Dataset Information

Culture-Independent Metagenomic Surveillance of Commercially Available Probiotics with High-Throughput Next-Generation Sequencing.

ABSTRACT: Millions of people consume dietary supplements either following a doctor's recommendation or at their own discretion to improve their overall health and well-being. This is a rapidly growing trend, with an associated and expanding manufacturing industry to meet the demand for new health-related products. In this study, we examined the contents and microbial viability of several popular probiotic products on the United States market. Culture-independent methods are proving ideal for fast and efficient analysis of foodborne pathogens and their associated microbial communities but may also be relevant for analyzing probiotics containing mixed microbial constituents. These products were subjected to next-generation whole-genome sequencing and analyzed by a custom in-house-developed k-mer counting method to validate manufacturer label information. In addition, the batch variability of respective products was examined to determine if any changes in their formulations and/or the manufacturing process occurred. Overall, the products we tested adhered to the ingredient claims and lot-to-lot differences were minimal. However, there were a few discrepancies in the naming of closely related Lactobacillus and Bifidobacterium species, whereas one product contained an apparent Enterococcus contaminant in two of its three lots. With the microbial contents of the products identified, we used traditional PCR and colony counting methods to comparatively assess our results and verify the viability of the microbes in these products with regard to the labeling claims. Of all the supplements examined, only one was found to be inaccurate in viability. Our use of next-generation sequencing as an analytical tool clearly demonstrated its utility for quickly analyzing commercially available products containing multiple microbes to ensure consumer safety. IMPORTANCE The rapidly growing supplement industry operates without a formal premarket approval process. Consumers rely on product labels to be accurate and true. Those products containing live microbials report both identity and viability on most product labels. This study used next-generation sequencing technology as an analytical tool in conjunction with classic culture methods to examine the validity of the labels on supplement products containing live microbials found in the United States marketplace. Our results show the importance of testing these products for identity, viability, and potential contaminants, as well as introduce a new culture-independent diagnostic approach for testing these products. Podcast: A podcast concerning this article is available.

SUBMITTER: Patro JN

PROVIDER: S-EPMC4894680 | biostudies-literature | 2016 Mar-Apr

REPOSITORIES: biostudies-literature

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Culture-Independent Metagenomic Surveillance of Commercially Available Probiotics with High-Throughput Next-Generation Sequencing.

Patro Jennifer N JN Ramachandran Padmini P Barnaba Tammy T Mammel Mark K MK Lewis Jada L JL Elkins Christopher A CA

mSphere 20160330 2

Millions of people consume dietary supplements either following a doctor's recommendation or at their own discretion to improve their overall health and well-being. This is a rapidly growing trend, with an associated and expanding manufacturing industry to meet the demand for new health-related products. In this study, we examined the contents and microbial viability of several popular probiotic products on the United States market. Culture-independent methods are proving ideal for fast and effi ...[more]

PMID: 27303722

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Project description:BackgroundThe aims of this study were to (1) evaluate the efficacy and safety of targeted antibiotics for the treatment of culture-negative prosthetic joint infection based on metagenomic next-generation sequencing results and (2) verify the accuracy and reliability of metagenomic next-generation sequencing for identifying pathogens related to culture-negative prosthetic joint infection.MethodsNinety-seven consecutive PJI patients, including 27 patients with culture-negative prosthetic joint infection, were treated surgically at our center. Thirteen of the 27 culture-negative prosthetic joint infection patients, who were admitted before June 2017 and treated with empirical antibiotics, comprised the empirical antibiotic group (EA group), and the other 14 patients, who were admitted after June 2017 and treated with targeted antibiotics according to their metagenomic next-generation sequencing results, were classified as the targeted antibiotic group (TA group). The short-term infection control rate, incidence of antibiotic-related complications and costs were compared between the two groups.ResultsTwo of the patients in the EA group experienced debridement and prolonged antimicrobial therapy due to wound infection after the initial revision surgery. No recurrent infections were observed in the TA group; however, no significant difference in the infection control rate was found between the two groups (83.33% vs 100%, P?=?0.217). More cases of antibiotic-related complications were recorded in the EA group (6 cases) than in the TA group (1 case), but the difference was not statistically significant (P?=?0.0697). The cost of antibiotics obtained for the EA group was 20,168.37 Yuan (3236.38-45,297.16), which was higher than that found for the TA group (10,164.16 Yuan, 2959.54-16,661.04, P?=?0.04).ConclusionsTargeted antibiotic treatment for culture-negative prosthetic joint infection based on metagenomic next-generation sequencing results is associated with a favorable outcome, and metagenomic next-generation sequencing is a reliable tool for identifying pathogens related to culture-negative prosthetic joint infection.

Project description:BackgroundCombinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges.Methodology/principal findingsWe gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs approximately 250-fold for generating 10(6) ligand sequences.Conclusions/significanceOur analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger sequencing and is proposed as the method of choice over a broad range of phage display applications in vitro, in cells, and in vivo.

Project description:BackgroundMetagenomic next-generation sequencing (mNGS) technology has been widely used to diagnose various infections. Based on the most common pathogen profiles, targeted mNGS (tNGS) using multiplex PCR has been developed to detect pathogens with predesigned primers in the panel, significantly improving sensitivity and reducing economic burden on patients. However, there are few studies on summarizing pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China on large scale.MethodsFrom January 2021 to December 2023, bronchoalveolar lavage fluid (BALF) or sputum samples from 546 immunocompetent and immunocompromised patients with suspected community-acquired pneumonia were collected. Pathogen profiles in those patients on whom mNGS was performed were summarized. Additionally, we also evaluated the performance of tNGS in diagnosing pulmonary infections.ResultsCombined with results of mNGS and culture, we found that the most common bacterial pathogens were Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii in both immunocompromised and immunocompetent patients with high detection rates of Staphylococcus aureus and Enterococcus faecium, respectively. For fungal pathogens, Pneumocystis jirovecii was commonly detected in patients, while fungal infections in immunocompetent patients were mainly caused by Candida albicans. Most of viral infections in patients were caused by Human betaherpesvirus 5 and Human gammaherpesvirus 4. It is worth noting that, compared with immunocompetent patients (34.9%, 76/218), more mixed infections were found in immunocompromised patients (37.8%, 14/37). Additionally, taking final comprehensive clinical diagnoses as reference standard, total coincidence rate of BALF tNGS (81.4%, 48/59) was much higher than that of BALF mNGS (40.0%, 112/280).ConclusionsOur findings supplemented and classified the pathogen profiles of pulmonary infections in immunocompetent and immunocompromised patients in Jilin Province of China. Most importantly, our findings can accelerate the development and design of tNGS specifically used for regional pulmonary infections.

Project description:Importance:Identifying infectious causes of subacute or chronic meningitis can be challenging. Enhanced, unbiased diagnostic approaches are needed. Objective:To present a case series of patients with diagnostically challenging subacute or chronic meningitis using metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF) supported by a statistical framework generated from mNGS of control samples from the environment and from patients who were noninfectious. Design, Setting, and Participants:In this case series, mNGS data obtained from the CSF of 94 patients with noninfectious neuroinflammatory disorders and from 24 water and reagent control samples were used to develop and implement a weighted scoring metric based on z scores at the species and genus levels for both nucleotide and protein alignments to prioritize and rank the mNGS results. Total RNA was extracted for mNGS from the CSF of 7 participants with subacute or chronic meningitis who were recruited between September 2013 and March 2017 as part of a multicenter study of mNGS pathogen discovery among patients with suspected neuroinflammatory conditions. The neurologic infections identified by mNGS in these 7 participants represented a diverse array of pathogens. The patients were referred from the University of California, San Francisco Medical Center (n = 2), Zuckerberg San Francisco General Hospital and Trauma Center (n = 2), Cleveland Clinic (n = 1), University of Washington (n = 1), and Kaiser Permanente (n = 1). A weighted z score was used to filter out environmental contaminants and facilitate efficient data triage and analysis. Main Outcomes and Measures:Pathogens identified by mNGS and the ability of a statistical model to prioritize, rank, and simplify mNGS results. Results:The 7 participants ranged in age from 10 to 55 years, and 3 (43%) were female. A parasitic worm (Taenia solium, in 2 participants), a virus (HIV-1), and 4 fungi (Cryptococcus neoformans, Aspergillus oryzae, Histoplasma capsulatum, and Candida dubliniensis) were identified among the 7 participants by using mNGS. Evaluating mNGS data with a weighted z score-based scoring algorithm reduced the reported microbial taxa by a mean of 87% (range, 41%-99%) when taxa with a combined score of 0 or less were removed, effectively separating bona fide pathogen sequences from spurious environmental sequences so that, in each case, the causative pathogen was found within the top 2 scoring microbes identified using the algorithm. Conclusions and Relevance:Diverse microbial pathogens were identified by mNGS in the CSF of patients with diagnostically challenging subacute or chronic meningitis, including a case of subarachnoid neurocysticercosis that defied diagnosis for 1 year, the first reported case of CNS vasculitis caused by Aspergillus oryzae, and the fourth reported case of C dubliniensis meningitis. Prioritizing metagenomic data with a scoring algorithm greatly clarified data interpretation and highlighted the problem of attributing biological significance to organisms present in control samples used for metagenomic sequencing studies.

Dataset Information

Culture-Independent Metagenomic Surveillance of Commercially Available Probiotics with High-Throughput Next-Generation Sequencing.

Publications

Culture-Independent Metagenomic Surveillance of Commercially Available Probiotics with High-Throughput Next-Generation Sequencing.

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