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.

In vitro colonisation of the distal colon by Akkermansia muciniphila is largely mucin and pH dependent.

Host mucin is the main constituent of the mucus layer that covers the gut epithelium of the host, and an important source of glycans for the bacteria colonising the intestine. Akkermansia muciniphila is a mucin-degrading bacterium, abundant in the human gut, that is able to produce acetate and propionate during this degradation process. A. muciniphila has been correlated with human health in previous studies, but a mechanistic explanation is lacking. In this study, the main site of colonisation was characterised alongside additional conditions, such as differences in colon pH, prebiotic supplementation and variable mucin supply. To overcome the limitations of in vivo studies concerning variations in mucin availability and difficult access to proximal regions of the colon, a dynamic in vitro gut model (SHIME) was used. In this model, A. muciniphila was found to colonise the distal colon compartment more abundantly than the proximal colon ((±8 log copies/ml compared to ±4 log copies/ml) and the preference for the distal compartment was found to be pH-dependent. The addition of mucin caused a specific increase of A. muciniphila (±4.5 log increase over two days), far exceeding the response of other bacteria present, together with an increase in propionate. These findings suggest that colonisation and mucin degradation by A. muciniphila is dependent on pH and the concentration of mucin. Our results revealed the preference of A. muciniphila for the distal colon environment due to its higher pH and uncovered the quick and stable response of A. muciniphila to mucin supplementation.

MOLECULAR INSIGHTS ON THE FUNCTIONAL MICROBIAL COMMUNITY FROM ORGANIC AND MINERAL GROWING MEDIA AND ITS INTERACTION WITH AGROBACTERIUM RHIZOGENES.

Despite numerous preventative measures, the hairy roots syndrome is an increasing problem in greenhouse horticulture. A recent survey of 177 tomato, cucumber and eggplant growers in Flanders (Belgium) revealed an increase of this disease in the last two years, with about 26% of all the tomato crops showing the syndrome. In this study, we compared the physicochemical and microbial community characteristics of inorganic and organic growing media in relation to the presence of the causative agent of the hairy roots, the plant pathogen Agrobacterium rhizogenes. We aimed to identify how the microbial and environmental interactions influenced the development and spread of this disease in a soilless cultivation system. Multivariate statistical analysis performed to assess the characteristics of each growing media revealed key variables impacting the hosted microbial community. Thus, humidity, pH, potassium and conductivity were drivers of the differences among microbial community composition. High throughput sequencing analysis of the bacterial family abundance of the communities present in organic media indicated potential competitive interactions with A. rhizogenes. Based on our hypothesis that growing media hosted a particular microbiota with potential for modulating hairy roots, we determined how the environment in organic media is reshaped to avoid establishment of A. rhizogenes. Our methodology provides a comprehensive insight into the complex bacterial interactions in horticultural media, which may be potentially applied for the development of effective control strategies and decrease in economic losses.

Effects of sampling location and time, and host animal on assessment of bacterial diversity and fermentation parameters in the bovine rumen.

AIMS: To investigate, using culture-independent methods, whether the ruminal bacterial structure, population and fermentation parameters differed between sampling locations and time. METHODS AND RESULTS: The detectable bacteria and fermentation parameters in the digesta from five locations in the rumen of three cows at three time points were analysed. The PCR-denaturing gradient gel electrophoresis (PCR-DGGE) profiles were similar among digesta samples from five locations (95.4%) and three time points (93.4%) within cows; however, a lower similarity was observed for samples collected from different host animals (85.5%). Rumen pH and concentration of volatile fatty acids (VFA) were affected by time points of sampling relative to feeding. CONCLUSIONS: The detectable bacterial structure in the rumen is highly conserved among different locations and over time, while the quantity of individual bacterial species may change diurnally in response to the feeding. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supplies the fundamental understanding of the microbial ecology in the rumen, which is essential for manipulation of ruminal microflora and subsequent improvement in animal production.