Low microbial biomass within the reproductive tract of mid-lactation dairy cows: A study approach.

The microbiome from the reproductive tract is being investigated for its putative effect on fertility, embryo development, and health status of the human or animal host postpartum. Besides the presence of a vaginal microbiome, recent studies have claimed the existence and putative role of the uterine microbiome. Yet, the extremely low bacterial numbers and high eukaryotic/prokaryotic DNA ratio make this a highly challenging environment to study with next-generation sequencing (NGS) techniques. Here, we describe the methodological challenges that are typically encountered when performing an accurate analysis of low microbial biomass samples, illustrated by data of our own observational study. In terms of the research question, we compared the microbial composition throughout different parts of the reproductive tract of clinically healthy, mid-lactation Holstein-Friesian cows. Samples were collected from 5 dairy cows immediately after killing. Swabs were taken from the vagina, and from 4 pre-established locations of the uterine endometrium. In addition to the conventional DNA extraction blank controls, sterile swabs rubbed over disinfected disposable gloves and the disinfected surface of the uterus (tunica serosa) before incision were taken as sampling controls. The DNA extraction, DNA quantification, quantitative PCR of the 16S rRNA genes, and 16S rRNA gene sequencing were performed. In terms of NGS data analysis, we performed prevalence-based filtering of putative contaminant operational taxonomic units (OTU) using the decontam R package. Although the bacterial composition differed between the vagina and uterus, no differences in bacterial community structure (α and ß diversity) were found among the different locations in the uterus. At phylum level, uterine samples had a greater relative abundance of Proteobacteria, and a lesser relative abundance of Firmicutes than vaginal samples. The number of shared OTU between vagina and uterus was limited, suggesting the existence of bacterial transmission routes other than the transcervical one to the uterus. The mid-lactation bovine genital tract is a low microbial biomass environment, which makes it difficult to distinguish between its constitutive versus contaminant microbiome. The integration of key controls is therefore strictly necessary to decrease the effect of accidentally introduced contaminant sequences and improve the reliability of results in samples with low microbial biomass.

Microbiological, chemical and sensory spoilage analysis of raw Atlantic cod (Gadus morhua) stored under modified atmospheres.

During fish spoilage, microbial metabolism leads to the production of volatile organic compounds (VOCs), characteristic off-odors and eventual consumer rejection. The aim of the present study was to contribute to the development of intelligent packaging technologies by identifying and quantifying VOCs that indicate spoilage of raw Atlantic cod (Gadus morhua) under atmospheres (%v/v CO2/O2/N2) 60/40/0, 60/5/35 and air. Spoilage was examined by microbiological, chemical and sensory analyses over storage time at 4 or 8 °C. Selected-ion flow-tube mass spectrometry (SIFT-MS) was used for quantifying selected VOCs and amplicon sequencing of the 16S rRNA gene was used for the characterization of the cod microbiota. OTUs classified within the Photobacterium genus increased in relative abundance over time under all storage conditions, suggesting that Photobacterium contributed to spoilage and VOC production. The onset of exponential VOC concentration increase and sensory rejection occurred at high total plate counts (7-7.5 log). Monitoring of early spoilage thus calls for sensitivity for low VOC concentrations.

Community structure, population dynamics and diversity of fungi in a full-scale membrane bioreactor (MBR) for urban wastewater treatment.

Community structure, population dynamics and diversity of fungi were monitored in a full-scale membrane bioreactor (MBR) operated throughout four experimental phases (Summer 2009, Autumn 2009, Summer 2010 and Winter, 2012) under different conditions, using the 18S-rRNA gene and the intergenic transcribed spacer (ITS2-region) as molecular markers, and a combination of temperature-gradient gel electrophoresis and 454-pyrosequencing. Both total and metabolically-active fungal populations were fingerprinted, by amplification of molecular markers from community DNA and retrotranscribed RNA, respectively. Fingerprinting and 454-pyrosequencing evidenced that the MBR sheltered a dynamic fungal community composed of a low number of species, in accordance with the knowledge of fungal diversity in freshwater environments, and displaying a medium-high level of functional organization with few numerically dominant phylotypes. Population shifts were experienced in strong correlation with the changes of environmental variables and operation parameters, with pH contributing the highest level of explanation. Phylotypes assigned to nine different fungal Phyla were detected, although the community was mainly composed of Ascomycota, Basidiomycota and Chytridiomycota/Blastocladiomycota. Prevailing fungal phylotypes were affiliated to Saccharomycetes and Chytridiomycetes/Blastocladiomycetes, which displayed antagonistic trends in their relative abundance throughout the experimental period. Fungi identified in the activated sludge were closely related to genera of relevance for the degradation of organic matter and trace-organic contaminants, as well as genera of dimorphic fungi potentially able to produce plant operational issues such as foaming or biofouling. Phylotypes closely related to genera of human and plant pathogenic fungi were also detected.