based on weighted UniFrac and Bray–Curtis dissimilarity. DADA2 tended to find more ASVs than the other two denoising pipelines when analyzing both the real soil data and two other host-associated datasets, suggesting that it could be better at finding rare organisms, but at the expense of possible false positives. The open-reference OTU clustering approach identified considerably more OTUs in comparison to the number of ASVs from the denoising pipelines in all datasets tested. The three denoising approaches were significantly different in their run times, with UNOISE3 running greater than 1,200 and 15 times faster than DADA2 and Deblur, respectively. Our findings indicate that, although all pipelines result in similar general community structure, the number of ASVs/OTUs and resulting alpha-diversity metrics varies considerably and should be considered when attempting to identify rare organisms from possible background noise

The impact of chemerin or chemokine-like receptor 1 loss on the mouse gut microbiome

Chemerin is an adipocyte derived signalling molecule (adipokine) that serves as a ligand activator of Chemokine-like receptor 1(CMKLR1). Chemerin/CMKLR1 signalling is well established to regulate fundamental processes in metabolism and inflammation. The composition and function of gut microbiota has also been shown to impact the development of metabolic and inflammatory diseases such as obesity, diabetes and inflammatory bowel disease. In this study, we assessed the microbiome composition of fecal samples isolated from wildtype, chemerin, or CMKLR1 knockout mice using Illumina-based sequencing. Moreover, the knockout mice and respective wildtype mice used in this study were housed at different universities allowing us to compare facility-dependent effects on microbiome composition. While there was no difference in alpha diversity within samples when compared by either facility or genotype, we observed a dramatic difference in the presence and abundance of numerous taxa between facilities. There were minor differences in bacterial abundance between wildtype and chemerin knockout mice, but significantly more differences in taxa abundance between wildtype and CMKLR1 knockout mice. Specifically, CMKLR1 knockout mice exhibited decreased abundance of Akkermansia and Prevotella, which correlated with body weight in CMKLR1 knockout, but not wildtype mice. This is the first study to investigate a linkage between chemerin/CMKLR1 signaling and microbiome composition. The results of our study suggest that chemerin/CMKLR1 signaling influences metabolic processes through effects on the gut microbiome. Furthermore, the dramatic difference in microbiome composition between facilities might contribute to discrepancies in the metabolic phenotype of CMKLR1 knockout mice reported by independent groups. Considered altogether, these findings establish a foundation for future studies to investigate the relationship between chemerin signaling and the gut microbiome on the development and progression of metabolic and inflammatory disease

Antibiotic and antifungal use in pediatric leukemia and lymphoma patients are associated with increasing opportunistic pathogens and decreasing bacteria responsible for activities that enhance colonic defense

Due to decreased immunity, both antibiotics and antifungals are regularly used in pediatric hematologic-cancer patients as a means to prevent severe infections and febrile neutropenia. The general effect of antibiotics on the human gut microbiome is profound, yielding decreased diversity and changes in community structure. However, the specific effect on pediatric oncology patients is not well-studied. The effect of antifungal use is even less understood, having been studied only in mouse models. Because the composition of the gut microbiome is associated with regulation of hematopoiesis, immune function and gastrointestinal integrity, changes within the patient gut can have implications for the clinical management of hematologic malignancies. The pediatric population is particularly challenging because the composition of the microbiome is age dependent, with some of the most pronounced changes occurring in the first three years of life. We investigated how antibiotic and antifungal use shapes the taxonomic composition of the stool microbiome in pediatric patients with leukemia and lymphoma, as inferred from both 16S rRNA and metagenome data. Associations with age, antibiotic use and antifungal use were investigated using multiple analysis methods. In addition, multivariable differential abundance was used to identify and assess specific taxa that were associated with multiple variables. Both antibiotics and antifungals were linked to a general decline in diversity in stool samples, which included a decrease in relative abundance in butyrate producers that play a critical role in host gut physiology (e.g., Faecalibacterium, Anaerostipes, Dorea, Blautia),. Furthermore, antifungal use was associated with a significant increase in relative abundance of opportunistic pathogens. Collectively, these findings have important implications for the treatment of leukemia and lymphoma patients. Butyrate is important for gastrointestinal integrity; it inhibits inflammation, reinforces colonic defense, mucosal immunity. and decreases oxidative stress. The routine use of broad-spectrum anti-infectives in pediatric oncology patients could simultaneously contribute to a decline in gastrointestinal integrity and colonic defense while promoting increases in opportunistic pathogens within the patient gut. Because the gut microbiome has been linked to both short-term clinical outcomes, and longer-lasting health effects, systematic characterization of the gut microbiome in pediatric patients during, and beyond, treatment is warranted

Human Microbiome: A Genetic Bazaar for Microbes?

SummaryA recent study suggests that lateral gene transfer has been particularly intense among human-associated microbes. What can this tell us about our relationship with our internal microbial world

DataSheet_2_The gastrointestinal antibiotic resistome in pediatric leukemia and lymphoma patients.xlsx

IntroductionMost children with leukemia and lymphoma experience febrile neutropenia. These are treated with empiric antibiotics that include β-lactams and/or vancomycin. These are often administered for extended periods, and the effect on the resistome is unknown.MethodsWe examined the impact of repeated courses and duration of antibiotic use on the resistome of 39 pediatric leukemia and lymphoma patients. Shotgun metagenome sequences from 127 stool samples of pediatric oncology patients were examined for abundance of antibiotic resistance genes (ARGs) in each sample. Abundances were grouped by repeated courses (no antibiotics, 1-2 courses, 3+ courses) and duration (no use, short duration, long and/or mixed durationg) of β-lactams, vancomycin and “any antibiotic” use. We assessed changes in both taxonomic composition and prevalence of ARGs among these groups.ResultsWe found that Bacteroidetes taxa and β-lactam resistance genes decreased, while opportunistic Firmicutes and Proteobacteria taxa, along with multidrug resistance genes, increased with repeated courses and/or duration of antibiotics. Efflux pump related genes predominated (92%) among the increased multidrug genes. While we found β-lactam ARGs present in the resistome, the taxa that appear to contain them were kept in check by antibiotic treatment. Multidrug ARGs, mostly efflux pumps or regulators of efflux pump genes, were associated with opportunistic pathogens, and both increased in the resistome with repeated antibiotic use and/or increased duration.ConclusionsGiven the strong association between opportunistic pathogens and multidrug-related efflux pumps, we suggest that drug efflux capacity might allow the opportunistic pathogens to persist or increase despite repeated courses and/or duration of antibiotics. While drug efflux is the most direct explanation, other mechanisms that enhance the ability of opportunistic pathogens to handle environmental stress, or other aspects of the treatment environment, could also contribute to their ability to flourish within the gut during treatment. Persistence of opportunistic pathogens in an already dysbiotic and weakened gastrointestinal tract could increase the likelihood of life-threatening blood borne infections. Of the 39 patients, 59% experienced at least one gastrointestinal or blood infection and 60% of bacteremia’s were bacteria found in stool samples. Antimicrobial stewardship and appropriate use and duration of antibiotics could help reduce morbidity and mortality in this vulnerable population.</p

DataSheet_1_The gastrointestinal antibiotic resistome in pediatric leukemia and lymphoma patients.docx

IntroductionMost children with leukemia and lymphoma experience febrile neutropenia. These are treated with empiric antibiotics that include β-lactams and/or vancomycin. These are often administered for extended periods, and the effect on the resistome is unknown.MethodsWe examined the impact of repeated courses and duration of antibiotic use on the resistome of 39 pediatric leukemia and lymphoma patients. Shotgun metagenome sequences from 127 stool samples of pediatric oncology patients were examined for abundance of antibiotic resistance genes (ARGs) in each sample. Abundances were grouped by repeated courses (no antibiotics, 1-2 courses, 3+ courses) and duration (no use, short duration, long and/or mixed durationg) of β-lactams, vancomycin and “any antibiotic” use. We assessed changes in both taxonomic composition and prevalence of ARGs among these groups.ResultsWe found that Bacteroidetes taxa and β-lactam resistance genes decreased, while opportunistic Firmicutes and Proteobacteria taxa, along with multidrug resistance genes, increased with repeated courses and/or duration of antibiotics. Efflux pump related genes predominated (92%) among the increased multidrug genes. While we found β-lactam ARGs present in the resistome, the taxa that appear to contain them were kept in check by antibiotic treatment. Multidrug ARGs, mostly efflux pumps or regulators of efflux pump genes, were associated with opportunistic pathogens, and both increased in the resistome with repeated antibiotic use and/or increased duration.ConclusionsGiven the strong association between opportunistic pathogens and multidrug-related efflux pumps, we suggest that drug efflux capacity might allow the opportunistic pathogens to persist or increase despite repeated courses and/or duration of antibiotics. While drug efflux is the most direct explanation, other mechanisms that enhance the ability of opportunistic pathogens to handle environmental stress, or other aspects of the treatment environment, could also contribute to their ability to flourish within the gut during treatment. Persistence of opportunistic pathogens in an already dysbiotic and weakened gastrointestinal tract could increase the likelihood of life-threatening blood borne infections. Of the 39 patients, 59% experienced at least one gastrointestinal or blood infection and 60% of bacteremia’s were bacteria found in stool samples. Antimicrobial stewardship and appropriate use and duration of antibiotics could help reduce morbidity and mortality in this vulnerable population.</p

Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa

Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children’s cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes