Novel Method To Identify Source-Associated Phylogenetic Clustering Shows thatListeria monocytogenesIncludes Niche-Adapted Clonal Groups with Distinct Ecological Preferences

Abstract

While phylogenetic and cluster analyses are often used to define clonal groups within bacterial species, the identification of clonal groups that are associated with specific ecological niches or host species remains a challenge. We used Listeria monocytogenes, which causes invasive disease in humans and different animal species and which can be isolated from a number of environments including food, as a model organism to develop and implement a two-step statistical approach to the identification of phylogenetic clades that are significantly associated with different source populations, including humans, animals, and food. If the null hypothesis that the genetic distances for isolates within and between source populations are identical can be rejected (SourceCluster test), then particular clades in the phylogenetic tree with significant overrepresentation of sequences from a given source population are identified (TreeStats test). Analysis of sequence data for 120 L. monocytogenes isolates revealed evidence of clustering between isolates from the same source, based on the phylogenies inferred from actA and inlA (P ‫؍‬ 0.02 and P ‫؍‬ 0.07, respectively; SourceCluster test). Overall, the TreeStats test identified 10 clades with significant (P < 0.05) or marginally significant (P < 0.10) associations with defined sources, including human-, animal-, and food-associated clusters. Epidemiological and virulence phenotype data supported the fact that the source-associated clonal groups identified here are biologically valid. Overall, our data show that (i) the SourceCluster and TreeStats tests can identify biologically meaningful source-associated phylogenetic clusters and (ii) L. monocytogenes includes clonal groups that have adapted to infect specific host species or colonize nonhost environments.Recent advances in DNA sequencing technologies have made DNA sequence-based subtyping methods such as multilocus sequence typing (MLST) widely available and affordable (2, 4), and as a result, a rich source of data for evolutionary and population genetics analyses of pathogenic and nonpathogenic bacteria is available (30). Traditional phylogenetic methods are frequently used to re-create the evolutionary history of a group of bacterial isolates and identify clonal groups of isolates that share a recent common ancestor. These methods also allow preliminary identification of clonal groups that show obvious associations with specific ecological niches, hosts, or disease symptoms in affected hosts. For example, MLST studies with Campylobacter jejuni showed that some clonal complexes within this species are exclusively or predominantly associated with specific host populations, including clonal complexes that were associated with human hosts and specific animal host species, such as sheep or poultry (5, 6). Similarly, Luan et al. (13) used MLST analysis to describe a specific clonal complex among invasive group B Streptococcus isolates which was associated with neonatal infections but which rarely caused invasive disease in adult...

Prevalence and Distribution of Listeria monocytogenes inlA Alleles Prone to Phase Variation and inlA Alleles with Premature Stop Codon Mutations among Human, Food, Animal, and Environmental Isolates

“…While all lineage I and lineage III isolates characterized to date carry the A 2 GA 4 allele (28,29), only a few lineage II strains carry the A 2 GA 4 allele; the majority of lineage II strains carry the A 7 or A 6 allele. Collectively, the observations from these studies suggest that (i) disease-associated (i.e., lineage I and III) L. monocytogenes strains have evolved to carry an A 2 GA 4 allele that facilitates stable expression of the key virulence factor InlA, (ii) generalist strains (i.e., lineage II strains) have evolved a phase variation mechanism for inlA, allowing for selective silencing of inlA in environments where it may not be needed (e.g., food processing environments or foods), and (iii) some lineage II strains have acquired an A 2 GA 4 allele by point mutation or by horizontal gene transfer, suggesting that a subpopulation of lineage II strains have adapted to a disease-associated lifestyle, consistent with reports of lineage II isolates being identified from human and animal disease cases (30).…”

Significant Shift in Median Guinea Pig Infectious Dose Shown by an Outbreak-AssociatedListeria monocytogenesEpidemic Clone Strain and a Strain Carrying a Premature Stop Codon Mutation ininlA

“…Previous molecular epidemiology studies suggested that L. monocytogenes contains at least two distinct subpopulations that differ in virulence, including (i) epidemic clone strains, which have been associated with the majority of listeriosis outbreaks worldwide and which are overrepresented among sporadic cases in the United States, and (ii) strains carrying virulence-attenuating PMSC mutations in inlA, which are common in RTE foods but which are associated with human disease only on very rare occasions (12,14,25,26,27,35,43,44,46,47,48). Current L. monocytogenes risk assessments (8,42,50), which assume that an L. monocytogenes population with nonuniform variation in virulence is present in RTE foods, therefore likely underestimate the virulence of epidemic clone strains and overestimate the virulence of strains carrying a PMSC in inlA.…”

“…Previous molecular epidemiology studies suggest that L. monocytogenes genetic lineages and clonal groups within those lineages differ in their associations with human disease and isolation from foods (12,15,25,26,27,39,40,47,48,49). For example, three highly clonal L. monocytogenes strains within lineage I (termed epidemic clones I, Ia, and II) that belong to serotype 4b have been linked to the majority of listeriosis epidemics worldwide (9,17) and have frequently been isolated from sporadic listeriosis cases in the United States (12).…”

Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum

“…Identification of 16S rDNA sequences associated with different source populations A distance matrix, generated by multiple alignments of 16S rDNA sequences for each isolate using phylogenetic analysis using parsimony (PAUP), was fed into SourceCluster to determine whether isolates from the same group (ICD, CCD, Healthy) clustered by sequence (Nightingale et al, 2006). In order to find specific branches in the phylogenetic tree, in which isolates from a specific group are more abundant or scarce than expected by chance, we used TreeStats.…”