Implicated Food Products for Listeriosis and Changes in Serovars of Listeria monocytogenes Affecting Humans in Recent Decades.

Listeriosis is a foodborne disease with a high fatality rate, and infection is mostly transmitted through ready-to-eat (RTE) foods contaminated with Listeria monocytogenes, such as gravad/smoked fish, soft cheeses, and sliced processed delicatessen (deli) meat. Food products/dishes stored in vacuum or in modified atmospheres and with extended refrigerator shelf lives provide an opportunity for L. monocytogenes to multiply to large numbers toward the end of the shelf life. Elderly, pregnant women, neonates, and immunocompromised individuals are particularly susceptible to L. monocytogenes. Listeriosis in humans manifests primarily as septicemia, meningitis, encephalitis, gastrointestinal infection, and abortion. In the mid 1990s and early 2000s a shift from L. monocytogenes serovar 4b to serovar 1/2a causing human listeriosis occurred, and serovar 1/2a is becoming more frequently linked to outbreaks of listeriosis, particularly in Europe and Northern America. Consumer lifestyle has changed, and less time is available for food preparation. Modern lifestyle has markedly changed eating habits worldwide, with a consequent increased demand for RTE foods; therefore, more RTE and take away foods are consumed. There is a concern that many Listeria outbreaks are reported from hospitals. Therefore, it is vitally important that foods (especially cooked and chilled) delivered to hospitals and residential homes for senior citizens and elderly people are reheated to at least 72°C: cold food, such as turkey deli meat and cold-smoked and gravad salmon should be free from L. monocytogenes. Several countries have zero tolerance for RTE foods that support the growth of Listeria.

Lineage II (Serovar 1/2a and 1/2c) Human Listeria monocytogenes Pulsed-Field Gel Electrophoresis Types Divided into PFGE Groups Using the Band Patterns Below 145.5 kb.

Among 504 clinical lineage II isolates of Listeria monocytogenes isolated during 1958-2010 in Sweden, 119 pulsed-field gel electrophoresis (PFGE) types (AscI) have been identified based on the number and distribution of all banding patterns in each DNA profile. In this study, these types were further divided into PFGE groups based on the configuration of small bands with sizes <145.5 kb. The 504 isolates included 483 serovar 1/2a isolates distributed into 114 PFGE types and 21 serovar 1/2c isolates distributed into 9 PFGE types; these were further divided into 21 PFGE groups. PFGE group, that is, configuration of small bands below 145.5 kb, and serovars were correlated. L. monocytogenes isolates belonging to PFGE groups A, B, C, E, F, H, K, L, M, S, V, W, Y, and Ö-6 to Ö-12 shared serovar 1/2a, with one exception. PFGE group E also included two PFGE types sharing serovar 1/2c and four PFGE types belonging to either serovar 1/2a or 1/2c. Isolates belonging to PFGE group N shared serovar 1/2c. In contrast to lineage I isolates, small fragments <33.3 kb were visible in all L. monocytogenes isolates belonging to lineage II. In the results from both the present and previous studies, the genomic region of small bands was genetically more conservative than in large bands. The distribution of these small bands established the relatedness of strains and defined a genetic marker for both lineages I and II, while also establishing their serogroup. The division of L. monocytogenes PFGE types into PFGE groups is advantageous as the profile of every new isolate can be identified easily and quickly through first studying the PFGE group affiliation of the isolate based on the smaller band patterns <145.5 kb, and then identifying the PFGE type based on the band patterns >145.5 kb.

Division of Human Listeria monocytogenes Pulsed-Field Gel Electrophoresis (PFGE) Types Belonging to Lineage I (Serovar 4b, 1/2b, and 3b) into PFGE Groups.

The 63 pulsed-field gel electrophoresis (PFGE) types identified among 427 clinical isolates of Listeria monocytogenes that were characterized in a previous study by serotyping and PFGE (AscI) could be further divided into 17 PFGE groups. While the 63 PFGE types, all part of lineage I, were established based on the number and distribution of all bands in each DNA profile, the 17 PFGE groups were based on the configuration of small bands with sizes <145.5 kb. The 30 PFGE types of L. monocytogenes serovar 4b isolates (n=334) were divided into 8 PFGE groups; the 32 PFGE types of serovar 1/2b isolates (n=90) and the serovar 3b isolates (n=3, 1 PFGE type) were divided into 9 PFGE groups. An association was observed between PFGE groups and serovars. L. monocytogenes isolates belonging to PFGE groups I, J, Q, R, X, Z, Ö-4, and Ö-5 all shared serovar 4b, whereas isolates from PFGE groups D, G, O, P, T, U, Ö-1, Ö-2, and Ö-3 shared serovar 1/2b. Small fragments <33.3 kb were nonvisible in all L. monocytogenes isolates. From the results of the present study, a procedure for accelerating the identification of PFGE types when analyzing new PFGE profiles can be suggested. Therefore, we propose a stepwise procedure to PFGE profiling by first identifying the PFGE group using the smaller band patterns <145.5 kb, and then determining PFGE types based on the band patterns >145.5 kb.

Investigation of a Listeria monocytogenes Chromosomal Immigration Control Region Reveals Diverse Restriction Modification Systems with Complete Sequence Type Conservation.

Listeria monocytogenes is a Gram-positive pathogen responsible for the severe foodborne disease listeriosis. A chromosomal hotspot between lmo0301 and lmo0305 has been noted to harbor diverse restriction modification (RM) systems. Here, we analyzed 872 L. monocytogenes genomes to better understand the prevalence and types of RM systems in this region, designated the immigration control region (ICR). Type I, II, III and IV RM systems were found in 86.1% of strains inside the ICR and in 22.5% of strains flanking the ICR. ICR content was completely conserved within the same multilocus sequence typing-based sequence type (ST), but the same RM system could be identified in diverse STs. The intra-ST conservation of ICR content suggests that this region may drive the emergence of new STs and promote clone stability. Sau3AI-like, LmoJ2 and LmoJ3 type II RM systems as well as type I EcoKI-like, and type IV AspBHI-like and mcrB-like systems accounted for all RM systems in the ICR. A Sau3AI-like type II RM system with specificity for GATC was harbored in the ICR of many STs, including all strains of the ancient, ubiquitous ST1. The extreme paucity of GATC recognition sites in lytic phages may reflect ancient adaptation of these phages to preempt resistance associated with the widely distributed Sau3AI-like systems. These findings indicate that the ICR has a high propensity for RM systems which are intraclonaly conserved and may impact bacteriophage susceptibility as well as ST emergence and stability.

Draft Genome Sequences of Heavy Metal-Resistant Listeria monocytogenes Strains of Sequence Type 14 (Clonal Complex 14) from Human Listeriosis Cases in Sweden.

Listeria monocytogenes of clonal complex 14 (CC14) is a potentially hypervirulent clone of serotype 1/2a but remains poorly characterized. We report the genome sequences of five sequence type 14 (ST14) (CC14) strains from human listeriosis cases in Sweden, which harbor a chromosomal heavy metal resistance island that is generally uncommon in serotype 1/2a.

Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing.

The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).

Characterization of human invasive isolates of Listeria monocytogenes in Sweden 1986-2007.

Since 1986, 68% of the Listeria monocytogenes isolates from human cases of invasive listeriosis in Sweden are available for retrospective studies. The aim of the present study was to characterize 601 human invasive isolates of L. monocytogenes in Sweden from 1986 to 2007 by using serotyping and pulsed-field gel electrophoresis. Since 1996, serovar 4b was permanently reduced to the second or third most common serovar in human cases in Sweden. During the latter period, 2000-2007, only 13% belonged to serovar 4b and 71% to 1/2a. The dendrogram, based on pulsovars, reveals two clusters with different serovars. Cluster 1 exhibits serovars 4b and 1/2b, whereas cluster 2 consists of serovar 1/2a. Serovar 1/2a seems to be more heterogeneous than serovar 4b.

Molecular grouping of Listeria monocytogenes based on the sequence of the inIB gene.

The major part of the gene inlB was sequenced in 24 strains of Listeria monocytogenes belonging to serovars 1/2a, 1/2b, 1/2c, 3b and 4b. A phylogenetic analysis based on the inlB nucleotide sequences showed that strains of serovars 1/2a and 1/2c were closely related, as well as those of serovars 1/2b and 3b. Strains sharing serovar 4b could be divided into two distinct groups. There were differences in amino-acid sequence between all serovars except between serovars 1/2b and 3b. Differences in amino-acid sequence were also seen within each of the serovars 1/2a and 4b. The data presented indicate that the inlB gene may be useful for typing purposes as an alternative or complement to serotyping.

Gram-positive and gram-negative bacteria induce different patterns of cytokine production in human mononuclear cells irrespective of taxonomic relatedness.

Upon bacterial stimulation, tissue macrophages produce a variety of cytokines that orchestrate the immune response that clears the infection. We have shown that Gram-positives induce higher levels of interleukin-12 (IL-12), interferon-gamma (IFN-gamma), and tumor necrosis factor (TNF) from human peripheral blood mononuclear cells (PBMCs) than do Gram-negatives, which instead induce more of IL-6, IL-8, and IL-10. Here, we study whether these patterns follows or crosses taxonomic borders. PBMCs from blood donors were incubated with UV-inactivated bacteria representing 37 species from five phyla. IL-12, TNF, IL-1beta, IL-6, IL-8, and IL-10 were measured in the supernatants after 24 h and IFN-gamma after 5 days. Irrespective of phylogenetic position, Gram-positive bacteria induced much more IL-12 (nine times more on average) and IFN-gamma (seven times), more TNF (three times), and slightly more IL-1beta (1.5 times) than did Gram-negatives, which instead induced more IL-6 (1.5 times), IL-8 (1.9 times), and IL-10 (3.3 times) than did Gram-positives. A notable exception was the Gram-positive Listeria monocytogenes, which induced very little IL-12, IFN-gamma, and TNF. The results confirm the fundamental difference in innate immune responses to Gram-positive and Gram-negative bacteria, which crosses taxonomic borders and probably reflects differences in cell wall structure.

Prevalence and fingerprinting of Listeria monocytogenes strains isolated from raw whole milk in farm bulk tanks and in dairy plant receiving tanks.

The incidence of Listeria species in raw whole milk from farm bulk tanks and from raw milk in storage at a Swedish dairy plant was studied. Listeria monocytogenes was found in 1.0% and Listeria innocua was found in 2.3% of the 294 farm bulk tank (farm tank) milk specimens. One farm tank specimen contained 60 CFU of L. monocytogenes ml(-1). L. monocytogenes was detected in 19.6% and L. innocua was detected in 8.5% of the milk specimens from the silo receiving tanks at the dairy (dairy silos). More dairy silo specimens were positive for both Listeria species during winter than during summer. Restriction enzyme analysis and pulsed-field gel electrophoresis were applied to 65 isolates of L. monocytogenes, resulting in 16 different clonal types. Two clonal types were shared by the farm tank milk and the dairy silo milk. All except one clonal type belonged to serovar 1/2a. In the dairy silo milk five clonal types were found more frequently and for a longer period than the others. No Listeria species were found in any other samples from the plant.