Aims To describe, at high resolution, the bacterial population dynamics and chemical transformations during the ensiling of alfalfa and subsequent exposure to air. Methods and Results Samples of alfalfa, ensiled alfalfa and silage exposed to air were collected and their bacterial population structures compared using 16S rRNA gene libraries containing approximately 1900 sequences each. Cultural and chemical analyses were also performed to complement the 16S gene sequence data. Sequence analysis revealed significant differences (P < 0·05) in the bacterial populations at each time point. The alfalfa-derived library contained mostly sequences associated with the Gammaproteobacteria (including the genera: Enterobacter, Erwinia and Pantoea); the ensiled material contained mostly sequences associated with the lactic acid bacteria (LAB) (including the genera: Lactobacillus, Pediococcus and Lactococcus). Exposure to air resulted in even greater percentages of LAB, especially among the genus Lactobacillus, and a significant drop in bacterial diversity. Conclusions In-depth 16S rRNA gene sequence analysis revealed significant bacterial population structure changes during ensiling and again during exposure to air. Significance and Impact of the Study This in-depth description of the bacterial population dynamics that occurred during ensiling and simulated feed out expands our knowledge of these processes.

Soil and soil biodiversity play critical roles in Nature's Contributions to People (NCP) # 10, defined as Nature's ability to regulate direct detrimental effects on humans, and on human-important plants and animals, through the control or regulation of particular organisms considered to be harmful. We provide an overview of pathogens in soil, focusing on human and crop pathogens, and discuss general strategies, and examples, of how soils' extraordinarily diverse microbial communities regulate soil-borne pathogens. We review the ecological principles underpinning the regulation of soil pathogens, as well as relationships between pathogen suppression and soil health. Mechanisms and specific examples are presented of how soil and soil biota are involved in regulating pathogens of humans and plants. We evaluate how specific agricultural management practices can either promote or interfere with soil's ability to regulate pathogens. Finally, we conclude with how integrating soil, plant, animal and human health through a ‘One Health’ framework could lead to more integrated, efficient and multifunctional strategies for regulating detrimental organisms and processes. This article is part of the theme issue ‘The role of soils in delivering Nature's Contributions to People’.

Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD) for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

Potent Botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit foodborne contamination and morbidity. In this report, we describe a rapid 96-well microfluidic double sandwich immunoassay for the sensitive detection of BoNT-A from animal sera. This BoNT microfluidic assay requires only 5 μL of serum, provides results in 75 min using a standard fluorescence microplate reader and generates minimal hazardous waste. The assay has a <30 pg·mL⁻¹ limit of detection (LOD) of BoNT-A from spiked human serum. This sensitive microfluidic BoNT-A assay offers a fast and simplified workflow suitable for the detection of BoNT-A from serum samples of limited volume in most laboratory settings.

Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A–H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s) for individual antibodies ranging from 10 to 48 × 10⁻¹¹ M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested.

Listeria monocytogenes is a foodborne pathogen that causes high rates of hospitalization and mortality in people infected. Contamination of fresh, ready to eat produce by this pathogen is especially troubling because of the ability of this bacterium to grow on produce under refrigeration temperatures. In this study, we created a library of over 8,000 plant phyllosphere associated bacteria (PPAB) and screened them for the ability to inhibit the growth of L. monocytogenes in an in vitro fluorescence based assay. One isolate, later identified as Bacillus amyloliquefaciens ALB65, was able to inhibit the fluorescence of L. monocytogenes by > 30-fold in vitro. B. amyloliquefaciens ALB65 was also able to grow, persist and reduce the growth of L. monocytogenes by > 1.5 log CFU on cantaloupe melon rinds inoculated with 5×10³ CFU at 30°C, and was able to completely inhibit its growth at temperatures below 8°C. DNA sequence analysis of the B. amyloliquefaciens ALB65 genome revealed 6 gene clusters that are predicted to encode genes for antibiotic production; however, no plant or human virulence factors were identified. These data suggest that B. amyloliquefaciens ALB65 is an effective and safe biological control agent for the reduction of L. monocytogenes growth on intact cantaloupe melons and possibly other types of produce. IMPORTANCEListeria monocytogenes is estimated by the Centers for Disease Control and Prevention and the US Food and Drug Administration to cause disease in approximately 1,600 – 2,500 people in the United States every year. The largest known outbreak of listeriosis in the United States was associated with intact cantaloupe melons in 2011, resulting in 147 hospitalizations and 33 deaths. In this study, we demonstrated that Bacillus amyloliquefaciens ALB65 is an effective biological control agent for the reduction of L. monocytogenes growth on intact cantaloupe melons under both pre- and post-harvest conditions. Furthermore, we demonstrated that B. amyloliquefaciens ALB65 can completely inhibit the growth of L. monocytogenes during cold storage (< 8°C).