Molecular Epidemiology of Invasive Listeriosis due to Listeria monocytogenes in a Spanish Hospital over a Nine-Year Study Period, 2006-2014

Abstract

We investigated the pathogenicity, invasiveness, and genetic relatedness of 17 clinical Listeria monocytogenes stains isolated over a period of nine years (2006-2014). All isolates were phenotypically characterised and growth patterns were determined. The antimicrobial susceptibility of L. monocytogenes isolates was determined in E-tests. Invasion assays were performed with epithelial HeLa cells. Finally, L. monocytogenes isolates were subtyped by PFGE and MLST. All isolates had similar phenotypic characteristics (β-haemolysis and lecithinase activity), and three types of growth curve were observed. Bacterial recovery rates after invasion assays ranged from 0.09% to 7.26% (1.62 ± 0.46). MLST identified 11 sequence types (STs), and 14 PFGE profiles were obtained, indicating a high degree of genetic diversity. Genetic studies unequivocally revealed the occurrence of one outbreak of listeriosis in humans that had not previously been reported. This outbreak occurred in October 2009 and affected three patients from neighbouring towns. In conclusion, the molecular epidemiological analysis clearly revealed a cluster (three human cases, all ST1) of not previously reported listeriosis cases in northwestern Spain. Our findings indicate that molecular subtyping, in combination with epidemiological case analysis, is essential and should be implemented in routine diagnosis, to improve the tracing of the sources of outbreaks.

Figure 1

Growth patterns of 17 clinical isolates of Listeria monocytogenes causing invasive infections at the “Complejo Asistencial Universitario de León” from 2006 to 2014. Two of the isolates had growth patterns different from that of all the other isolates: isolate 6 (GP2) and isolate 7 (GP3), respectively.

Figure 2

Invasion assays for the 17 clinical isolates of Listeria monocytogenes in HeLa epithelial cells. The mean number of internalised bacteria as a percentage of the initial inoculum is shown on the y-axis. The error bars show the standard error of two independent experiments, each performed in duplicate. The wild-type L. monocytogenes P14 and noninvasive L. monocytogenes ΔprfA strains were included, to assess the reproducibility of the experiments.

Figure 3

Genetic relationships between 17 clinical isolates of Listeria monocytogenes, based upon comparison of pulsed-field gel electrophoresis profiles obtained with the restriction enzymes ApaI and AscI. The dendrogram was produced with a Dice similarity coefficient matrix, using the unweighted pair group method with arithmetic mean (UPGMA). Tolerance and optimisation values were set to 1.5%. Clusters are arbitrarily designated A to C. Scale bar indicates similarity values.

Figure 4

Multilocus sequence typing of 17 Listeria monocytogenes isolates from sporadic cases of human listeriosis in Spain during the 2006–2014 period. The sequence types were clustered according to the sequence of the abcz housekeeping gene, using a minimum spanning tree (MST) tool available from the Pasteur Institute MLST database (http://www.pasteur.fr/recherche/genopole/PF8/mlst/). The STs from genetic lineages I and III (a) and genetic lineage II (b) found in this study are underlined. Sporadic listeriosis cases and outbreaks in Spain (2012–2014) listed in the Pasteur Institute MLST database are shown in boxes outlined with dotted lines. L. monocytogenes sample origins and PFGE profiles are included in each MST. The coloured zones surrounding groups of STs indicate clonal complexes (CC) differing by only one gene from other members of the group.