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.

The Cellient™ Automated Cell Block System (Hologic) can be used to analyze cells of HPV-positive cervical scrapes staining positive with the biomarker p16. For this study fourteen cervical scrapes of Tanzanian women infected with HIV testing positive for HPV were selected. The paraffin Cellient sections were stained with the Papanicolaou method, with hematoxylin eosin (HE), and with the biomarker p16. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections (cut from paraffin blocks prepared from the residual cervical sample) were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. HPV16 was found in six scrapes and HPV52 in four. In the Papanicolaou sections, it was easy to detect dyskeratotic cells. Eleven of the 14 cases were p16 positive and five contained p16 positive dyskeratocytes. Of the 10 ASCUS scrapes, two contained p16 positive CIN 1 epithelial fragments. All four HSIL cases contained p16 positive CIN 3 epithelial fragments. In HIV-positive HPV-positive women, the Cellient system resulted in high quality histology sections with perfect p16 images of dyskeratocytes.

The Cellient system for cytohistology to analyze p16 positive dyskeratocytes in paraffin sections of HPV-positive cervical scrapes

In June 23±25, 1986, a major international symposium was held in Goteborg, Sweden [Eden CS, Kass EH (eds) (1989) Host-parasite interactions in urinary tract infections (UTIs), University of Chicago Press, Chicago]. The conference was a who's who of people in the ®eld at that time: Drs. Kass, Bailey, Kunim, Ronald, Svanborg Eden, Asscher, Leer, Kaijser, Roberts, Stamm, Kallenius, Schaeer, Ofek, Korhonen, Bruce, Maskell, Salit, Andriole, Smellie, Winberg, Elliott, Domingue, Topley, Uehling and others. The primary focus at the time was the adhesion of pathogens to uroepithelial cells and the role of adhesins in pyelonephritis and catheter infections. There was satisfaction with the use of antibiotics as therapeutic and prophylactic agents, and some studies to suggest that infection could be prevented using antiadhesin vaccines and lactobacilli. Over 13 years later, a lot has changed. Sadly, Drs. Bailey and Kass have died, and a number of others have retired or left the ®eld, such that few of these investigators remain focused on UTI research. Meanwhile, antibiotic resistance has increased, the anti-adhesin work has so far not led to vaccine production, and UTI is still a major cause of disease in an estimated 300 million patients worldwide each year. The management of asymptomatic bacteriuria is still somewhat controversial, and post-antibiotic yeast infections are all too common. Infections associated with medical devices and spinal cord injury continue to kill a signi®cant number of people, and government granting agencies repeatedly fail to place any emphasis on funding research in these areas. However, some progress has been made in the last 13 years and indeed there appears to be a rebirth of interest into the study of this common disease. We have a better understanding of how microbes adhere to cells and materials, and how they form, survive and communicate within bio®lms. We recognize that adhesion is not the only factor in pathogenesis, and that antibiotic therapy should not be the only weapon. Consumer awareness is driving alternative therapies such as cranberry juice, and more conclusive data is now available on the potential of probiotic lactobacilli as a means to prevent recurrences. The present volume of the journal features some of the key people working in this ®eld, and some of the major breakthroughs which have been made, and which will lead to improvements in diagnosis, treatment and prevention of UTI. Dr. Wade Bushman and I thank these authors for their valuable contributions. We hope that their insight will enhance the reader's knowledge and understanding of the area, and stimulate students, scientists and physicians to make further inroads in the management of chronic and acute UTI in the years ahead.

Urinary tract infection

The present invention discloses methods of using bacterial profiles of breast samples for the diagnosis and prognosis of breast cancer. The present invention discloses also methods of using microorganisms in the prevention, risk reduction and treatment of breast cancer.

Bacteria for the prevention, treatment and diagnosis of breast cancer


 The popularity of using probiotics has surged, since they became widely accepted as safe and help improve general health. Inevitably, some of these products are used after expiration when microbial cell viability is below the recommended effective dose. Given that probiotics must be live microorganisms administered in adequate amounts, the aim of this study was to measure viability in expired products and assess how packaging and storage conditions impact efficacy, if at all. Thirty-three expired probiotic products were evaluated, of which 26 were stored in conditions recommended by the manufacturer. The viable microbial cells were enumerated and representative isolates identified by 16S and internally transcribed spacer rRNA gene sequencing. While the products had a mean past expiration time of 11.32 (1–22) years, 22 still had viable contents, and 5 were within or above the original product cell count claim. Product formulation and the number of species present did not appear to impact the stability of the products. However, overall packaging type, storage conditions and time since expiry were found to affect viability. All products with viable cells had the strain stipulated on the label. Despite some selected probiotic products retaining viability past their expiry date (indicating long-term storage is possible), the total counts were mostly well below that required for efficacious use as recommended by the manufacturer. Consuming expired probiotics may not yield the benefits for which they were designed.

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Gregor Reid

Papers

The Cellient system for cytohistology to analyze p16 positive dyskeratocytes in paraffin sections of HPV-positive cervical scrapes

Urinary tract infection

Bacteria for the prevention, treatment and diagnosis of breast cancer

Expired probiotics: what is really in your cabinet?

Micronutrient supplemented probiotic yogurt for HIV-infected adults taking HAART in London, Canada