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

Abstract

Epidemiological studies and analysis of putative virulence genes have shown that Listeria monocytogenes has diverged into several phylogenetic divisions. We hypothesize that similar divergence has occurred for many genes that influence niche-specific fitness and virulence and that identifying these differences may offer new opportunities for the detection, treatment, and control of this important pathogen. To explore this issue further, we developed a microarray composed of fragmented DNA taken from 10 strains of L. monocytogenes. We then hybridized genomic DNA from 50 different strains to replicate arrays and analyzed the resulting hybridization patterns. A simple Euclidean distance metric permitted the reconstruction of previously described genetic relationships between serotypes, and only four microarray probes were needed to discriminate between the most important serotypes (1/2a, 1/2b, 1/2c, and 4). We calculated an index of linkage equilibrium from the microarray data and confirmed that L. monocytogenes has a strongly clonal population structure (I A ‫؍‬ 3.85). Twenty-nine informative probes were retrieved from the library and sequenced. These included genes associated with repairing UV-damaged DNA, salt tolerance, biofilm formation, heavy metal transport, ferrous iron transport, and teichoic acid synthesis. Several membrane-bound lipoproteins and one internalin were identified, plus three phage sequences and six sequences with unknown function. Collectively, these data confirm that many genes have diverged between lineages of L. monocytogenes. Furthermore, these results demonstrate the value of mixed-genome microarrays as a tool for deriving biologically useful information and for identifying and screening genetic markers for clinically important microbes.Listeria monocytogenes is considered a significant food-borne pathogen due to its potential for high mortality rates and because it is a difficult pathogen to control in production environments (29). 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). We hypothesize that similar divergence has occurred for many other genes that influence niche-specific fitness and virulence. If this hypothesis is correct, then identifying these differences may offer new insights into the ecology of L. monocytogenes as well as new opportunities for the detection, treatment, and control of this important pathogen.Differential fitness and virulence are likely to be controlled by either differential gene expression or the presence of lineage-specific genes or allelic variants. This paper foc...

Selective Discrimination of Listeria monocytogenes Epidemic Strains by a Mixed-Genome DNA Microarray Compared to Discrimination by Pulsed-Field Gel Electrophoresis, Ribotyping, and Multilocus Sequence Typing

“…A shotgun library was constructed by mixing equal molar amounts of genomic DNAs from 20 strains (six serotypes) of L. monocytogenes (4,9). A 2,000-probe screening microarray was constructed from the clone library and screened for polymorphic probes by hybridizing the genomic DNAs from 80 strains of L. monocytogenes to the array.…”

“…DNA subtyping with DNA microarrays may provide an improved alternative to resolve genetic differences that exist among isolates (4,9,28). This technique has the added advantage that, unlike PFGE, ribotyping, and MLST, it can identify specific or unique genes associated with strains of interest.…”

“…This technique has the added advantage that, unlike PFGE, ribotyping, and MLST, it can identify specific or unique genes associated with strains of interest. For example, Call and colleagues (9) demonstrated that certain strains of L. monocytogenes contained genes responsible for repairing UV-damaged DNA, salt tolerance, and biofilm formation, which would confer an advantage in certain ecological niches such as food production environments.…”

Gene Scanning of an Internalin B Gene Fragment Using High-Resolution Melting Curve Analysis as a Tool for Rapid Typing of Listeria monocytogenes

“…12,13 Phylogenetic groups have been correlated with serotypes: PG I.1 with serotype 1/2a, 3a; I.2 with 1/2c, 3c; II.1 with 4b, 4d, 4e; II.2 with 1/2b, 3b, 7; and III with 4a, 4c. 10 In cases of outbreak, the discriminatory power of multiplex PCR serotyping methods is insufficient, and it is necessary to type isolates by pulsed-field gel electrophoresis (PFGE), which is the current gold standard for L. monocytogenes typing. The PFGE method is time-consuming, however, and is difficult to standardize, which hampers interlaboratory exchange and comparison of typing results.…”

Review. Specific detection of Listeria monocytogenes in foods using commercial methods: from chromogenic media to real-time PCR

“…This PCR test constitutes a rapid and practical alternative to laborious classical serotyping. In the future, groundbreaking methods such as microarrays will probably allow the screening of food contamination only by the virulent subtypes of L. monocytogenes, thus improving the prevention of food-borne human listeriosis (Call et al, 2003a).…”