Determination of Evolutionary Relationships of Outbreak-Associated Listeria monocytogenes Strains of Serotypes 1/2a and 1/2b by Whole-Genome Sequencing

Abstract

e We used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates of Listeria monocytogenes serotypes 1/2a and 1/2b. Isolates from 6 of 11 outbreaks fell outside the clonal groups or "epidemic clones" that have been previously associated with outbreaks, suggesting that epidemic potential may be widespread in L. monocytogenes and is not limited to the recognized epidemic clones. Pairwise comparisons between epidemiologically related isolates within clonal complexes showed that genome-level variation differed by 2 orders of magnitude between different comparisons, and the distribution of point mutations (core versus accessory genome) also varied. In addition, genetic divergence between one closely related pair of isolates from a single outbreak was driven primarily by changes in phage regions. The evolutionary analysis showed that the changes could be attributed to horizontal gene transfer; members of the diverse bacterial community found in the production facility could have served as the source of novel genetic material at some point in the production chain. The results raise the question of how to best utilize information contained within the accessory genome in outbreak investigations. The full magnitude and complexity of genetic changes revealed by genome sequencing could not be discerned from traditional subtyping methods, and the results demonstrate the challenges of interpreting genetic variation among isolates recovered from a single outbreak. Epidemiological information remains critical for proper interpretation of nucleotide and structural diversity among isolates recovered during outbreaks and will remain so until we understand more about how various population histories influence genetic variation. Listeria monocytogenes is a bacterial pathogen that is almost exclusively transmitted by food. Invasive listeriosis typically presents as sepsis or meningoencephalitis in older adults, those with certain chronic illnesses, and people undergoing immunosuppression. Infections during pregnancy can cause fever and other nonspecific symptoms in the mother with severe outcomes such as fetal loss, premature labor, and neonatal illness and death. Although listeriosis is relatively rare (ϳ1,600 cases occur annually in the United States), approximately 20% of cases are fatal and outbreaks are not uncommon. There are 13 known serotypes of L. monocytogenes, though the majority of human illnesses are caused by serotypes 1/2a, 1/2b, and 4b (1, 2). Molecular subtyping methods have differentiated L. monocytogenes isolates into 4 distinct genetic lineages, with isolates of serotypes 4b and 1/2b typically belonging to lineage I (LI) and isolates of serotype 1/2a typically belonging to lineage II (LII) (3). Strains of lineages III and IV rarely cause listeriosis in humans. Historically, isolates of serotype 4b have caused the greatest proportion of listeriosis outbreaks and the largest number of cases per outbreak (2). In 2011, however, serotypes 1/2a and 1/2b were implic...

Implementation of Nationwide Real-time Whole-genome Sequencing to Enhance Listeriosis Outbreak Detection and Investigation

“…Although PFGE has proved remarkably useful in detecting listeriosis clusters, it lacks the discriminatory capacity and phylogenetic basis of more advanced methods [13]. Genome insertions, deletions, rearrangement, and point mutations at a restriction enzyme site can cause Lm isolates that are highly related genetically to appear different by PFGE.…”

Looking Backward To Move Forward: the Utility of Sequencing Historical Bacterial Genomes

“…A subsequent study used whole-genome sequencing to determine the evolutionary relationships among 20 outbreak-associated clinical isolates of Listeria monocytogenes (36). For placement of these 20 genomes into the larger context of L. monocytogenes genetic diversity, other isolates were included in the study; of particular interest were 9 SLCC isolates from 1924 to 1983.…”

“…For placement of these 20 genomes into the larger context of L. monocytogenes genetic diversity, other isolates were included in the study; of particular interest were 9 SLCC isolates from 1924 to 1983. The use of these isolates allowed an L. monocytogenes phylogeny to be constructed that clustered the 20 genomes into 10 distinct phylogenetic groups (36). From this study, an evolutionary framework was generated to better understand how L. monocytogenes outbreak-associated serotypes 1/2a and 1/2b were related to one another and to other clones.…”

Genome-Wide Profiling of Enterotoxigenic Staphylococcus aureus Strains Used for the Production of Naturally Contaminated Cheeses

“…Whole genome sequencing (WGS) provides a comprehensive view of the genome structure and gene content of microorganisms [14][15][16], and is now gradually replacing well-recognised, standardised protocols like PulseNet for its higher sensitivity, specificity, and inter-laboratory comparability, as well as timely resolution when compared with traditional molecular methods [17,18]. WGS has been applied for genomic comparison of microorganisms associated with foodborne outbreaks and for surveillance of high-burden diseases for almost a decade [15], allowing the identification of pathogens in the foods responsible for the infections [15,[19][20][21]. In fact, precise gene identifications and detailed virulence portraits can be determined in a single session of sequencing, while bioinformatics analysis for foodborne pathogens provides valuable resources for databases and collections of genomic features [22].…”