Formalin-Fixed Paraffin-Embedded (FFPE) samples are not a beneficial replacement for frozen tissues in fetal membrane microbiota research.

Formalin-Fixed Paraffin-Embedded (FFPE) tissues are routinely collected, archived, and used for clinical diagnosis, including maternal and neonatal health. Applying FFPE samples to microbiota research would be beneficial to reduce preparation, storage and costs associated with limited available frozen samples. This research aims to understand if FFPE fetal membrane samples are comparable to frozen tissues, which are the current gold standard for DNA microbiota analysis. Extracted DNA from nine matched paired patients were sequenced by Illumina sequencing of the V4 16S rRNA gene region. This included duplicate frozen amnion and chorion fetal membrane rolls or FFPE combined amniochorionic samples. Negative controls of surrounding wax blocks and DNA extraction reagents were processed alongside samples using identical methods. DNA quality and quantity was assessed by NanoDrop, agarose gel electrophoresis and Bioanalyzer. Decontam and SourceTracker were integrated into microbiota analysis to identify the presence of contaminating sources. The bacterial profile and nine genera differed between FFPE and frozen fetal membranes. There were no differences in bacterial profiles between FFPE samples and corresponding wax negative controls, with 49% of bacteria in FFPE fetal membrane samples matched to the source origin of paraffin wax, and 40% originating from DNA extraction reagent sources. FFPE samples displayed high fragmentation and low quantity of extracted DNA compared to frozen samples. The microbiota of FFPE fetal membrane samples is influenced by processing methods, with the inability to differentiate between the microbiota of the tissue sample and the surrounding wax block. Illumina sequencing results of FFPE and frozen fetal membrane samples should not be compared using the methods employed here. Variation could be influenced by limitations including storage time, DNA extraction and purification methods. To utilise FFPE fetal membrane samples in microbiota research then contamination prevention and detection methods must be included into optimised and standardised protocols, with recommendations presented here.

Invasive Non-Native Crustacean Symbionts: Diversity and Impact.

Invasive non-native species (INNS) pose a risk as vectors of parasitic organisms (Invasive Parasites). Introducing invasive parasites can result in ecological disturbances, leading to biodiversity loss and native species illness/mortality, but occasionally can control INNS limiting their impact. Risks to human health and the economy are also associated with INNS and invasive parasites; however, we understand little about the diversity of symbiotic organisms co-invading alongside INNS. This lack of clarity is an important aspect of the 'One Health' prerogative, which aims to bridge the gap between human, wildlife, and ecosystem health. To explore symbiont diversity associated with the invasive crustacean group (including: crab, lobster, crayfish, shrimp, amphipod, isopod, copepod, barnacle, other) (n = 323) derived from 1054 aquatic invertebrates classed as INNS across databases, we compile literature (year range 1800-2017) from the native and invasive range to provide a cumulative symbiont profile for each species. Our search indicated that 31.2% of INN crustaceans were known to hold at least one symbiont, whereby the remaining 68.8% had no documented symbionts. The symbiont list mostly consisted of helminths (27% of the known diversity) and protists (23% of the known diversity), followed by bacteria (12%) and microsporidians (12%). Carcinus maenas, the globally invasive and extremely well-studied green crab, harboured the greatest number of symbionts (n = 72). Additional screening is imperative to become more informed on invasive symbiont threats. We reveal that few studies provide truly empirical data that connect biodiversity loss with invasive parasites and suggest that dedicated studies on available systems will help to provide vital case studies. Despite the lack of empirical data, co-invasive parasites of invasive invertebrates appear capable of lowering local biodiversity, especially by causing behavioural change and mortality in native species. Alternatively, several invasive parasites appear to protect ecosystems by controlling the impact and population size of their invasive host. We provide a protocol that could be followed to explore symbiont diversity in invasive groups as part of our case studies. The consequence of limited parasite screening of INNS, in addition to the impacts invasive parasites impart on local ecologies, are explored throughout the review. We conclude in strong support of the 'One Health' prerogative and further identify a need to better explore disease in invasion systems, many of which are accountable for economic, human health and ecological diversity impacts.

Fetal membrane bacterial load is increased in histologically confirmed inflammatory chorioamnionitis: A retrospective cohort study.

INTRODUCTION: It is widely debated whether fetal membranes possess a genuine microbiome, and if bacterial presence and load is linked to inflammation. Chorioamnionitis is an inflammation of the fetal membranes. This research focussed on inflammatory diagnosed histological chorioamnionitis (HCA) and aimed to determine whether the bacterial load in fetal membranes correlates to inflammatory response, including histological staging and inflammatory markers in HCA. METHODS: Fetal membrane samples were collected from patients with preterm spontaneous labour and histologically phenotyped chorioamnionitis (HCA; n = 12), or preterm (n = 6) and term labour without HCA (n = 6). The bacterial profile of fetal membranes was analysed by sequencing the V4 region of the 16S rRNA gene. Bacterial load was determined using qPCR copy number/mg of tissue. The association between bacterial load and bacterial profile composition was assessed using correlation analysis. RESULTS: Bacterial load was significantly greater within HCA amnion (p = 0.002) and chorion (p = 0.042), compared to preterm birth without HCA. Increased bacterial load was positively correlated with increased histological staging (p = 0.001) and the expression of five inflammatory markers; IL8, TLR1, TLR2, LY96 and IRAK2 (p=<0.050). Bacterial profiles were significantly different between membranes with and without HCA in amnion (p = 0.012) and chorion (p = 0.001), but no differences between specific genera were detected. DISCUSSION: Inflammatory HCA is associated with infection and increased bacterial load in a dose response relationship. Bacterial load is positively correlated with HCA severity and the TLR signalling pathway. Further research should investigate the bacterial load threshold required to generate an inflammatory response in HCA.

Detecting macroecological patterns in bacterial communities across independent studies of global soils.

The emergence of high-throughput DNA sequencing methods provides unprecedented opportunities to further unravel bacterial biodiversity and its worldwide role from human health to ecosystem functioning. However, despite the abundance of sequencing studies, combining data from multiple individual studies to address macroecological questions of bacterial diversity remains methodically challenging and plagued with biases. Here, using a machine-learning approach that accounts for differences among studies and complex interactions among taxa, we merge 30 independent bacterial data sets comprising 1,998 soil samples from 21 countries. Whereas previous meta-analysis efforts have focused on bacterial diversity measures or abundances of major taxa, we show that disparate amplicon sequence data can be combined at the taxonomy-based level to assess bacterial community structure. We find that rarer taxa are more important for structuring soil communities than abundant taxa, and that these rarer taxa are better predictors of community structure than environmental factors, which are often confounded across studies. We conclude that combining data from independent studies can be used to explore bacterial community dynamics, identify potential 'indicator' taxa with an important role in structuring communities, and propose hypotheses on the factors that shape bacterial biogeography that have been overlooked in the past.

Cesarean or Vaginal Birth Does Not Impact the Longitudinal Development of the Gut Microbiome in a Cohort of Exclusively Preterm Infants.

The short and long-term impact of birth mode on the developing gut microbiome in neonates has potential implications for the health of infants. In term infants, the microbiome immediately following birth across multiple body sites corresponds to birth mode, with increased Bacteroides in vaginally delivered infants. We aimed to determine the impact of birth mode of the preterm gut microbiome over the first 100 days of life and following neonatal intensive care unit (NICU) discharge. In total, 867 stool samples from 46 preterm infants (21 cesarean and 25 vaginal), median gestational age 27 weeks, were sequenced (V4 region 16S rRNA gene, Illumina MiSeq). Of these, 776 samples passed quality filtering and were included in the analysis. The overall longitudinal alpha-diversity and within infant beta-diversity was comparable between cesarean and vaginally delivered infants. Vaginally delivered infants kept significantly more OTUs from 2 months of life and following NICU discharge, but OTUs lost, gained, and regained were not different based on birth mode. Furthermore, the temporal progression of dominant genera was comparable between birth modes and no significant difference was found for any genera following adjustment for covariates. Lastly, preterm gut community types (PGCTs) showed some moderate differences in very early life, but progressed toward a comparable pattern by week 5. No PGCT was significantly associated with cesarean or vaginal birth. Unlike term infants, birth mode was not significantly associated with changes in microbial diversity, composition, specific taxa, or overall microbial development in preterm infants. This may result from the dominating effects of NICU exposures including the universal use of antibiotics immediately following birth and/or the lack of Bacteroides colonizing preterm infants.

Impacts of organic and conventional crop management on diversity and activity of free-living nitrogen fixing bacteria and total bacteria are subsidiary to temporal effects.

A three year field study (2007-2009) of the diversity and numbers of the total and metabolically active free-living diazotophic bacteria and total bacterial communities in organic and conventionally managed agricultural soil was conducted using the Nafferton Factorial Systems Comparison (NFSC) study, in northeast England. Fertility management appeared to have little impact on both diazotrophic and total bacterial communities. However, copy numbers of the nifH gene did appear to be negatively impacted by conventional crop protection measures across all years suggesting diazotrophs may be particularly sensitive to pesticides. Impacts of crop management were greatly overshadowed by the influence of temporal effects with diazotrophic communities changing on a year by year basis and from season to season. Quantitative analyses using qPCR of each community indicated that metabolically active diazotrophs were highest in year 1 but the population significantly declined in year 2 before recovering somewhat in the final year. The total bacterial population in contrast increased significantly each year. It appeared that the dominant drivers of qualitative and quantitative changes in both communities were annual and seasonal effects. Moreover, regression analyses showed activity of both communities was significantly affected by soil temperature and climatic conditions.

Diversity and activity of free-living nitrogen-fixing bacteria and total bacteria in organic and conventionally managed soils.

Agricultural soils are heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, the molecular ecology of the total bacterial and free-living nitrogen-fixing communities in soils from the Nafferton Factorial Systems Comparison (NFSC) study in northeast England were examined. The field experiment was factorial in design, with organic versus conventional crop rotation, crop protection, and fertility management factors. Soils were sampled on three dates (March, June, and September) in 2007. Total RNA was extracted from all soil samples and reverse transcribed. Denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were used to analyze nifH and 16S rRNA genes in order to study free-living diazotrophs and the total bacterial community, respectively. Crop rotation was shown to have a significant effect on total bacterial diversity (and that of free-living N fixers) (P ≤ 0.001). On all three dates, nifH activity was higher in the conventional crop rotation. In contrast, qPCR analysis of free-living N fixers indicated significantly higher levels of activity in conventionally fertilized plots in June (P = 0.0324) and in plots with organic crop protection in September (P = 0.0143). To our knowledge, the effects of organic and conventional farming systems on free-living diazotrophs have never been studied. An increased understanding of the impacts of management practices on free-living N fixers could allow modifications in soil management practices to optimize the activity of these organisms.