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Bats are known to be reservoirs for a variety of mammalian pathogens, including viruses, fungi, and bacteria. Many of the studies examining the microbial community inhabiting bats have investigated bacterial taxa found within specific bat tissues and isolated bat guano pellets, but relatively few studies have explored bacterial diversity within bat guano piles. In large bat caves, bat guano can accumulate over time, creating piles several meters deep and forming complex interactions with coprophagous organisms in a habitat with low light and oxygen. As the guano decays, the nutrient composition changes, but the bacterial communities deep within the pile have not been characterized. Here, we assess the bacterial communities across varying depths within the guano pile using both culture-independent and culture-dependent methods. We found that although similar taxa are found throughout the guano pile, the relative abundances of taxa within the pile shift, allowing certain taxa to dominate the bacterial community at varying depths. We also identified potential bacterial functions being performed within the bat guano as various depths within the pile and found little variation in terms of the dominant predicted functions, suggesting that although the relative abundances of bacterial taxa are changing, the functions being performed are similar. Additionally, we cultured 15 different bacterial species, including 2 not present in our culture-independent analysis, and discuss the pathogenicity potential of these taxa. This study represents the first characterization of the bacterial community from the extreme environment within a bat guano pile and demonstrates the potential for bat caves as resources for identifying new bacterial species.

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In commercial agriculture, populations and interactions of rhizosphere microflora are potentially affected by the use of specific agrichemicals, possibly by affecting gene expression in these organisms. To investigate this, we examined changes in bacterial gene expression within the rhizosphere of glyphosate-tolerant corn (Zea mays) and soybean (Glycine max) in response to long-term glyphosate (PowerMAX™, Monsanto Company, MO, USA) treatment. A long-term glyphosate application study was carried out using rhizoboxes under greenhouse conditions with soil previously having no history of glyphosate exposure. Rhizosphere soil was collected from the rhizoboxes after four growing periods. Soil microbial community composition was analyzed using microbial phospholipid fatty acid (PLFA) analysis. Total RNA was extracted from rhizosphere soil, and samples were analyzed using RNA-Seq analysis. A total of 20-28 million bacterial sequences were obtained for each sample. Transcript abundance was compared between control and glyphosate-treated samples using edgeR. Overall rhizosphere bacterial metatranscriptomes were dominated by transcripts related to RNA and carbohydrate metabolism. We identified 67 differentially expressed bacterial transcripts from the rhizosphere. Transcripts downregulated following glyphosate treatment involved carbohydrate and amino acid metabolism, and upregulated transcripts involved protein metabolism and respiration. Additionally, bacterial transcripts involving nutrients, including iron, nitrogen, phosphorus, and potassium, were also affected by long-term glyphosate application. Overall, most bacterial and all fungal PLFA biomarkers decreased after glyphosate treatment compared to the control. These results demonstrate that long-term glyphosate use can affect rhizosphere bacterial activities and potentially shift bacterial community composition favoring more glyphosate-tolerant bacteria.

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The etiological agent of necrotic enteritis (NE) is Clostridium perfringens (CP), which is an economically significant problem for broiler chicken producers worldwide. Traditional use of in-feed antibiotic growth promoters to control NE disease have resulted in the emergence of antibiotic resistance in CP strains. Identification of probiotic bacteria strains as an alternative to antibiotics for the control of intestinal CP colonization is crucial. Two experiments were conducted to determine changes in intestinal bacterial assemblages in response to CP infection and in-feed bacitracin methylene disalicylate (BMD) in broiler chickens. In each experiment conducted in battery-cage or floor-pen housing, chicks were randomly assigned to the following treatment groups: 1) BMD-supplemented diet with no CP challenge (CM), 2) BMD-free control diet with no CP challenge (CX), 3) BMD-supplemented diet with CP challenge (PCM), or 4) BMD-free control diet with CP challenge (PCX). The establishment of CP infection was confirmed, with the treatment groups exposed to CP having a 1.5- to 2-fold higher CP levels (P < 0.05) compared to the non-exposed groups. Next-generation sequencing of PCR amplified 16S rRNA genes, was used to perform intestinal bacterial diversity analyses pre-challenge, and at 1, 7, and 21 d post-challenge. The results indicated that the intestinal bacterial assemblage was dominated by members of the phylum Firmicutes in all treatments before and after CP challenge, especially the Lactobacillaceae and Clostridiales families. In addition, we observed post-challenge emergence of members of the Enterobacteriaceae and Streptococcaceae in the non-medicated PCX treatment, and emergence of the Enterococcaceae in the medicated PCM treatment. This study highlights the bacterial interactions that could be important in suppressing or eliminating CP infection within the chicken intestine. Future studies should explore the potential to use commensal strains of unknown Clostridiales, Lactobacillaceae, Enterobacteriaceae, Streptococcaceae, and Enterococcaceae in effective probiotic formulations for the control of CP and NE disease.

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Glyphosate is one of the most widely used herbicides in agriculture with predictions that 1.35 million metric tons will be used annually by 2017. With the advent of glyphosate tolerant (GT) cropping more than 10 years ago, there is now concern for non-target effects on soil microbial communities that has potential to negatively affect soil functions, plant health, and crop productivity. Although extensive research has been done on short-term response to glyphosate, relatively little information is available on long-term effects. Therefore, the overall objective was to investigate shifts in the rhizosphere bacterial community following long-term glyphosate application on GT corn and soybean in the greenhouse. In this study, rhizosphere soil was sampled from rhizoboxes following 4 growth periods, and bacterial community composition was compared between glyphosate treated and untreated rhizospheres using next-generation barcoded sequencing. In the presence or absence of glyphosate, corn and soybean rhizospheres were dominated by members of the phyla Proteobacteria, Acidobacteria, and Actinobacteria. Proteobacteria (particularly gammaproteobacteria) increased in relative abundance for both crops following glyphosate exposure, and the relative abundance of Acidobacteria decreased in response to glyphosate exposure. Given that some members of the Acidobacteria are involved in biogeochemical processes, a decrease in their abundance could lead to significant changes in nutrient status of the rhizosphere. Our results also highlight the need for applying culture-independent approaches in studying the effects of pesticides on the soil and rhizosphere microbial community.

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Microbial succession during leaf breakdown was investigated in a small forested stream in west-central Georgia, USA, using multiple culture-independent techniques. Red maple (Acer rubrum) and water oak (Quercus nigra) leaf litter were incubated in situ for 128 days, and litter breakdown was quantified by ash-free dry mass (AFDM) method and microbial assemblage composition using phospholipid fatty acid analysis (PLFA), ribosomal intergenic spacer analysis (RISA), denaturing gradient gel electrophoresis (DGGE), and bar-coded next-generation sequencing of 16S rRNA gene amplicons. Leaf breakdown was faster for red maple than water oak. PLFA revealed a significant time effect on microbial lipid profiles for both leaf species. Microbial assemblages on maple contained a higher relative abundance of bacterial lipids than oak, and oak microbial assemblages contained higher relative abundance of fungal lipids than maple. RISA showed that incubation time was more important in structuring bacterial assemblages than leaf physicochemistry. DGGE profiles revealed high variability in bacterial assemblages over time, and sequencing of DGGE-resolved amplicons indicated several taxa present on degrading litter. Next-generation sequencing revealed temporal shifts in dominant taxa within the phylum Proteobacteria, whereas γ-Proteobacteria dominated pre-immersion and α- and ß-Proteobacteria dominated after 1 month of instream incubation; the latter groups contain taxa that are predicted to be capable of using organic material to fuel further breakdown. Our results suggest that incubation time is more important than leaf species physicochemistry in influencing leaf litter microbial assemblage composition, and indicate the need for investigation into seasonal and temporal dynamics of leaf litter microbial assemblage succession.

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The use of fluorescence in situ hybridization (FISH) to identify and enumerate specific bacteria within a mixed culture or environmental sample has become a powerful tool in combining microscopy with molecular phylogenetic discrimination. However, processing a large number of samples in parallel can be difficult because the bacterial cells are typically fixed and hybridized on microscope slides rather than processed in solution. In addition, gram-positive cells and certain environmental samples present a unique challenge to achievement of adequate cell fixation and uniform hybridization for optimal FISH analysis. Here, we describe a protocol for FISH in solution that can be performed entirely in suspension, in a microcentrifuge tube format, prior to microscopy. This protocol can be applied to both gram-positive and -negative cells, as well as complex microbial assemblages. The method employs a rapid technique for performing multiple hybridizations simultaneously, which may be used to qualitatively assess the presence of specific phylogenetic groups in bacterial cultures or environmental samples, and/or directly quantify fluorescence by fluorometry or flow cytometry.

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