Research on the human microbiome has yielded numerous insights into health and disease, but also has resulted in a wealth of experimental artifacts. Here, we present suggestions for optimizing experimental design and avoiding known pitfalls, organized in the typical order in which studies are carried out. We first review best practices in experimental design and introduce common confounders such as age, diet, antibiotic use, pet ownership, longitudinal instability, and microbial sharing during cohousing in animal studies. Typically, samples will need to be stored, so we provide data on best practices for several sample types. We then discuss design and analysis of positive and negative controls, which should always be run with experimental samples. We introduce a convenient set of non-biological DNA sequences that can be useful as positive controls for high-volume analysis. Careful analysis of negative and positive controls is particularly important in studies of samples with low microbial biomass, where contamination can comprise most or all of a sample. Lastly, we summarize approaches to enhancing experimental robustness by careful control of multiple comparisons and to comparing discovery and validation cohorts. We hope the experimental tactics summarized here will help researchers in this exciting field advance their studies efficiently while avoiding errors.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0267-5) contains supplementary material, which is available to authorized users.

IntroductionChronic lymphocytic leukemia (CLL) follows either an indolent or an aggressive course 1 and clinical decompensation is often accompanied by the appearance of new or increasing numbers of genetic aberrations associated with shorter survival, "clonal evolution." 2 The mechanism(s) responsible for the generation of these genetic abnormalities are not defined in CLL, which is not the case in certain other human cancers, especially lymphoid malignancies of germinal center (GC) origin, in which activation-induced deaminase (AID) appears to be pathogenic. [3][4][5] AID is required for the beneficial generation of Ab diversity in normal B lymphocytes by inducing IGV somatic hypermutation (SHM) and helps in the development of protective effector mechanisms by mediating IGH class-switch recombination (CSR). 6,7 These beneficial on-target AID activities occur primarily during a GC reaction and involve conversion of cytidine to uridine on single-stranded DNA at the IG locus. Such on-target actions in CLL B cells have been a matter of interest for several years, primarily because the presence or absence of IGHV mutations (which require AID) in CLL cells is closely linked to clinical outcome. Patients with leukemic clones with minimal (Ͻ 2% difference from germline) or no mutation in the IGHV (unmutated CLL [U-CLL]) have a far worse prognosis than patients with IGHV-mutated CLL (M-CLL). 8,9 Despite this SHM-based subcategorization of CLL cases, some clones exhibit ongoing IGHV diversification in vivo and in vitro, [10][11][12] with an antigen-driven pattern present in some cases, 13 and up to 50% of patients exhibit molecular evidence for intraclonal isotype CSR. [14][15][16][17][18] AID activity focused elsewhere 19 ) can lead to mutations, deletions, or translocations outside of the IG locus, as in GC-derived lymphomas. [3][4][5] However, such a role for AID in CLL has been questioned for several reasons: (1) although circulating CLL cells can express AID mRNA, [20][21][22] the number of such cells is exceedingly low (0.01%-0.2%) 22 ; (2) AID protein synthesis by these same cells has not been demonstrated 18,[20][21][22][23] ; (3) demonstration of the full range of AID functions is lacking in CLL, for example, by failure of cells to demonstrate SHM, especially for U-CLL clones, even on stimulation and induction of AID mRNA, 21 thereby creating the apparent paradox that U-CLL patients express more AID mRNA than M-CLL patients yet exhibit no or minimal SHM; and (4) despite association with several prognostic markers, 20,21,[24][25][26] no prospective analysis linking AID expression and disease severity has been performed.In the present study, we aimed to address these issues as a means of determining whether AID could be involved in the evolution of CLL to a more aggressive disease. We report that CLL cells are able to produce AID protein, but synthesis is The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and...

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an antibiotic resistance threat of the highest priority. Given the limited treatment options for this multidrug-resistant organism (MDRO), there is an urgent need for targeted strategies to prevent transmission. Here, we applied whole-genome sequencing to a comprehensive collection of clinical isolates to reconstruct regional transmission pathways and analyzed this transmission network in the context of statewide patient transfer data and patient-level clinical data to identify drivers of regional transmission. We found that high regional CRKP burdens were due to a small number of regional introductions, with subsequent regional proliferation occurring via patient transfers among health care facilities. While CRKP was predicted to have been imported into each facility multiple times, there was substantial variation in the ratio of intrafacility transmission events per importation, indicating that amplification occurs unevenly across regional facilities. While myriad factors likely influence intrafacility transmission rates, an understudied one is the potential for clinical characteristics of colonized and infected patients to influence their propensity for transmission. Supporting the contribution of high-risk patients to elevated transmission rates, we observed that patients colonized and infected with CRKP in high-transmission facilities had higher rates of carbapenem use, malnutrition, and dialysis and were older. This report highlights the potential for regional infection prevention efforts that are grounded in genomic epidemiology to identify the patients and facilities that make the greatest contribution to regional MDRO prevalence, thereby facilitating the design of precision interventions of maximal impact.

Introductory Paragraph Initial microbial colonization and later succession in the gut of human infants are linked to health and disease later in life. The timing of the appearance of the first gut microbiome, and the consequences for the early life metabolome, are just starting to be defined. Here we evaluated the gut microbiome, proteome, and metabolome in 88 African American newborns using fecal samples collected in the first few days of life. Gut bacteria became detectable using molecular methods by 16 hours after birth. Detailed analysis of the three most common species, Escherichia coli , Enterococcus faecalis , and Bacteroides vulgatus , did not suggest a genomic signature for neonatal gut colonization. The appearance of bacteria was associated with reduced abundance of approximately 50 human proteins, decreased levels of free amino acids, and an increase in products of bacterial fermentation, including acetate and succinate. Using flux balance modeling and in vitro experiments, we provide evidence that fermentation of amino acids provides a mechanism for the initial growth of Escherichia coli , the most common early colonizer, under anaerobic conditions. These results provide a deep characterization of the first microbes in the human gut and show how the biochemical environment is altered by their appearance.