Genomic characterization of Listeria monocytogenes strains involved in a multistate listeriosis outbreak associated with cantaloupe in US

Abstract

A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray, Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data (in the form of present or absent calls of specific DNA sequences) separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks.

Figure 1. PFGE profiles of Listeria monocytogenes cantaloupe outbreak strains.

AscI patterns are shown in panel A: lane 2: GX6A16.0099 (III); lane 3: GX6A16.0019 (II); lane 4: GX6A16.0001 (IV); lane 5: GX6A16.0029 (I) and ApaI patterns in panel B lane 2: GX6A12.0001 (III); lane 3: GX6A12.0227 (II); lane 4: GX6A12.0001 (IV); lane 5: GX6A12.0069 (I). Lane 1 in both panels is Salmonella braenderup H9812 standard (digested by XbaI). The Roman numerals represent the PFGE pattern combination.

Figure 2. Distribution of Listeria monocytogenes isolates producing PFGE pattern combinations.

I- IV from human (grey) or nonhuman (green) sources. Only isolates from August 25–November 15, 2011 (duration of the outbreak) are included.

Figure 3. Relatedness analysis of the compatible parsimony informative genes from the 15 cantaloupe associated outbreak isolates, a shrimp isolate (LS693) and 15 L. monocytogenes isolates from several unrelated listeriosis episodes/outbreaks.

The tree was generated from the concatenated gene contents using neighbor joining with the uncorrected p distance. The numbers in red color indicate the cantaloupe associated outbreak and the shrimp isolates. The boxes indicate the close-up portions of the cantaloupe outbreak strains (green; serotype 1/2b and blue; serotype 1/2a). Scale bar represents number of gene differences (present or absent) per gene site. The Roman numerals represent the PFGE pattern combination.

Figure 4. Relatedness analysis of the compatible parsimony informative genes from the 9 cantaloupe associated outbreak isolates, a shrimp isolate (LS693) and 7 unrelated L. monocytogenes isolates belonging to serotype 1/2a .

The tree was generated from the concatenated gene contents using neighbor joining with the uncorrected p distance. The numbers highlighted in red indicate the cantaloupe associated outbreak and the shrimp isolates. Scale bar represents number of gene differences (present or absent) per gene site. The Roman numerals represent the PFGE pattern combination.

Figure 5. Diagrammatic representation of the distribution of unique probe-sets of the serotype 1/2a 2011 cantaloupe outbreak strains from different PFGE pattern combination groups based on their functions.

A) Probe sets uniquely present in the strains from the PFGE pattern combination I and absent in both PFGE pattern combination III and IV. B) Probe sets uniquely present in the strains from the PFGE pattern combination III and IV and absent in the PFGE pattern combination I. The numbers of probe-sets were indicated in the periphery of the pie-chart.

Figure 6. Hierarchical clustering based on the Robust Multi Array (RMA) analysis.

Strains highlighted in red indicated the serotype 1/2a strains from the 2011 Cantaloupe outbreak.

Figure 7. Comparison of the RMA summarized probe-set intensities.

(A) pattern combination I isolates - LS742 and LS743; (B) pattern combination I (LS742) and pattern combination III (LS746); (C) pattern combination I (LS742) and pattern combination IV (LS747); (D) pattern combination III (LS746) and pattern combination IV(LS747). Red dots indicate RMA summarized probe-set intensity differences of less than or equal to 2-fold between two strains. Blue dots indicate RMA summarized probe-set intensity differences of more than 2-fold between the two strains.

Figure 8. Diagrammatic representation of the distribution of unique probe-sets of the serotype 1/2a, PFGE pattern combination IV 2011 and absent in the PFGE pattern combination I and III strains based on their functions.

The numbers of probe-sets were indicated in the periphery of the pie-chart.

Figure 9. Relatedness analysis of the compatible parsimony informative genes from the 7 cantaloupe outbreak and 8 unrelated L. monocytogenes strains of serotype 1/2b .

The tree was generated from the concatenated gene contents using neighbor joining with the uncorrected p distance. Strains highlighted in red indicated the cantaloupe outbreak and unmatched strains. Scale bar represents number of gene differences (present or absent) per gene site.

Figure 10. Hierarchical clustering based on the Robust Multi Array (RMA) analysis.

Strains highlighted in red indicated the serotype 1/2b strains from the 2011 Cantaloupe outbreak.