Combined Ribotyping and Random Multiprimer DNA Analysis To Probe the Population Structure of Listeria monocytogenes

Abstract

To improve our understanding of the genetic links between strains originating from food and strains responsible for human diseases, we studied the genetic diversity and population structure of 130 epidemiologically unrelated Listeria monocytogenes strains. Strains were isolated from different sources and ecosystems in which the bacterium is commonly found. We used rRNA gene restriction fragment length polymorphism analysis with two endonucleases and random multiprimer DNA analysis with seven oligonucleotide primers to study multiple genetic features of each strain. We used three clustering methods to identify genetic links between individual strains and to determine the precise genetic structure of the population. The combined results confirmed that L. monocytogenes strains can be divided into two major phylogenetic divisions. The method used allowed us to demonstrate that the genetic structure and diversity of the two phylogenetic divisions differ. Division I is the most homogeneous and can easily be divided into subgroups with dissimilarity distances of less than 0.30. Each of these subgroups mainly, or exclusively, contains a single serotype (1/2b, 4b, 3b, or 4a). The serotype 4a lineage appears to form a branch that is highly divergent from the phylogenetic group containing serotypes 1/2b, 4b, and 3b. Division II contains strains of serotypes 1/2a, 1/2c, and 3a. It exhibits more genetic diversity with no peculiar clustering. The fact that division II is more heterogeneous than division I suggests that division II evolved from a common ancestor earlier than division I. A significant association was found between division I and human strains, suggesting that strains from division I are better adapted to human hosts. Listeria monocytogenes was first described by Murray in 1926 in guinea pigs and was recognized as a human pathogen over 70 years ago. Its clinical manifestations include meningitis, meningoencephalitis, bacteremia (27), and occasionally localized infections (16). Listeriosis is particularly dangerous for immunocompromised people and pregnant women, as fetuses may be affected by perinatal listeriosis (23). It is now known that humans become infected after ingesting contaminated food products (10). The expansion of the agro-food industry, together with the use of cold storage systems, has led to the contamination of a wide variety of foods with listeriae (10), resulting in large food-borne outbreaks. However, sporadic listeriosis remains the most frequent manifestation of the illness (2).Although we have a thorough understanding of the virulence of the bacterium and the physiopathology of the illness, the epidemiology of human listeriosis is not fully understood (26). All 13 L. monocytogenes serotypes can cause human listeriosis, but serotypes 1/2a, 1/2b, and 4b account for 95% of the cases that occur (22). The differential prevalence of these serotypes and the absence of clear links between particular forms of listeriosis and certain serotypes may be explained by studying the genetic structure of th...

Mixed-Genome Microarrays Reveal Multiple Serotype and Lineage-Specific Differences among Strains of Listeria monocytogenes

“…A recent analysis of a substracted genomic library demonstrated three unique hybridization patterns for serotype 1/2a (18). The findings in this latter report and a recent report by Mereghetti et al (22) are consistent with our findings of three clusters for serotype 1/2a.…”

“…BvrB is part of a locus that represses prfAdependent genes in a soil environment (4,32). Epidemiology data suggest that not all strains of L. monocytogenes are equally virulent in humans and that there may be strains that are more virulent in other animals (22,25,36). A number of our polymorphic probes have high BLASTx matches for gene products that may allow enhanced survival and virulence in the mammalian host.…”

“…The dendrogram (Fig. 1) illustrates two phylogenetic divisions that correspond to findings from multilocus enzyme electrophoresis (2, 25), pulsed-field gel electrophoresis (5), ribotyping (16), randomly amplified polymorphic DNA (22), and sequencing (11,34). Classification of isolates into these two divisions is confirmed both by serotyping and by a PCR-based classification system (Table 1) (19).…”

“…There are 13 recognized serotypes of L. monocytogenes, and most human infections involve serotypes 1/2a, 1/2b, and 4b, with the latter being associated with the largest food-borne outbreaks (25,34). Two major phylogenetic divisions clearly differentiate between the most commonly isolated L. monocytogenes serotypes (serotypes 1/2a and 1/2c versus 1/2b and 4b) (22,25). Analysis of putative virulence genes suggests that the evolution of somatic and flagellar antigens has paralleled the evolution of genes associated with virulence (11,33).…”

Pulsed-Field Gel Electrophoresis (PFGE) Analysis of Temporally Matched Listeria monocytogenes Isolates from Human Clinical Cases, Foods, Ruminant Farms, and Urban and Natural Environments Reveals Source-Associated as Well as Widely Distributed PFGE Types

“…Lineage III strains are rare but have been shown to be overrepresented among isolates from animal listeriosis cases (29). Molecular subtyping studies have been able to identify further specific L. monocytogenes subtypes and clonal groups that appear to be associated with human listeriosis outbreaks and are common among isolates from human listeriosis cases (23,29,30 Increasing evidence indicates that many human disease-associated subtypes, including those representing human epidemic clones, are not only found in foods and food-processing environments but may also be present in urban and natural environments as well as in farm environments (6,36,39,47). Molecular subtyping data have also shown that L. monocytogenes can persist in processing environments for considerable time periods (up to more than 10 years) (30,43) and that human listeriosis outbreaks can be traced back to persistent contamination by the outbreak subtype in the source plant.…”