Pan-genomic Characterization of Listeria monocytogenes Strains Associated with the 2011 Cantaloupe Outbreak

Introduction: Listeria monocytogenes is a Gram-positive foodborne pathogen responsible for listeriosis, an invasive bacterial infection in susceptible groups such as pregnant women, neonates and immuno-compromised adults. In September 2011, several listeriosis cases were first reported by the Colorado Department of Public Health and Environment (CDPHE). Follow-up investigation by the local, state and federal public health and regulatory agencies led to the discovery of a multistate listeriosis outbreak linked to the consumption of contaminated Rocky Ford cantaloupes. We have analyzed some environmental and food isolates implicated in this multi-state outbreak using a multiplex PCR and a microarray-based method to identify their serotypes and relatedness to each other and to other listeriosis outbreak strains.

Purpose: The objective for this study is to characterize genetic relatedness and determine the gene contents of the 2011 cantaloupe outbreak strains.

Methods: We have serotyped 27 L. monocytogenes strains associated with the 2011 cantaloupe outbreak using a combination of a slide-agglutination and a multiplex PCR assay. Seven strains from this outbreak and one unmatched strain from this group have further been analyzed using a microarray-based method (Listeria Gene-chip) to identify the genetic differences. The genomic DNAs isolated from these strains were hybridized onto the Listeria gene chip that was designed based on 24 L. monocytogenes publicly available genome sequences. The relatedness analysis was determined by comparing the gene content information of these cantaloupe outbreak strains against the database previously constructed from gene contents of 38 L. monocytogenes strains in our collection. 

Results: The genotypic microarray analysis indicated that the cantaloupe outbreak strains are clustered in two different groups dividing three strains into the serotype 1/2a and five strains into serotype 1/2b, in agreement with PCR serotyping results. However, the cantaloupe outbreak strains are more closely related to each other than to the other strains in our database. The detailed analysis also revealed that the genetic contents of these two groups of strains are very distinct. Among the outbreak strains all of the PFGE cluster 2 belonged to the serotype 1/2b while strains in PFGE cluster 3 and 4 belonged to serotype 1/2a. One strain, LS667 of the PFGE cluster 4 pattern also belonged to serotype 1/2b, which was confirmed by our microarray-based relatedness analysis.

Significance: We show that genotyping microarray grouped the L. monocytogenes strains associated in the 2011 cantaloupe outbreaks in two distinct genomic clusters−one containing 1/2a strains and the other containing 1/2b strains. Cantaloupe outbreak-related 1/2a and 1/2b strains appeared to be genetically distinct from the previously known 1/2a and 1/2b outbreak strains. In addition, the microarray analysis and PCR serotyping revealed that strains of the PFGE cluster 4 belonging to both serotypes 1/2a and 1/2b.