Genomic analysis of high copy-number sequences for the targeted detection of Listeria species using a flow-through surveillance system

Authors: Quinones, Beatriz; Yambao, Jaszemyn; DEGUZMAN, VERONICA - Snapdna; Lee, Bertram; MEDIN, DAVID - Snapdna

Submitted to: Archives of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/17/2021
Publication Date: 6/2/2021
Citation: Quinones, B., Yambao, J.C., Deguzman, V., Lee, B.G., Medin, D. 2021. Genomic analysis of high copy-number sequences for the targeted detection of Listeria species using a flow-through surveillance system. Archives Of Microbiology. 203:3667-3682. https://doi.org/10.1007/s00203-021-02388-2.
DOI: https://doi.org/10.1007/s00203-021-02388-2

Interpretive Summary: Bacterial and viral foodborne pathogens are responsible for a consistent level of human illness that poses a substantial public health and economic burden. Approximately one in six Americans contracts a foodborne illness each year with an estimated annual economic impact of approximately $16 billion. In particular, the human enteric pathogen Listeria monocytogenes is a significant cause of foodborne illness and is responsible for systemic listeriosis with an approximate 30% mortality rate in susceptible populations of pregnant women, neonates, elderly, or immunocompromised patients. Recently, high profile outbreaks of L. monocytogenes have been associated with deli meats, fresh produce, and ready-to-eat foods, and the annual economic impact of listeriosis in the United States is estimated at over $2.8 billion. L. monocytogenes is widespread in the environment; therefore, food production facilities are required to constantly monitor and control for the presence of Listeria species on the surfaces of the equipment at processing facilities. Recent studies have provided evidence that Listeria species can be considered as a broad indicator of the conditions potentially favorable for L. monocytogenes growth and survival in the environment. By using a broad indicator group, such as screening for all Listeria species at the genus level, increases the chances of finding L. monocytogenes niches and reacting in an effective manner to mitigate the prevalence of this pathogen in a food production facility. The primary obstacle to rapid food pathogen analysis today is bacterial enrichment followed by plate culturing on selective media. Depending on which pathogen is targeted for detection, enrichment can take from 8 hours to 24 hours or even longer for incubation times. Further, since culturing is not yet effective for all foodborne pathogens, standard culture-based methods cannot analyze all types of samples. In the present study, a comparative genomic analysis was conducted to examine multicopy sequences in the genome of Listeria species to aid in the development of molecular detection assays. The molecular detection assay was then incorporated with a flow through system that includes a sample preparation process that eliminates the need to culture bacteria. As a proof-of-concept, the flow through system was tested with environmental swab samples to be as representative and challenging as possible from distinct locations at a leafy greens processing facility to aid in the development of an integrated detection platform for the in-process surveillance of Listeria species.

Technical Abstract: The bacterial foodborne pathogen, Listeria monocytogenes, has been significantly implicated in high-profile outbreaks linked to fresh produce. The annual economic impact of listeriosis in the United States is estimated at over $2.8 billion. Given that L. monocytogenes is widespread in the environment, food production facilities constantly monitor and control for the presence of Listeria species on surfaces. To develop and validate an integrated detection platform for the in-process surveillance of foodborne pathogens, the present study conducted a comparative genomic analysis for evaluating high copy-number sequences to enable the targeted and specific detection of Listeria species. By conducting an in-silico analysis with a dynamic programming algorithm, simulated folding was performed to assess the accessibility of multicopy targeted regions in the ribosomal RNA for optimal detection. To reduce any false positives due to low levels of specificity in the oligonucleotide design, the complexity of the targeted regions was also further examined, and the selected sites for the design of the oligonucleotides were found to have an optimal complexity score between 1 and 0.8, indicating relative accessibility and complexity of regions within the targeted sequences. Validation experiments indicated that the probe-based assay had an RNA analytical sensitivity limit of less than 10 fg of Listeria RNA or less than 5 CFU/mL by using crude lysate as template (Fisher’s exact test, p<0.0001). No positive signals were detected when testing non-targeted environmental bacterial strains, such as Bacillus spp., Citrobacter spp. Enterobacter spp., and Pseudomonas spp. As a proof-of-concept for the development of the in-process detection system, a flow through system, consisting of aptamer-capture step, followed by sample concentration and mechanical lysis, was developed for the detection of Listeria species from environmental samples. Experimental results demonstrated that the prototype method achieved the detection of less than 5-10 cells (Fisher’s exact test, p<0.001) from spiked sponge-swab samples collected at a leafy greens processing facility. The supporting evidence revealed this integrated system rapidly detected Listeria at low cell concentrations without pre-enrichment steps from environmental monitoring samples.