Virus–Bacteria Interactions: Implications and Potential for the Applied and Agricultural Sciences

Eukaryotic virus–bacteria interactions have recently become an emerging topic of study due to multiple significant examples related to human pathogens of clinical interest. However, such omnipresent and likely important interactions for viruses and bacteria relevant to the applied and agricultural sciences have not been reviewed or compiled. The fundamental basis of this review is that these interactions have importance and deserve more investigation, as numerous potential consequences and applications arising from their discovery are relevant to the applied sciences. The purpose of this review is to highlight and summarize eukaryotic virus–bacteria findings in the food/water, horticultural, and animal sciences. In many cases in the agricultural sciences, mechanistic understandings of the effects of virus–bacteria interactions remain unstudied, and many studies solely focus on co-infections of bacterial and viral pathogens. Given recent findings relative to human viral pathogens, further research related to virus–bacteria interactions would likely result in numerous discoveries and beneficial applications

Generation of Nucleic Acid Aptamer Candidates against a Novel Calicivirus Protein Target

Human norovirus is the leading cause of foodborne illness globally. One of the challenges in detecting noroviruses is the identification of a completely broadly reactive ligand; however, all detection ligands generated to date target the viral capsid, the outermost of which is the most variable region of the genome. The VPg is a protein covalently linked to the viral genome that is necessary for replication but hitherto remains underexplored as a target for detection or therapeutics. The purpose of this work was to generate nucleic acid aptamers against human norovirus (Norwalk) and cultivable surrogate (Tulane) VPgs for future use in detection and therapeutics. Eight rounds of positive-SELEX and two rounds of counter-SELEX were performed. Five and eight unique aptamer sequences were identified for Norwalk and Tulane VPg, respectively, all of which were predicted to be stable (∆G \u3c −5.0) and one of which occurred in both pools. All candidates displayed binding to both Tulane and Norwalk VPg (positive:negative \u3e 5.0), and all but two of the candidates displayed very strong binding (positive:negative \u3e 10.0), significantly higher than binding to the negative control protein (p \u3c 0.05). Overall, this work reports a number of aptamer candidates found to be broadly reactive and specific for in vitro-expressed VPgs across genus that could be used for future application in detection or therapeutics. Future work characterizing binding of the aptamer candidates against native VPgs and in therapeutic applications is needed to further evaluate their application

Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII.4 strain

AbstractHuman noroviruses (NoV) are the leading cause of acute viral gastroenteritis worldwide. Significant antigenic diversity of NoV strains has limited the availability of broadly reactive ligands for design of detection assays. The purpose of this work was to produce and characterize single stranded (ss)DNA aptamers with binding specificity to human NoV using an easily produced NoV target—the P domain protein. Aptamer selection was done using SELEX (Systematic Evolution of Ligands by EXponential enrichment) directed against an Escherichia coli-expressed and purified epidemic NoV GII.4 strain P domain. Two of six unique aptamers (designated M1 and M6-2) were chosen for characterization. Inclusivity testing using an enzyme-linked aptamer sorbent assay (ELASA) against a panel of 14 virus-like particles (VLPs) showed these aptamers had broad reactivity and exhibited strong binding to GI.7, GII.2, two GII.4 strains, and GII.7 VLPs. Aptamer M6-2 exhibited at least low to moderate binding to all VLPs tested. Aptamers significantly (p<0.05) bound virus in partially purified GII.4 New Orleans outbreak stool specimens as demonstrated by ELASA and aptamer magnetic capture (AMC) followed by RT-qPCR. This is the first demonstration of human NoV P domain protein as a functional target for the selection of nucleic acid aptamers that specifically bind and broadly recognize diverse human NoV strains

Sample Preparation: The Forgotten Beginning

Advances in molecular technologies and automated instrumentation have provided many opportunities for improved detection and identification of microorganisms; however, the upstream sample preparation steps needed to apply these advances to foods have not been adequately researched or developed. Thus, the extent to which these advances have improved food microbiology has been limited. The purpose of this review is to present the current state of sample preparation, to identify knowledge gaps and opportunities for improvement, and to recognize the need to support greater research and development efforts on preparative methods in food microbiology. The discussion focuses on the need to push technological developments toward methods that do not rely on enrichment culture. Among the four functional components of microbiological analysis (i.e., sampling, separation, concentration, detection), the separation and concentration components need to be researched more extensively to achieve rapid, direct, and quantitative methods. The usefulness of borrowing concepts of separation and concentration from other disciplines and the need to regard the microorganism as a physicochemical analyte that may be directly extracted from the food matrix are discussed. The development of next-generation systems that holistically integrate sample preparation with rapid, automated detection will require interdisciplinary collaboration and substantially increased funding

Detection of Gram-Negative Histamine-Producing Bacteria in Fish: A Comparative Study

ABSTRACT Poisoning due to ingestion of foods with elevated levels of biogenic amines (histamine, putrescine, cadaverine, and tyramine) is well documented. Histamine fish poisoning largely is due to growth of naturally occurring bacteria associated with scombroid fish species. A rapid and reliable method is needed to screen for the presence of histamine-forming bacteria in fish. This study included a comparison of three methods for the detection of histamine-producing bacteria. A total of 152 histamineproducing and non-histamine-producing bacteria from multiple sources were screened using a modified Niven&apos;s agar method, a potentiometric method, and a PCR-based assay targeting a 709-bp fragment of the histidine decarboxylase gene. Histamine production by bacterial isolates was confirmed by high-performance liquid chromatography (HPLC). Bacterial strains were categorized as producing high amounts of histamine, low amounts of histamine, or no histamine. Of the 152 strains tested, 128 (84%) were positive with the Niven&apos;s agar method, 73 (48%) were positive with the potentiometric technique, and 74 (49%) were positive with the PCR assay. Overall, a 38% false-positive rate was observed with the modified Niven&apos;s agar method, although this method detected both low-histamine and high-histamine strains. There was a high degree of concordance (.99%) between results of the potentiometric and PCR methods, but neither of these methods detected low-histamine bacteria. These observations support the need for a simple and straightforward yet sensitive method for detecting histamine-producing bacteria in seafood and environmental samples

Evaluation of a novel chlorine dioxide-based packaging technology to reduce human enteric virus contamination on refrigerated tomatoes and blueberries

IntroductionChlorine dioxide (ClO2) is a promising antimicrobial with various food applications, one of those being inclusion in packaging. The purpose of this study was to evaluate a novel ClO2-based antimicrobial packaging system (InvisiShield™) for its efficacy against human norovirus (hNoV) and hepatitis A virus (HAV) in refrigerated fresh produce.MethodsGrape tomatoes or blueberries were placed in polypropylene trays and selectively inoculated with 6.0 log10 hNoV Genome Equivalent Copies (GEC; 20% stool suspension) or 6.2 log10 HAV GEC (cell culture lysate). Trays were heat sealed with a three-phase polymer film consisting of a base, channeling agent, and the ClO2 active (treatment); or control (no active) film and stored at 7°C for 24, 48 h, and 7 days. At each timepoint, the product was collected and processed for virus concentration using the sequential steps of elution and polyethylene glycol precipitation. Viruses in extracts were quantified using RNase-RT-qPCR.Results and discussionLog10 reductions (LR) in hNoV GEC for tomatoes were 2.2 ± 1.3, 2.9 ± 0.7, and 3.6 ± 0.3, after 24, 48 h and 7 days, respectively. For blueberries, hNoV LR were 1.4 ± 0.7, 1.7 ± 0.5, and 2.7 ± 0.2 GEC, respectively. Hepatitis A virus GEC LR were 0.4 ± 0.2, 1.0 ± 0.1, and 2.1 ± 0.7 for tomatoes, and 0.1 ± 0.2, 1.2 ± 0.4, and 3.2 ± 0.2 for blueberries, after 24, 48 h and 7 days, respectively. Position of the fruit in the tray did not affect inactivation (p &gt; 0.05). Sensory analysis on the treated tomato products revealed no statistically significant difference in appearance, flavor and texture attributes compared to the control. This novel ClO2-based antimicrobial packaging system effectively reduced concentrations of hNoV and HAV, as evaluated using reduction in GEC as proxy for infectivity, on grape tomatoes and blueberries after one day, with efficacy improving over 7 days of refrigerated storage. This technology shows promise as an antiviral treatment as applied to refrigerated fresh produce items

Contamination of Fresh Produce by Microbial Indicators on Farms and in Packing Facilities: Elucidation of Environmental Routes

ABSTRACT To improve food safety on farms, it is critical to quantify the impact of environmental microbial contamination sources on fresh produce. However, studies are hampered by difficulties achieving study designs with powered sample sizes to elucidate relationships between environmental and produce contamination. Our goal was to quantify, in the agricultural production environment, the relationship between microbial contamination on hands, soil, and water and contamination on fresh produce. In 11 farms and packing facilities in northern Mexico, we applied a matched study design: composite samples (n � 636, equivalent to 11,046 units) of produce rinses were matched to water, soil, and worker hand rinses during two growing seasons. Microbial indicators (coliforms, Escherichia coli, Enterococcus spp., and somatic coliphage) were quantified from composite samples. Statistical measures of association and correlations were calculated through Spearman’s correlation, linear regression, and logistic regression models. The concentrations of all microbial indicators were positively correlated between produce and hands ( � range, 0.41 to 0.75; P � 0.01). When E. coli was present on hands, the handled produce was nine times more likely to contain E. coli (P � 0.05). Similarly, when coliphage was present on hands, the handled produce was eight times more likely to contain coliphage (P � 0.05). There were relatively low concentrations of indicators in soil and water samples, and a few sporadic significant associations were observed between contamination of soil and water and contamination of produce. This methodology provides a foundation for future field studies, and results highlight the need for interventions surrounding farmworker hygiene and sanitation to reduce microbial contamination of farmworkers’ hands. IMPORTANCE This study of the relationships between microbes on produce and in the farm environment can be used to support the design of targeted interventions to prevent or reduce microbial contamination of fresh produce with associated reductions in foodborne illness. KEYWORDS environmental microbiology, food-borne pathogens, produc

Detection of Listeria monocytogenes from a Model Food by Fluorescence Resonance Energy Transfer-Based PCR with an Asymmetric Fluorogenic Probe Set

It has been shown that fluorescence resonance energy transfer (FRET)-based PCR, including the TaqMan assay and molecular beacons, has potential for rapid detection of pathogens. In these promising real-time detection assays a single internal oligonucleotide probe labeled on both the 5′ (reporter) and 3′ (quencher) ends is used for selective generation of fluorescence. In this paper, we describe the use of a previously reported novel probe design for FRET-based PCR detection of Listeria monocytogenes in pure culture and in a model food commodity. In the assay described here an asymmetric probe set is used; this probe set consists of a long 5′ fluorescein-labeled reporter probe and a short, complementary 3′ DABCYL-labeled quencher oligonucleotide, which are used in a 5′ nuclease amplification and detection assay. By using the listeriolysin O (hly) and p60 (iap) genes as amplification targets, the performance of two primer-probe sets in amplification and subsequent detection of target DNA was evaluated. In studies performed with pure cultures of L. monocytogenes, the PCR profiles indicated that the relative change in fluorescence intensity was correlated with both the initial number of cells and the accumulation of specific amplicons for both hly and iap gene fragments. Experiments were also done to determine the applicability of the method to the detection of L. monocytogenes by targeting hly DNA and its short-lived mRNA product in a model food commodity. Twenty-five-milliliter samples of reconstituted nonfat dry milk (NFDM) were seeded with L. monocytogenes and processed to concentrate the bacteria by centrifugation, and this was followed by nucleic acid extraction and amplification with hly-specific primers. Endpoint detection of PCR and reverse transcription-PCR amplicons could be achieved at inoculum levels of 10(3) and 10(4) CFU of L. monocytogenes/25 ml of NFDM, respectively. This study demonstrated that this asymmetric FRET-based amplification and detection protocol provides an alternative approach for endpoint detection of nucleic acid amplification products as applied to detection of pathogens in a model food