Wilhelm Tham

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

ABSTRACT 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.

Occurrence of Listeria monocytogenes in soft and semi-soft cheeses in retail outlets in Sweden.

Samples of 333 retail cheeses produced in or imported into Sweden were examined for the presence of Listeria monocytogenes. Listeria monocytogenes was isolated from 6% of the cheese samples. Cheeses made from raw milk were more frequently contaminated with L. monocytogenes (42%) than cheeses made from heat-treated milk (2%). The incidence of the organism in whole cheeses and pre-cut wedges was similar (6%). L. monocytogenes was only found in imported cheeses (18 from France, and one from Germany and Italy, respectively). The numbers of L. monocytogenes varied from < 1 x 10(2) to 1 x 10(5) cfu/g. All L. monocytogenes strains belonged to serogroup 1/2, except isolates from two samples that belonged to serogroup 4.

Febrile gastroenteritis after eating on-farm manufactured fresh cheese : an outbreak of listeriosis?

An outbreak of febrile gastroenteritis affected consumers of on-farm manufactured dairy products from a summer farm in Sweden. Symptoms included diarrhoea, fever, stomach cramps and vomiting in 88, 60, 54 and 21% of cases identified. The median incubation period was 31 h. A cohort study with 33 consumers showed an attack rate of 52% and an association between the total amount of product eaten and illness (P=0.07). Twenty-seven of 32 (84%) stool samples cultured for Listeria monocytogenes tested positive, although there was no association between clinical disease and the isolation of L. monocytogenes. In addition, gene sequences for VTEC and ETEC were detected in 6 and 1 subjects, respectively. Bacteriological analysis of cheese samples revealed heavy contamination with L. monocytogenes and coagulase positive staphylococci in all of them and gene markers for VTEC in one of them. Molecular profiles for L. monocytogenes isolated from dairy products, stool samples and an abscess from 1 patient who developed septic arthritis were identical. Results of both microbiological and epidemiological analyses point to L. monocytogenes as the most likely cause of this outbreak. The finding of markers for VTEC in some humans and cheese samples means that a mixed aetiology at least in some cases cannot be conclusively ruled out.

Lessons from an outbreak of listeriosis related to vacuum-packed gravad and cold-smoked fish.

The first lesson learned from this outbreak was that vacuum-packed rainbow trout is not only an excellent medium for the growth of Listeria monocytogenes, but may also cause human listeriosis. Another lesson is that one single fish processing plant may spread multiple clonal types of L. monocytogenes by selling contaminated products to consumers. Thus, when investigating fish-borne outbreaks of listeriosis one should identify and type several isolates of L. monocytogenes from each food and environmental sample, since multiple clonal types might be present. The outbreak described in this paper involved at least eight human cases, three clonal types of L. monocytogenes, and lasted for 11 months. During the outbreak investigation, L. monocytogenes was also isolated from another brand of rainbow trout found in the refrigerator of one of the patients. These latter isolates belonged to a clonal type not associated with the outbreak. However, this clonal type is of considerable interest since it has been associated with foodborne outbreaks of listeriosis in several countries, and is also the second most common clonal type among human clinical isolates of L. monocytogenes in Sweden. Besides the described outbreak, it is likely that vacuum-packed, cold-smoked and gravad rainbow trout have been involved in additional cases of foodborne listeriosis in Sweden.

The clones of Listeria monocytogenes detected in food depend on the method used

S. LONCAREVIC, W. THAM AND M.‐L. DANIELSSON‐THAM. 1996. Restriction enzyme analysis (REA) with pulsed‐field gel electrophoresis (PFGE) has been used to characterize and compare Listeria monocytogenes strains isolated from foods by two methods, an enrichment procedure and a direct plating procedure. In total 151 isolates from nine foods were investigated. In six of the foods (101 strains investigated) only one clone of L. monocytogenes was found irrespective of the method used. In three foods (50 strains investigated) the direct plating procedure yielded more clones than the enrichment procedure. At the most, five clones were detected in the same food. The results presented here indicate that direct plating from the food reveals more L. monocytogenes clones than revealed by an enrichment procedure.

A case of foodborne listeriosis in Sweden.

A 70‐year‐old woman fell seriously ill overnight with meningitis and was admitted to hospital. Cerebrospinal fluid culture yielded Listeria monocytogenes. One of the first problems in solving a human case of listeriosis suspected to be foodborne is to find the foods likely to have been transmitting L. monocytogenes. Two enrichment procedures and a direct plating procedure were used for isolation of the bacteria from different food items collected from the patients refrigerator, local retail store and producer. Samples of vacuum‐packed products of sliced pork brawn, sliced cooked medwurst and berliner wurst of the same brand harboured L. monocytogenes. Serotyping and restriction enzyme analysis (REA) with pulsed‐field gel electrophoresis (PFGE) were used to characterize and compare 41 isolates, including the human strain. At least three clones were present in the foods investigated, and one of these was identical to the human clone. This clone was present in samples of medwurst from the patients refrigerator and the local retail store. This is, to our knowledge, the first proven foodborne case of listeriosis reported in Sweden.

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.

Pyrosequencing as a Method for Grouping of Listeria monocytogenes Strains on the Basis of Single-Nucleotide Polymorphisms in the inlB Gene

ABSTRACT By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene. Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups.

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.