Studies on Simultaneous Enrichment and Detection of Escherichia coli O157:H7 during Sample Shipment.

The USDA-FSIS has zero tolerance for E. coli O157:H7 in raw ground beef. Currently, FSIS collects samples from beef processing facilities and ships them overnight to regional testing laboratories. Pathogen detection requires robust methods that employ an initial 15-24 h culture enrichment. This study assessed the potential of using the ΦV10nluc phage-based luminescence detection assay during enrichment while the sample is in transit. Parameters including phage concentrations, temperature, and media-to-sample ratios were evaluated. Results in liquid media showed that 1.73× 103 pfu/mL of ΦV10nluc was able to detect 2 CFU in 10 h. The detection of E. coli O157:H7 was further evaluated in kinetic studies using ratios of 1:3, 1:2, and 1:1 ground beef sample to enrichment media, yielding positive results for as little as 2-3 CFU in 325 g ground beef in about 15 h at 37 °C. These results suggest that this approach is feasible, allowing the detection of a presumptive positive upon arrival of the sample to the testing lab. As the current cargo hold controlled temperature is required to be 15-25 °C, the need for elevated temperature should be easily addressed. If successful, this approach could be expanded to other pathogens and foods.

Bacteriophage-Based Biosensors: A Platform for Detection of Foodborne Bacterial Pathogens from Food and Environment.

Foodborne microorganisms are an important cause of human illness worldwide. Two-thirds of human foodborne diseases are caused by bacterial pathogens throughout the globe, especially in developing nations. Despite enormous developments in conventional foodborne pathogen detection methods, progress is limited by the assay complexity and a prolonged time-to-result. The specificity and sensitivity of assays for live pathogen detection may also depend on the nature of the samples being analyzed and the immunological or molecular reagents used. Bacteriophage-based biosensors offer several benefits, including specificity to their host organism, the detection of only live pathogens, and resistance to extreme environmental factors such as organic solvents, high temperatures, and a wide pH range. Phage-based biosensors are receiving increasing attention owing to their high degree of accuracy, specificity, and reduced assay times. These characteristics, coupled with their abundant supply, make phages a novel bio-recognition molecule in assay development, including biosensors for the detection of foodborne bacterial pathogens to ensure food safety. This review provides comprehensive information about the different types of phage-based biosensor platforms, such as magnetoelastic sensors, quartz crystal microbalance, and electrochemical and surface plasmon resonance for the detection of several foodborne bacterial pathogens from various representative food matrices and environmental samples.

Petri-plate, bacteria, and laser optical scattering sensor.

Classical microbiology has paved the path forward for the development of modern biotechnology and microbial biosensing platforms. Microbial culturing and isolation using the Petri plate revolutionized the field of microbiology. In 1887, Julius Richard Petri invented possibly the most important tool in microbiology, the Petri plate, which continues to have a profound impact not only on reliably isolating, identifying, and studying microorganisms but also manipulating a microbe to study gene expression, virulence properties, antibiotic resistance, and production of drugs, enzymes, and foods. Before the recent advances in gene sequencing, microbial identification for diagnosis relied upon the hierarchal testing of a pure culture isolate. Direct detection and identification of isolated bacterial colonies on a Petri plate with a sensing device has the potential for revolutionizing further development in microbiology including gene sequencing, pathogenicity study, antibiotic susceptibility testing , and for characterizing industrially beneficial traits. An optical scattering sensor designated BARDOT (bacterial rapid detection using optical scattering technology) that uses a red-diode laser, developed at the beginning of the 21st century at Purdue University, some 220 years after the Petri-plate discovery can identify and study bacteria directly on the plate as a diagnostic tool akin to Raman scattering and hyperspectral imaging systems for application in clinical and food microbiology laboratories.

Receptor-targeted engineered probiotics mitigate lethal Listeria infection.

Probiotic bacteria reduce the intestinal colonization of pathogens. Yet, their use in preventing fatal infection caused by foodborne Listeria monocytogenes (Lm), is inconsistent. Here, we bioengineered Lactobacillus probiotics (BLP) to express the Listeria adhesion protein (LAP) from a non-pathogenic Listeria (L. innocua) and a pathogenic Listeria (Lm) on the surface of Lactobacillus casei. The BLP strains colonize the intestine, reduce Lm mucosal colonization and systemic dissemination, and protect mice from lethal infection. The BLP competitively excludes Lm by occupying the surface presented LAP receptor, heat shock protein 60 and ameliorates the Lm-induced intestinal barrier dysfunction by blocking the nuclear factor-κB and myosin light chain kinase-mediated redistribution of the major epithelial junctional proteins. Additionally, the BLP increases intestinal immunomodulatory functions by recruiting FOXP3+T cells, CD11c+ dendritic cells and natural killer cells. Engineering a probiotic strain with an adhesion protein from a non-pathogenic bacterium provides a new paradigm to exclude pathogens and amplify their inherent health benefits.

Design and application of a portable luminometer for bioluminescence detection.

The silicon photomultiplier (SiPM) for low light detection has many advantages when compared to existing photon counting detectors, such as high sensitivity, low cost, robustness, and compact hardware. To facilitate the use of SiPM as a portable, field deployable device, an electrical circuit was designed consisting of an amplifier, comparator, and microcontroller. In addition, a 3D printing was used to create a portable cradle for housing the SiPM. To evaluate its detection ability, a laser experiment and bioluminescent experiments, including Pseudomonas fluorescens M3A detection, E. coli O157:H7 PhiV10nluc lysogen detection, and a luminescence-based detection of E. coli O157:H7 in ground meat using the engineered luminescent-based reporter phage PhiV10nluc, were conducted. In the same experimental setting, our previously developed smartphone-based luminometer called the bioluminescent-based analyte quantitation by smartphone and a conventional photomultiplier tube-based benchtop luminometer were used to compare detection levels and applicability for supporting luminescent phage-based pathogen detection. Results showed that the SiPM provides better performance in terms of time to detection and SNR and could be used as the light detection component of the PhiV10nluc phage-based detection format.

The effect of a novel low temperature-short time (LTST) process to extend the shelf-life of fluid milk.

Pasteurization has long been the standard method to extend the shelf-life of dairy products, as well as a means to reduce microbial load and the risk of food-borne pathogens. However, the process has limitations, which include cost effectiveness, high energy input, and reduction of product quality/organoleptic characteristics. In an effort to reduce these limitations and extend shelf-life, this study examined a novel low temperature, short time (LTST) method in which dispersed milk in the form of droplets was treated with low heat/pressure variation over a short treatment time, in conjunction with pasteurization. Lactobacillus fermentum and Pseudomonas fluorescens Migula were exposed to conventional pasteurization treatments with and without LTST. Using these organisms, the LTST addition was able to reduce microbial load below detection limits; 1.0 × 10(1) cfu/mL, from approximately 1.2 × 10(8) and 1.0 × 10(7) cfu/mL for L. fermentum and P. fluorescens Migula, respectively. In addition, the shelf-life of the treated, raw, and uninoculated product was prolonged from 14 to 35 days, compared with standard pasteurization, to as long as 63 days with the LTST amendment. Sensory analysis of samples also demonstrated equal or greater preference for LTST + pasteurization treated milk when compared to pasteurization alone (α = 0.05). Conventional pasteurization was effective at reducing the above mentioned microorganisms by as much as 5.0 log10 cfu/mL. However, LTST was able to achieve 7.0-8.0 log10 cfu/mL reduction of the same microorganisms. In addition, BActerial Rapid Detection using Optical scattering Technology detected and identified microorganisms isolated both pre- and post-treatment, of which the only organisms surviving LTST were Bacillus spp. Increased lethality, improved shelf-life, and equal or better organoleptic characteristics without increased energy consumption demonstrate the effectiveness of the incorporation of LTST. The improved shelf-life may potentially have major impacts in the dairy industry in terms of shipping and overall sustainability.

Effect of steric hindrance on the properties of antibacterial and biocompatible copolymers.

The development of polymers that are both bactericidal and biocompatible would have many applications and are currently of substantial research interest. It is well known that polymers of alkyl-quaternized poly(4-vinylpyridine) are known to be effective against a wide range of microbes: when copolymerized with monomers that form biocompatible materials, they has also been shown to possess biocompatible properties. However, the relationship of the various physical and chemical properties of these polymers and copolymers with the antibacterial and biocompatible properties remains poorly understood: maximizing the selectivity and performance of these materials is absolutely needed before they have the potential for commercial applications. Maximizing the performance will require a complete understanding of the effect of physical and chemical adjustments on these quaternized polymer bactericides. This article seeks to explore and characterize the effect of one specific property, steric hindrance, on the copolymers' antibacterial and biocompatible properties. We have thus synthesized and characterized a new class of copolymers from 2-vinylpyridine and poly(ethylene glycol) methyl ether methacrylate, measured its bactericidal and biocompatible properties, and compared its performance to chemically similar but sterically different polymer bactericides. This work thereby enables both a greater understanding of the properties of the 2-vinylpyridine copolymers and an improved understanding of the material properties that are vital for the development of antibacterial polymers.

Structure-activity relationships of antibacterial and biocompatible copolymers.

The development of polymers that are both bactericidal and biocompatible would have many applications and are currently of research interest. Following the development of strongly bactericidal copolymers of 4-vinylpyridine and poly(ethylene glycol) methyl ether methacrylate, biocompatibility assays have been completed on these materials to measure their potential biocompatibility. In this article, a new methodology for measuring protein interaction was developed for water-soluble polymers by coupling proteins to surfaces and then measuring the adsorption of copolymers onto these surfaces. Ellipsometry was then used to measure the thickness of adsorbed polymers as a measurement of biocompatibility. These results were then compared and correlated with the results of other biocompatibility assays previously conducted on these polymers, affording a greater understanding of the biocompatibility of the copolymers as well as improving the understanding of the effect of hydrophilic and hydrophobic groups that is vital for the development of these materials.

Application of a high throughput bioluminescence-based method and mathematical model for the quantitative comparison of polymer microbicide efficiency.

Quaternized poly(4-vinyl pyridine)-based copolymers are known to be effective against a wide range of bacteria and possess biocompatible properties. Extensive testing of a wide range of copolymers is necessary to further explore and enhance the biocidal properties. However, testing is hampered by labor-intensive bacteria testing techniques. The present paper presents a new testing method, based on bioluminescent reporter strains to enable fast evaluation of bactericidal properties. The reported method enables us to create real-time characterization of bacteria behavior with far less labor than required through traditional testing methods. A mathematical model was also developed to characterize the change in bacteria populations exposed to biocides and to enable the quantitative comparison of minimum bactericidal concentrations.

Sequence analysis of Escherichia coli O157:H7 bacteriophage PhiV10 and identification of a phage-encoded immunity protein that modifies the O157 antigen.

Bacteriophage PhiV10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the PhiV10 genome is 39 104 bp long and contains 55 predicted genes. PhiV10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage epsilon15 and a prophage from E. coli APEC O1. The attachment site of PhiV10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. PhiV10 encodes an O-acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block PhiV10 superinfection, indicating that the O157 antigen is most likely the PhiV10 receptor.

Chlorine dioxide oxidation of dihydronicotinamide adenine dinucleotide (NADH).

The oxidation of dihydronicotinamide adenine dinucleotide (NADH) by chlorine dioxide in phosphate buffered solutions (pH 6-8) is very rapid with a second-order rate constant of 3.9 x 10(6) M(-1) s(-1) at 24.6 degrees C. The overall reaction stoichiometry is 2ClO2(*) per NADH. In contrast to many oxidants where NADH reacts by hydride transfer, the proposed mechanism is a rate-limiting transfer of an electron from NADH to ClO2(*). Subsequent sequential fast reactions with H(+) transfer to H2O and transfer of an electron to a second ClO2(*) give 2ClO2(-), H3O(+), and NAD(+) as products. The electrode potential of 0.936 V for the ClO2(*)/ClO2(-) couple is so large that even 0.1 M of added ClO2(-) (a 10(3) excess over the initial ClO2(*) concentration) fails to suppress the reaction rate.

Hemocompatibility of hydrophilic antimicrobial copolymers of alkylated 4-vinylpyridine.

Quaternized poly(vinylpyridine) (PVP) is a polymer with inherent antimicrobial properties that is effective against Gram-positive bacteria, Gram-negative bacteria, viruses, and yeast cells. However, quaternized PVP has poor biocompatibility, which prevents its use in biomaterial applications. Copolymerization was examined as a method of modifying the structure to incorporate biocompatibility. Polyethyleneglycol methyl ether methacrylate (PEGMA) and hydroxyethyl methacrylate (HEMA) are polymers generally known to be biocompatible and thus were chosen as comonomers. Random copolymers of 4-vinylpyridine and PEGMA or HEMA were synthesized via free radical polymerization and quaternized with bromohexane. Copolymer biocompatibility was characterized by interaction with human red blood cells to analyze hemolysis. Hemolysis of human red blood cells was conducted on insoluble films and on water-soluble polymers in a serial dilution study. Hemolysis results demonstrated that blood compatibility does not depend on PEG chain length in PEGMA incorporated copolymers. Results indicate a critical weight ratio of PEGMA to VP in copolymers separating the no-hemolysis regime from 100% hemolysis.

Synergistic activity of hydrophilic modification in antibiotic polymers.

Quaternized poly(vinylpyridine) is known to kill up to 99% of drug-resistant gram-positive and -negative bacteria but shows minimal biocompatibility. We report enhanced bactericidal activity of vinylpyridine through copolymerization with hydroxyethyl methacrylate and poly(ethylene gycol) methyl ether methacrylate. Copolymers with increasing comonomer content were synthesized by radical polymerization and quaternized with hexylbromide. We assessed the effects of the changes in polymer composition on the bactericidal activity of the surface activity using a bioluminescent pathogenic strain of Escherichia coli (O157:H7). By recording the photoluminescence emitted by these bacteria in contact with the copolymers, it was shown that several of the copolymers possess better antibacterial efficiency than quaternized poly(vinylpyridine). Results indicate that several of the copolymers synthesized possess antibacterial activity approximately 20 times greater than the pure quaternized poly(vinylpyridine) homopolymer, while only containing 1 wt % hexylated pyridinium. This behavior is explained by the increased surface wettability of the copolymers containing lesser amounts of poly(vinylpyridine), as bactericidal behavior correlates to the hydrophilicity of the system as measured by contact angles. A hydrophilicity based design-paradigm can significantly improve both the efficacy and the biocompatibility of antibacterial materials.

A molecular beacon DNA microarray system for rapid detection of E. coli O157:H7 that eliminates the risk of a false negative signal.

A DNA hybridization based optical detection platform for the detection of foodborne pathogens has been developed with virtually zero probability of the false negative signal. This portable, low-cost and real-time assaying detection platform utilizes the color changing molecular beacon as a probe for the optical detection of the target sequence. The computer-controlled detection platform exploits the target hybridization induced change of fluorescence color due to the Förster (fluorescence) resonance energy transfer (FRET) between a pair of spectrally shifted fluorophores conjugated to the opposite ends of a beacon (oligonucleotide probe). Unlike the traditional fluorophore-quencher beacon design, the presence of two fluorescence molecules allows to actively visualize both hybridized and unhybridized states of the beacon. This eliminates false negative signal detection characteristic for the fluorophore-quencher beacon where bleaching of the fluorophore or washout of a beacon is indistinguishable from the absence of the target DNA sequence. In perspective, the two-color design allows also to quantify the concentration of the target DNA in a sample down to < =1 ng/microl. The new design is suitable for simultaneous reliable detection of hundreds of DNA target sequences in one test run using a series of beacons immobilized on a single substrate in a spatial format.

Molecular characterization of autoinduction of bioluminescence in the Microtox indicator strain Vibrio fischeri ATCC 49387.

Repeated attempts to clone the luxI from Vibrio fischeri ATCC 49387 failed to produce a clone carrying a functional LuxI. Sequence data from the clones revealed the presence of a polymorphism when compared with previously published luxI sequences, prompting further characterization of bioluminescence regulation in V. fischeri ATCC 49387. Further investigation of V. fischeri ATCC 49387 revealed that its LuxI protein lacks detectable LuxI activity due to the presence of a glutamine residue at position 125 in the deduced amino acid sequence. Specific bioluminescence in V. fischeri ATCC 49387 increases with increasing cell density, indicative of a typical autoinduction response. However, conditioned medium from this strain does not induce bioluminescence in an ATCC 49387 luxR-plux-based acyl homoserine lactone reporter strain, but does induce bioluminescence in ATCC 49387. It has been previously shown that a V. fischeri MJ-1 luxI mutant exhibits autoinduction of bioluminescence through N-octanoyl-L-homoserine lactone, the product of the AinS autoinducer synthase. However, a bioreporter based on luxR-plux from V. fischeri ATCC 49387 responded poorly to conditioned medium from V. fischeri ATCC 49387 and also responded poorly to authentic N-octanoyl-DL-homoserine lactone. A similar MJ-1-based bioreporter showed significant induction under the same conditions. A putative ainS gene cloned from ATCC 49387, unlike luxI from ATCC 49387, expresses V. fischeri autoinducer synthase activity in Escherichia coli. This study suggests that a regulatory mechanism independent of LuxR and LuxI but possibly involving AinS is responsible for the control of autoinduction of bioluminescence in V. fischeri ATCC 49387.