Biocontrol of Escherichia coli O157:H7 by Enterobacter asburiae AEB30 on intact cantaloupe melons.

Escherichia coli O157:H7 causes >73,000 foodborne illnesses in the United States annually, many of which have been associated with fresh ready-to-eat produce including cantaloupe melons. In this study, we created a produce-associated bacterial (PAB) library containing >7500 isolates and screened them for the ability to inhibit the growth of E. coli O157:H7 using an in vitro fluorescence-based growth assay. One isolate, identified by 16S and whole-genome sequence analysis as Enterobacter asburiae, was able to inhibit the growth of E. coli by ~30-fold in vitro and produced zones of inhibition between 13 and 21 mm against 12 E. coli outbreak strains in an agar spot assay. We demonstrated that E. asburiae AEB30 was able to grow, persist and inhibit the growth of E. coli on cantaloupe melons under simulated pre- and post-harvest conditions. Analysis of the E. asburiae AEB30 genome revealed an operon encoding a contact-dependent growth inhibition (CDI) system that when mutated resulted in the loss of E. coli growth inhibition. These data suggest that E. asburiae AEB30 is a potential biocontrol agent to prevent E. coli contamination of cantaloupe melons in both pre- and post-harvest environments and that its mode of action is via a CDI system.

Complete genome sequence of Citrobacter braakii ASE1 generated by PacBio sequencing.

We report the complete genome sequence of Citrobacter braakii ASE1 generated by the PacBio Sequel II platform. This bacterium was isolated from the soil of a lettuce farm in Salinas, CA, USA, in 2020. The genome consists of a single circular chromosome of 5,021,820 bp with a 52.2% GC content.

Use of Pantoea agglomerans ASB05 as a biocontrol agent to inhibit the growth of Salmonella enterica on intact cantaloupe melons.

AIMS: To identify biocontrol agents to prevent the growth of Salmonella serotype Enterica on cantaloupe melons during the pre- and postharvest periods. METHODS AND RESULTS: We created a produce-associated bacterial library containing 8736 isolates and screened it using an in-vitro fluorescence inhibition assay to identify bacteria that inhibit the growth of S. Enterica. One isolate, Pantoea agglomerans ASB05, was able to grow, persist, and inhibit the growth of S. Enterica on intact cantaloupe melons under simulated pre- and postharvest conditions. We also demonstrated that the growth inhibition of S. Enterica by P. agglomerans ASB05 was due to the production of a phenazine type antibiotic. CONCLUSIONS: Pantoea agglomerans ASB05 is an effective biocontrol agent for the prevention of S. Enterica growth on intact cantaloupe melons in both the pre- and postharvest environments.

Sensitive and selective detection of peanut allergen Ara h 1 by ELISA and lateral flow immunoassay.

The Ara h1 protein is a peanut allergen and it provides a useful biomarker for the detection of peanut protein. In this manuscript, we describe the generation of monoclonal antibodies (MAbs) against the Ara h1 protein and their development into sensitive and selective immunoassays for peanut detection. Our enzyme-linked immunosorbent assay (sELISA) detects a peanut meal standard with a sensitivity of 10 ng/mL and 500 ng/mL by lateral flow immunoassay (LFIA). MAb Ara h1 binding epitopes were identified, and immunoassay detection was limited to peanut meal varieties irrespective of thermal treatment. No binding was observed from tree nut meals (100-0.4 µg/mL). Peanut allergen detection during food manufacturing can limit the incidence of product recall resulting from cross-contact contamination or improper labeling of finished food products. Detection of Ara h1 by immunoassay can provide a cost-effective method for rapid surveillance of peanut during food production and prior to consumption.

Complete Genome Sequence of Enterobacter asburiae Strain AEB30, Determined Using Illumina and PacBio Sequencing.

The complete genome sequence of Enterobacter asburiae strain AEB30 is presented. The strain was isolated from store-bought ginger in Albany, CA, in 2016.

Role of soil in the regulation of human and plant pathogens: soils' contributions to people.

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'.

Complete Genome Sequence of Pantoea agglomerans ASB05 Using Illumina and PacBio Sequencing.

We present the complete genome sequence of Pantoea agglomerans ASB05 and three associated plasmids, generated using a combination of the Illumina and PacBio platforms. P. agglomerans ASB05 was isolated from fresh cherries purchased in Albany, CA, in 2016.

A rapid and sensitive lateral flow immunoassay (LFIA) for the detection of gluten in foods.

The gluten protein found in a variety of cereal grains is a food allergen that can elicit a spectrum of immuno-inflammatory responses in people. Consumer awareness has prompted changes in food labeling requirements, expanded gluten-free food product availability and increased demand for effective gluten testing methodologies. To meet the challenges associated with gluten testing from diverse and complex foods we developed a lateral flow immunoassay (LFIA) using a pair of novel gliadin monoclonal antibodies (MAbs). Using a visual gold reporter, we show sensitive gluten detection (150 ng/mL) from complex food substrates using a fast (<5 min) and easy testing methodology. In this report we characterize the binding properties of a cohort of newly generated gliadin monoclonal antibodies suitable for gluten detection using multiple assay formats and introduce a novel plug-n-play test strip platform with integrated test components in a single-use format.

Bacillus amyloliquefaciens ALB65 Inhibits the Growth of Listeria monocytogenes on Cantaloupe Melons.

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 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 six 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 U.S. Food and Drug Administration to cause disease in approximately 1,600 to 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 postharvest conditions. Furthermore, we demonstrated that B. amyloliquefaciens ALB65 can completely inhibit the growth of L. monocytogenes during cold storage (<8°C).

Complete Genomic Sequences of Three Salmonella enterica subsp. enterica Serovar Muenchen Strains from an Orchard in San Joaquin County, California.

We present here the complete genome sequences of three Salmonella enterica subsp. enterica serovar Muenchen strains, LG24, LG25, and LG26. All three strains were isolated from almond drupes grown in an orchard in San Joaquin County, California, in 2016. These genomic sequences are nonidentical and will contribute to our understanding of S. enterica genomics.

Use of Phyllosphere-Associated Lactic Acid Bacteria as Biocontrol Agents To Reduce Salmonella enterica Serovar Poona Growth on Cantaloupe Melons.

Foodborne illness associated with fresh, ready-to-eat produce continues to be a significant challenge to public health. In this study, we created a phyllosphere-associated lactic acid bacteria (PLAB) library and screened it via a high-throughput in vitro fluorescent assay to identify bacteria capable of inhibiting the growth of the pathogenic bacterium Salmonella enterica. One isolate, 14B4, inhibited the growth of S. enterica by >45-fold in vitro; it was able to grow and persist on the surfaces of cantaloupe melons at both ambient (25°C) and refrigerator (5°C) temperatures. Isolate 14B4 inhibited the growth of S. enterica on the surfaces of cantaloupes by >3 log when incubated at 25°C for 24 h and by >4 log when the cantaloupes were stored at 5°C for 3 days and the temperature was shifted to 25°C for 2 days. Genomic DNA sequence analysis of isolate 14B4 revealed that it was Lactococcus lactis and that it did not contain any known antibiotic biosynthesis gene clusters, antibiotic resistance genes, or genes encoding any known virulence factors. Organic acid analysis revealed that L. lactis produces substantial amounts of lactic acid, which is likely the inhibitory substance that reduced the growth of Salmonella on the cantaloupes.

Rapid Detection of Staphylococcal Enterotoxin-B by Lateral Flow Assay.

A cohort of monoclonal antibodies (mAbs) were generated against Staphylococcal enterotoxin-B (SEB) and selected by double sandwich enzyme-linked immunosorbent assay (ELISA) for solution capture of the toxin. Clonal hybridoma cell lines were established and a pair of anti-SEB mAbs selected for the development of a sandwich ELISA. Immobilized 3D6 mAb (IgG1, kappa) when paired with 4C9 mAb (IgG1, kappa) conjugated to horseradish peroxidase generates a typical dose-response curve with an EC50 of 24.8 ng/mL for purified SEB using chemiluminescent detection. These mAbs bind SEB by Western blot and ELISA binding to classical enterotoxin serotypes show that the 3D6 mAb binds both SEB and the SEC1 serotypes, whereas 4C9 binds only SEB. These mAbs effectively port onto lateral flow test strips with a visual detection sensitivity for SEB of 5 ng/mL in <10 minutes using a 4C9 conjugated to a 40 nm gold reporter.

Dosage-dependent effects of monensin on the rumen microbiota of lactating dairy cattle.

We examined the dose-dependent effects of feeding lactating dairy cows a standard diet supplemented with monensin at 175, 368, or 518 mg cow-1 day-1 on the rumen microbiota. For each dosage, 3 animals were randomly assigned into groups and fed the same basal total mixed ration diet supplemented with monensin, at the respective dose. After 20 days, rumen samples were taken and the effect on the microbiota was examined by 16S rRNA gene sequence analysis and qPCR. At the lowest dose no significant change in 16S rRNA gene sequences associated with any bacterial phyla was observed; however, at the medium and high dosages, we observed significant reductions in sequences associated with gram-positive bacteria and significant increases in those associated with gram-negative bacteria that were dosage dependent. All dosages reduced the levels of sequences associated with methanogenic archaea in the rumen, with the medium dosage showing the largest decline. No significant difference was observed for the 18S rRNA gene sequences associated with protozoa in any of the libraries. Our results indicate that with this diet the medium dosage of monensin was most efficacious for the reduction in methanogenic archaea in the rumen of lactating dairy.

Complete Genome Sequence of Lactococcus lactis subsp. lactis Strain 14B4, Which Inhibits the Growth of Salmonella enterica Serotype Poona In Vitro.

We present here the complete genome sequence of Lactococcus lactis strain 14B4, isolated from almond drupes in northern California. This strain was observed to inhibit the growth of Salmonella enterica serotype Poona strain RM3363 in vitro.

Complete Genome Sequences of Three Bacillus amyloliquefaciens Strains That Inhibit the Growth of Listeria monocytogenes In Vitro.

Here, we report the complete genome sequences of three Bacillus amyloliquefaciens strains isolated from alfalfa, almond drupes, and grapes that inhibited the growth of Listeria monocytogenes strain 2011L-2857 in vitro We also report multiple gene clusters encoding secondary metabolites that may be responsible for the growth inhibition of L. monocytogenes.

Identification and verification of hybridoma-derived monoclonal antibody variable region sequences using recombinant DNA technology and mass spectrometry.

Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody. It is the VR that define the unique antigen binding properties and proper sequence identification is essential for functional evaluation and performance of recombinant antibodies (rAb). This determination can be achieved by sequence analysis of immunoglobulin (Ig) transcripts obtained from a monoclonal antibody (MAb) producing hybridoma and subsequent expression of a rAb. However the polyploidy nature of a hybridoma cell often results in the added expression of aberrant immunoglobulin-like transcripts or even production of anomalous antibodies which can confound production of rAb. An incorrect VR sequence will result in a non-functional rAb and de novo assembly of Ig primary structure without a sequence map is challenging. To address these problems, we have developed a methodology which combines: 1) selective PCR amplification of VR from both the heavy and light chain IgG from hybridoma, 2) molecular cloning and DNA sequence analysis and 3) tandem mass spectrometry (MS/MS) on enzyme digests obtained from the purified IgG. Peptide analysis proceeds by evaluating coverage of the predicted primary protein sequence provided by the initial DNA maps for the VR. This methodology serves to both identify and verify the primary structure of the MAb VR for production as rAb.

Affinity Purification of Antibodies.

Antibodies are provided in a variety of formats that include antiserum, hybridoma culture supernatant, or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facilitate assay reproducibility, economy, and reduced interference of nonspecific components as well as improved storage, stability, and bio-conjugation. Although not always necessary, the relative simplicity of antibody purification using commercially available protein-A, protein-G, or protein-L resins with basic chromatographic principles warrants purification when antibody source material is available in sufficient quantity. Here, we define three simple methods using immobilized (1) protein-A, (2) protein-G, and (3) protein-L agarose beads to yield highly purified antibody.

Detection of shiga toxins by lateral flow assay.

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.

Rapid and selective detection of botulinum neurotoxin serotype-A and -B with a single immunochromatographic test strip.

Botulinum neurotoxins (BoNT) are the most potent toxins known. Produced by Clostridium botulinum, BoNTs are classified into seven, antigenically distinct serotypes, designated A-G. The toxin acts to inhibit acetylcholine release, resulting in paralysis and death. Naturally occurring foodborne disease is most often the result of improper canning of foods, while wound botulism, associated with injection drug users, is on the rise. Because of its potency, BoNTs have also been identified as targets for use by bioterrorists. The 'gold standard' of detection of BoNTs is the mouse bioassay, an expensive and time consuming test that requires specialized equipment and trained personnel. There is a need for a rapid, sensitive diagnostic for BoNTs that could be used by minimally trained personnel in the event of a foodborne outbreak or a bioterrorist threat. Here, we describe the use of a single lateral flow device (LFD) that can detect and distinguish between BoNT/A and B, two of the four serotypes that are known to intoxicate humans and together represent >80% of naturally occurring illness. The device could detect as little as 5 ng/mL of purified BoNT/A and 10 ng/mL of BoNT/B in 2% and 1% milk, respectively. In undiluted apple juice, 25 ng/mL of BoNT/A and 10 ng/mL of BoNT/B could be detected. No cross reactivity between BoNT/A and B antibodies was observed. The LFD described here is easy to use, requires no specialized training or equipment, and can identify and distinguish between BoNT/A and /B serotypes. These attributes make this rapid diagnostic device a potentially valuable tool in the fields of food safety and homeland security.

A rapid method to improve protein detection by indirect ELISA.

The enzyme-linked immunosorbant assay (ELISA) is a rapid, high-throughput, quantitative immunoassay for the selective detection of target antigens. The general principle behind an ELISA is antibody mediated capture and detection of an antigen with a measurable substrate. Numerous incarnations of the ELISA have resulted in its commercialization for sensitive diagnostic applications using a variety of detection platforms. Many of these applications require a pair of antibodies necessary for the capture and detection of a specific antigen (cELISA) in defined substrates. However, the availability of cELISA for target antigens is limited and thus restricts the use of this technique for quantitative measure of antigens during discovery. Alternatively, the indirect ELISA (iELISA) requires only a single antibody directed against a target antigen that has been immobilized to a surface. Unlike the cELISA, which uses an immobilized capture antibody that can bind a native antigen in solution followed by a detector antibody that binds captured antigen, the iELISA uses an antibody the binds directly to an immobilized antigen for detection. Although the iELISA may lack the sensitivity of a cELISA, its requirement of only a single antigen specific antibody makes it a simple technique for evaluating the relative difference in the level of target protein expression between samples. However, many antibodies that work effectively to detect protein antigens in other immunoassays such as Western blotting or immunohistochemistry fail to work in microplate based iELISA. Although these alternate immunoassay methods are useful for qualitative determination of target antigens, they provide limited quantitative information, limiting the assessment of sample specific differences in protein expression. We hypothesized that protein conformation following adsorption on the plastic surface of microplates impedes antibody epitope binding and this restriction could be overcome by a short chemical denaturation step. In this report we define a rapid method to assess the utility of an antibody for iELISA application and demonstrate a significant improvement in both qualitative and quantitative protein detection after chemical denaturation using defined assay conditions.