Development of a Bacteriophage-based Portable Biosensor for the Detection of Shiga-toxin Producing Escherichia coli (STEC) Strains in Food and Environmental Matrices

A fast and reliable on-site foodborne pathogens screening can reduce the incidence of foodborne illnesses, hospitalizations and economic loss. It can also circumvent conventional laboratory-based tests with minimal sample treatments and shorter turnaround time. Rapid detection of biological hazards has been largely dependent on immunological agents (ie antibodies). Antibodies are expensive to manufacture and experience cross-reactivity, instability with shorter shelf life. Our aim was to improve the screening process of Shiga-toxin producing Escherichia coli (STEC) strains in food and environmental matrices by developing a novel, inexpensive handheld bacteriophage-based amperometric biosensor that can directly detect live STEC cells.This biosensor development began by isolating STEC-specific bacteriophages from natural environmental samples (ie cow manure and surface water) hence, constructing a comprehensive bacteriophage isolates collection targeting an array of significant STEC serogroups. As an alternative to antibodies, purified bacteriophages could be easily and inexpensively propagated in a standard laboratory. Isolated bacteriophages were morphologically characterized while its physiologic behavior and specific host interactions were also investigated. The results indicated that majority of STEC-specific bacteriophages belong to Myoviridae and Siphoviridae families. Suitable bacteriophages for biosensor purposes were selected on the basis of the presence of head and tail and absence of virulence genes (stx1/stx2). Chemical modification via site-specific biotinylation of bacteriophage heads was performed prior to its biosensor incorporation. The results showed that biotinylation of bacteriophages did not reduce biofunctionality. Representative STEC O26, O157, and O179-specific biotinylated bacteriophages were immobilized onto the surface of streptavidin-modified screen-printed carbon electrodes (SPCE) to capture their target STEC cells. After STEC cells were bound to the capture elements, another set of biotinylated bacteriophages labeled with streptavidivin-horseradish peroxidase were added forming stable binding complexes which were then subjected to amperometric detection. The sandwich-type bacteriophage-based detection approach allowed live STEC cells rapid detection in microvolume samples (50 µL) via amperometric readouts (∆ current) between target and non-target bacteria in pure culture setup and complex matrices. With its simplicity and reliability, this technology can immensely assist the food industry and regulatory inspectors to efficiently maintain food safety in a fraction of the cost of traditional method

Simultaneous Colorimetric Detection of a Variety of Salmonella spp. in Food and Environmental Samples by Optical Biosensing Using Oligonucleotide-Gold Nanoparticles

Optical biosensors for rapid detection of significant foodborne pathogens are steadily gaining popularity due to its simplicity and sensitivity. While nanomaterials such as gold nanoparticles (AuNPs) are commonly used as signal amplifiers for optical biosensors, AuNPs can also be utilized as a robust biosensing platform. Many reported optical biosensors were designed for individual pathogen detection in a single assay and have high detection limit (DL). Salmonella spp. is one of the major causative agents of foodborne sickness, hospitalization and deaths. Unfortunately, there are around 2,000 serotypes of Salmonella worldwide, and rapid and simultaneous detection of multiple strains in a single assay is lacking. In this study, a comprehensive and highly sensitive simultaneous colorimetric detection of nineteen (19) environmental and outbreak Salmonella spp. strains was achieved by a novel optical biosensing platform using oligonucleotide-functionalized AuNPs. A pair of newly designed single stranded oligonucleotides (30-mer) was displayed onto the surface of AuNPs (13 nm) as detection probes to hybridize with a conserved genomic region (192-bases) of ttrRSBCA found on a broad range of Salmonella spp. strains. The sandwich hybridization (30 min, 55°C) resulted in a structural formation of highly stable oligonucleotide/AuNPs-DNA complexes which remained undisturbed even after subjecting to an increased salt concentration (2 M, final), thus allowing a direct discrimination via color change of target (red color) from non-target (purplish-blue color) reaction mixtures by direct observation using the naked eye. In food matrices (blueberries and chicken meat), nineteen different Salmonella spp. strains were concentrated using immunomagnetic separation and then simultaneously detected in a 96-well microplate by oligonucleotide-functionalized AuNPs after DNA preparation. Successful oligonucleotide/AuNPs-DNA hybridization was confirmed by gel electrophoresis while AuNPs aggregation in non-target and control reaction mixtures was verified by both spectrophotometric analysis and TEM images. Results showed that the optical AuNP biosensing platform can simultaneously screen nineteen (19) viable Salmonella spp. strains tested with 100% specificity and a superior detection limit of <10 CFU/mL or g for both pure culture and complex matrices setups. The highly sensitive colorimetric detection system can significantly improve the screening and detection of viable Salmonella spp. strains present in complex food and environmental matrices, therefore reducing the risks of contamination and incidence of foodborne diseases

Advances, applications, and limitations of portable and rapid detection technologies for routinely encountered foodborne pathogens

Traditional foodborne pathogen detection methods are highly dependent on pre-treatment of samples and selective microbiological plating to reliably screen target microorganisms. Inherent limitations of conventional methods include longer turnaround time and high costs, use of bulky equipment, and the need for trained staff in centralized laboratory settings. Researchers have developed stable, reliable, sensitive, and selective, rapid foodborne pathogens detection assays to work around these limitations. Recent advances in rapid diagnostic technologies have shifted to on-site testing, which offers flexibility and ease-of-use, a significant improvement from traditional methods’ rigid and cumbersome steps. This comprehensive review aims to thoroughly discuss the recent advances, applications, and limitations of portable and rapid biosensors for routinely encountered foodborne pathogens. It discusses the major differences between biosensing systems based on the molecular interactions of target analytes and biorecognition agents. Though detection limits and costs still need further improvement, reviewed technologies have high potential to assist the food industry in the on-site detection of biological hazards such as foodborne pathogens and toxins to maintain safe and healthy foods. Finally, this review offers targeted recommendations for future development and commercialization of diagnostic technologies specifically for emerging and re-emerging foodborne pathogens

Discovery of Shiga Toxin-Producing Escherichia coli (STEC)-Specific Bacteriophages From Non-fecal Composts Using Genomic Characterization

Composting is a complex biodegradable process that converts organic materials into nutrients to facilitate crop yields, and, if well managed, can render bactericidal effects. Majority of research focused on detection of enteric pathogens, such as Shiga toxin-producing Escherichia coli (STEC) in fecal composts. Recently, attention has been emphasized on bacteriophages, such as STEC-specific bacteriophages, associated with STEC from the fecal-contaminated environment because they are able to sustain adverse environmental condition during composting process. However, little is known regarding the isolation of STEC-specific bacteriophages in non-fecal composts. Thus, the objectives were to isolate and genomically characterize STEC-specific bacteriophages, and to evaluate its association with STEC in non-fecal composts. For bacteriophage isolation, the samples were enriched with non-pathogenic E. coli (3 strains) and STEC (14 strains), respectively. After purification, host range, plaque size, and phage morphology were examined. Furthermore, bacteriophage genomes were subjected to whole-genome sequencing using Illumina MiSeq and genomic analyses. Isolation of top six non-O157 and O157 STEC utilizing culture methods combined with PCR-based confirmation was also conducted. The results showed that various STEC-specific bacteriophages, including vB_EcoM-Ro111lw, vB_EcoM-Ro121lw, vB_EcoS-Ro145lw, and vB_EcoM-Ro157lw, with different but complementary host ranges were isolated. Genomic analysis showed the genome sizes varied from 42kb to 149kb, and most bacteriophages were unclassified at the genus level, except vB_EcoM-Ro111lw as FelixO1-like viruses. Prokka predicted less than 25% of the ORFs coded for known functions, including those essential for DNA replication, bacteriophage structure, and host cell lysis. Moreover, none of the bacteriophages harbored lysogenic genes or virulence genes, such as stx or eae. Additionally, the presence of these lytic bacteriophages was likely attributed to zero isolation of STEC and could also contribute to additional antimicrobial effects in composts, if the composting process was insufficient. Current findings indicate that various STEC-specific bacteriophages were found in the non-fecal composts. In addition, the genomic characterization provides in-depth information to complement the deficiency of biological features regarding lytic cycle of the new bacteriophages. Most importantly, these bacteriophages have great potential to control various serogroups of STEC

The morphology of the STEC-specific phages isolated from Salinas area.

<p>(A) O157-specific bacteriophage, (B) O145-specific bacteriophage, (C) O45-specific bacteriophage, (D) O179-specific bacteriophage.</p

Samples with positive of STEC-specific phages and STEC strains throughout sampling period displayed by sample month.

<p>Samples with positive of STEC-specific phages and STEC strains throughout sampling period displayed by sample month.</p

Shiga toxin-producing <i>E</i>. <i>coli</i> (STEC) strains isolated from different sources by U.S. Department of Agriculture ARS used for free STEC-specific phage isolation.

<p>Shiga toxin-producing <i>E</i>. <i>coli</i> (STEC) strains isolated from different sources by U.S. Department of Agriculture ARS used for free STEC-specific phage isolation.</p

Geographical location of the watershed sites where the samples were collected in the area of Salinas Valley.

<p>Sample sites are labeled with a six-letter acronym in black type (locations with agriculture impact) or red type (locations with human impact). Insert is an expanded view of the area near the city of Salinas.</p

Summary of phage and STEC isolation data from water samples collected from Salinas, CA.

<p>The acronym is used for each sample site with the number of water samples collected. Red oval shape with red letter indicates isolation of STEC-specific phage, and green rectangular shape with green letters indicates STEC bacterial isolation.</p