Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.

Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.

An expert panel was convened in October 2013 by the International Scientific Association for Probiotics and Prebiotics (ISAPP) to discuss the field of probiotics. It is now 13 years since the definition of probiotics and 12 years after guidelines were published for regulators, scientists and industry by the Food and Agriculture Organization of the United Nations and the WHO (FAO/WHO). The FAO/WHO definition of a probiotic--"live microorganisms which when administered in adequate amounts confer a health benefit on the host"--was reinforced as relevant and sufficiently accommodating for current and anticipated applications. However, inconsistencies between the FAO/WHO Expert Consultation Report and the FAO/WHO Guidelines were clarified to take into account advances in science and applications. A more precise use of the term 'probiotic' will be useful to guide clinicians and consumers in differentiating the diverse products on the market. This document represents the conclusions of the ISAPP consensus meeting on the appropriate use and scope of the term probiotic.

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Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic

From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites

Motivation The Illumina paired-end sequencing technology can generate reads from both ends of target DNA fragments, which can subsequently be merged to increase the overall read length. There already exist tools for merging these paired-end reads when the target fragments are equally long. However, when fragment lengths vary and, in particular, when either the fragment size is shorter than a single-end read, or longer than twice the size of a single-end read, most state-of-the-art mergers fail to generate reliable results. Therefore, a robust tool is needed to merge paired-end reads that exhibit varying overlap lengths because of varying target fragment lengths. Results We present the PEAR software for merging raw Illumina paired-end reads from target fragments of varying length. The program evaluates all possible paired-end read overlaps and does not require the target fragment size as input. It also implements a statistical test for minimizing false-positive results. Tests on simulated and empirical data show that PEAR consistently generates highly accurate merged paired-end reads. A highly optimized implementation allows for merging millions of paired-end reads within a few minutes on a standard desktop computer. On multi-core architectures, the parallel version of PEAR shows linear speedups compared with the sequential version of PEAR. Availability and implementation PEAR is implemented in C and uses POSIX threads. It is freely available at http://www.exelixis-lab.org/web/software/pear.

PEAR: a fast and accurate Illumina Paired-End reAd mergeR

Bacterial biofilms in nature and disease.

Effects ofCiprofloxacin, Norfloxacin, andOfloxacin on InVitro Adhesion andSurvival ofPseudomonas aeruginosa AK1on Urinary Catheters

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Lactic Acid Bacteria: Microbiological and Functional Aspects, 3rd Revised and Expanded Edition Edited by Seppo Salminen, Atte von Wright, and Arthur Ouwehand New York: Marcel Dekker, 2004. 633 pp. $229.95 (cloth)

Numerous diseases occur in the genitourinary tract of men, women, and children. These disorders cover the gamut of infection, incontinence, cancer, inflammatory problems, and anatomical malfunctions in the urethra, bladder, kidney, prostate, vulva, vagina, cervix, and uterus. The potential for probiotics to counter these ailments is not restricted to infection, but it is in this area that most of the research to date has been done. This chapter begins with conditions under which there is somewhat of a rationale for probiotics to affect the urogenital tract, but for which actual data are sparse. The main goal of probiotics applications is to prevent genitourinary tract infections by populating the vagina with lactobacilli after antifungal agents have reduced the yeast count. Probiotics repopulate the genitourinary tract, compete with other bacteria, and are flushed from the host, necessitating regular, repeated administration. A study was performed to examine the ability of probiotics to prevent bacterial vaginosis (BV) and yeast vaginitis in patients receiving antibiotic therapy. Widespread and growing interest in the use of probiotics for urogenital health has served as an impetus for numerous research studies.

Probiotics and Diseases of the Genitourinary Tract

Chronic kidney disease (CKD) disproportionately affects populations in developing countries. In sub-Saharan Africa, CKD prevalence is high (12–23%) and is associated with cardiovascular manifestations. Uremic toxins, especially p-cresol and p-cresyl sulfate, are associated with the disease. Reducing uremic toxins in the body slows disease progression and improves patient outcomes. Probiotic Bifidobacterium breve HRVD521-US, B. animalis HRVD524-US, B. longum SD-BB536-JP, and B. longum SD-CECT7347-SP internalize p-cresol and improve longevity in vivo. In 2002, Tanzanian communities were taught to produce probiotic yogurt (Fiti®) supplemented with Lacticaseibacillus rhamnosus GR-1. This has expanded to over 100 community producers across the country. To produce yogurt that could reduce the burden of CKD by sequestering uremic toxins, we decided to test the addition of p-cresol-clearing bifidobacterial strains. By repeating the Fiti® production process performed in Tanzanian communities and adding a bifidobacterial strain, we found that they were successfully incorporated into the yogurt without any detrimental effect on sensory properties or viable counts. Three of the four strains significantly reduced p-cresol when added to a simulated colonic environment. In conclusion, this study has shown that Fiti® sachets provided to Tanzanian communities to produce yogurt can be supplemented with strains that can potentially confer additional health benefits. 

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Supplementing Yogurt with Probiotic Bifidobacteria to Counter Chronic Kidney Disease

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Gregor Reid

Papers

Effects ofCiprofloxacin, Norfloxacin, andOfloxacin on InVitro Adhesion andSurvival ofPseudomonas aeruginosa AK1on Urinary Catheters

Lactic Acid Bacteria: Microbiological and Functional Aspects, 3rd Revised and Expanded Edition Edited by Seppo Salminen, Atte von Wright, and Arthur Ouwehand New York: Marcel Dekker, 2004. 633 pp. $229.95 (cloth)

Probiotics and Diseases of the Genitourinary Tract

Supplementing Yogurt with Probiotic Bifidobacteria to Counter Chronic Kidney Disease

How probiotics and the microbiome will change medical practice