Microevolution and Gain or Loss of Mobile Genetic Elements of Outbreak-Related Listeria monocytogenes in Food Processing Environments Identified by Whole Genome Sequencing Analysis

Abstract

Whole genome sequencing (WGS) analyses have been instrumental in traceback investigations of Listeria monocytogenes (Lm). To demonstrate how long-read sequencing analysis can capture and describe relationships among isolates from clinical, food, and environmental sources, we analyzed 366 long-read-and shotgun-sequenced isolates from 16 Lm outbreak strains associated with cantaloupe, leafy green, stone fruit, caramel apple, mung bean sprout, multiple cheese products, multiple ice cream products, and their production environments. The analyses demonstrated that outbreak strains could be distributed in different areas and zones of food production environments through persistent or repeated contamination. Multi-strain and multi-clone contamination were common. Further, WGS could differentiate among isolates collected at different time points or from different production lines in the same facility, revealing microevolution events in processing environments. Our comparison between complete and shotgun genomes showed that isolates of the same outbreak strain diversified mostly by gain/loss of plasmids and chromosome-borne prophages that constitute 2 to 5% of the chromosome. In contrast, other genes missing in the shotgun genomes were randomly scattered, constituting ∼0.5% of the chromosome. Among different outbreak strains of the same CC, most gene-scale differences were due to gain/loss of mobile genetic elements, such as plasmids, chromosome-borne prophages, a Tn916 like transposon, and Listeria Genomic Island 2. The nucleotide variations in the same prophage and the same plasmid shared among isolates of the same outbreak strain were limited, which enabled different WGS tools to unambiguously cluster isolates of the same outbreak strain. In some outbreak strains, correlation between prophage gain/loss and single nucleotide polymorphism (SNP) accumulations in the genome backbone were observed.

Characterization of Mobile Genetic Elements Using Long-Read Sequencing for Tracking Listeria monocytogenes from Food Processing Environments

“…Comparative genomics and evolutionary analysis on a small portion of prophage regions next to the phage insertion sites of the 1998 and 2002 Lm ECII outbreak isolates indicated that the prophage region might be conserved among isolates that were associated with the same outbreak, which represented a short-term evolution scenario [ 7 , 8 ]; these studies did not review the entire prophage regions. A similar phenomenon was observed in isolates from ECs associated with other listeriosis outbreaks that were linked to different types of foods when the entire prophage regions of these isolates were analyzed [ 9 ]. Thus, prophage regions might be important genetic markers to determine epidemiological relatedness and to identify resident strains.…”

“…The mutation rate on 33 CC321 strains from lineage II was 1.15 × 10 −7 (95% HPD, 0.79 × 10 −7 to 1.52 × 10 −7 ) substitutions/site/year [ 34 ]. In another study that calculated substitution rates among isolates associated with specific outbreaks, the average substitution rates were slightly higher, 5.5 × 10 −7 to 5.8 × 10 −7 substitutions/site/year for CC6 isolates associated with two different outbreaks linked to contaminated cheese products [ 9 ]. In our study, the time between the most recent common ancestor and the outbreak recognition was 30 and 52 years for the 1998 and 2002 U.S. outbreak, respectively.…”

“…In our study, the time between the most recent common ancestor and the outbreak recognition was 30 and 52 years for the 1998 and 2002 U.S. outbreak, respectively. It was two years for the South African outbreak, and it was three and four years for the two previously analyzed cheese-associated outbreaks caused by ECII or CC6 isolates [ 9 ]. However, we have to interpret these results with caution because the selection of isolates would impact the estimate of the time of emergence.…”

“…Plasmid-borne resistance genes, such as bcrABC which confers resistance to BC, were previously reported on a putative transposon harbored by a plasmid of H7550, a 1998 listeriosis outbreak isolate, suggesting evidence of a role for MGEs contributing to adaptation and persistence of Lm strains in the meat or poultry processing facilities [ 12 ]. The gene cassette, bcrABC , was also observed in plasmids of multiple isolates from a 2017 outbreak associated with ice cream in Florida, but the cassette was not observed in plasmids of strains from other listeriosis outbreaks analyzed in that study [ 9 ]. Other plasmid-borne resistance genes, such as qacA, qacC, emrE, and emrC, were observed in plasmids of Lm strains isolated from the retail environment in the U.S. [ 15 , 16 ].…”

Harnessing Whole Genome Sequence Data for Facility-Specific Signatures for Listeria monocytogenes: A Case Study With Turkey Processing Plants in the United States

“…Such persistence is mediated by several adaptations, including the ability to form persistent biofilms, tolerance to sanitizers such as quaternary ammonium compounds, growth at low temperatures, and phage resistance (Kathariou, 2002;Gandhi and Chikindas, 2007;Kim et al, 2008;Carpentier and Cerf, 2011;Kathariou et al, 2017). The increasing employment of whole genome sequencing (WGS) has significantly facilitated the assessment of L. monocytogenes persistence in individual food processing plants, greatly improving the resolution afforded by previous tools such as pulsed-field gel electrophoresis or the seven-locus multilocus sequence typing (MLST) scheme (Fagerlund et al, 2016(Fagerlund et al, , 2020Knudsen et al, 2017;Hurley et al, 2019;Stoller et al, 2019;Harrand et al, 2020;Yang et al, 2020).…”