Portuguese chouriço de vinho is made by drying coarsely minced meat and fat that has been previously marinated with wine (usually red), salt, and garlic for 1 to 2 days at a low temperature (4 to 8 °C). This procedure may improve the microbiological safety of the product. The aim of this study was to evaluate the behavior of three pathogens in this product, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus, to establish the minimum period of drying and maturation necessary to render safe products. The pathogens were inoculated in the chouriço de vinho batter. A factorial design was used to study the following variables in the fermentation process: (i) the presence or absence of an indigenous Lactobacillus sakei starter culture; (ii) the presence or absence of fermentable carbohydrates; and (iii) the salt level (1.5 or 3%). The samples were analyzed 24 h after the preparation of the batter (at stuffing); after 7, 15, and 30 days of drying; and after 30 days of storage at 4 °C under vacuum. Under all of the conditions studied, the levels of the three pathogens decreased during the drying period. In the early stages of drying, the addition of L. sakei starter culture and/or carbohydrates resulted in lower levels of gram-positive pathogens. After 15 days of drying, populations of all pathogens decreased by ca. 2 log in all samples. At that sampling time, L. monocytogenes was undetectable in the chouriço de vinho with L. sakei starter culture and carbohydrates. The mean count of S. aureus after 15 days of drying was below 1 log CFU/g. After 30 days of drying, no pathogens were detected. The drying period could be shortened to 15 days when considering only the gram-positive pathogens studied and the use of a starter culture and carbohydrates. Due to the low infective dose of Salmonella spp., the product should be considered safe after 30 days, when this pathogen became undetectable.
Serious infection with the bacterium L. monocytogenes mainly manifests as sepsis and/or meningitis. A particular entity is Listeria brain stem encephalitis, which is characterized by progressive brain stem deficits. The condition is fatal unless early treated. The purpose of the present study was to assess the incidence of brain stem encephalitis in a population-based listeriosis material. Medical records from 212 of the 240 patients with serious listeriosis reported in Norway from 1977 to 2000, as well as autopsy material from 8 of these patients, were available. This material was searched for clinical and neuropathological evidence of brain stem infection. Findings indicating brain stem encephalitis were present in 19 of the 172 patients with adult listeriosis (11%) but none of the 40 pregnancy-related listeriosis cases. None of the 19 patients had been diagnosed with Listeria brain stem infection originally. We conclude that brain stem encephalitis is relatively common in this Norwegian listeriosis material.
Listeria monocytogenes, a psychrotrophic organism capable of growing at refrigeration temperatures, is of major concern in extended shelf life, refrigerated foods. Considering that as much as 80-90% of human listeriosis cases are linked to the ingestion of contaminated food, human cases are predominantly seen in high-risk individuals, including organ-transplant recipients, patients with AIDS and HIV-infected individuals, pregnant women, cancer patients, and the elderly. In 2001, the Canadian Listeriosis Reference Service (LRS) was created by the Bureau of Microbial Hazards (Health Canada) and the National Microbiology Laboratory (now part of the Public Health Agency of Canada). Major goals of the LRS include investigation of listeriosis cases and maintenance of a national collection of isolates. The LRS intends to create a comprehensive molecular epidemiological database of all isolates in Canada for use as a resource for outbreak investigations, research and other microbiological investigations. The PFGE profiles are being established and stored for clinical, food, environmental, and possibly animal strains of L. monocytogenes. The LRS pursues research activities for investigation and implementation of other molecular methods for characterizing L. monocytogenes isolates. Ribotyping, Multi-locus Sequence Typing (MLST), Variable Number of Tandem Repeats (VNTR), Multi-locus virulence sequence typing (MLVA), microarray- based technologies and sequence-based typing schemes, are being investigated on selected diversity sets. The LRS has also used PFGE typing for outbreak investigations. The molecular epidemiological data, timely coordination and exchange of information should help to reduce the incidence of listeriosis in Canada. In Canada, listeriosis is not a national notifiable disease, except for the province of Quebec, where it has been since 1999. The LRS, Canadian Public Health Laboratory Network, and federal epidemiologists are currently working on making human listeriosis notifiable throughout Canada.
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 patient's 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 patient's refrigerator and the local retail store. This is, to our knowledge, the first proven foodborne case of listeriosis reported in Sweden.
Public health authorities place a high priority on investigating listeriosis outbreaks, and these epidemiological investigations remain challenging. Some approaches have been described in the literature to address these challenges. This review of listeriosis clusters and outbreaks investigated in the Province of Quebec (Quebec) highlights investigative approaches that contributed to identifying the source of these outbreaks.
The Laboratoire de Santé Publique du Québec (LSPQ) implemented pulsed-field gel electrophoresis (PFGE) molecular subtyping in 1997 to identify Listeria monocytogenes clusters among isolates from invasive listeriosis cases identified throughout Quebec. A cluster was defined as three cases or more with the same or similar PFGE profiles (=3 band difference) occurring over a 4-month period. An investigation was initiated if the epidemiologic indicators suggested a common source. Listeriosis data from LSPQ's database were reviewed to identify and describe clusters detected from 1997 to 2011, including those that led to an outbreak investigation. Epidemiological reports prepared following each outbreak were also reviewed.
Eleven clusters were identified in the province by LSPQ between 1997 and 2011. Outbreak investigations were initiated for six clusters, four of which involved more than 10 cases. Factors that contributed to identifying the source for three of these outbreaks highlighted the value of (1) making all stakeholders (food safety and inspection services, public health authorities, and laboratories) aware of any ongoing investigation and sharing relevant information even if the source is not yet identified; (2) promptly collecting food samples identified and considered as possible vehicles of infection identified during the interview of a Listeria case; (3) collecting food items and/or environmental samples in locations reported in common by cases in the same cluster.
Multiple approaches should be considered when investigating L. monocytogenes clusters. Networks to facilitate continuous exchange of human and food data between public health and food safety partners should be encouraged.
A total of 84 strains of Listeria monocytogenes were analysed by multilocus enzyme electrophoresis at twelve enzyme loci. Eight enzyme loci were polymorphic with between 2 and 4 alleles per locus. Fourteen electrophoretic types (ETs) were identified. Among 62 human clinical isolates from Denmark, 8 different ETs were defined. Two ETs, designated ET 1 and ET 6, accounted for 77% of the human clinical isolates investigated. These ETs are identical with those responsible for several epidemics in Switzerland and in the United States. Comparison of 58 isolates of L. monocytogenes, typed by MEE, in relation to phage typing showed that phage typing was more discriminatory than MEE. The ability of MEE to distinguish between phage types of Epi-type and other phage types, however, was almost optimal. MEE typed 23 of 24 strains of Epi-type as belonging to ET 1. In contrast ET 1 was not found in 26 strains with phage types other than the Epi-type.
On the basis of specially developed scheme for the isolation of Listeria strains comprising 2 enrichment stages and the use of growth inhibitors, 128 L. monocytogenes cultures were isolated from clinical material, foodstuffs and sewage water. Highly virulent L.monocytogenes strains isolated from clinical material belonged to serovar 4b (54%) and 1/2a (38%), while those isolated from foodstuffs and sewage water belonged to 4b (74%). The restriction analysis of the chromosomal DNA of the isolated cultures with the use of restrictase EcoR1 on the basis of pulsed-field gel electrophoresis (PFGE) made it possible to distinguish Listeria strains in accordance with 5 types of restrictograms. The restrictograms of highly virulent L. monocytogenes strains, serovar 4b, belonged to types 1 and 2, while those of L. monocytogenes strains, serovar 1/2a, belonged to types 2 and 3. The comparative use of different methods for typing L. monocytogenes (sero-, phago-, bio- and resistotyping, the analysis of plasmid composition and restriction analysis) revealed that the combination of serotyping and restriction analysis on the basis of PFGE proved to be most promising for the characterization of the isolated L. monocytogenes strains and the assessment of their epidemic importance.
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.
In 1958-74 altogether 64 cases of bacteriologically verified infections of Listeria monocytogenes were diagnosed in Sweden in children, aged more than 27 days, and in adults. Immunosuppression predisposed to the disease. Thus, many patients had co-existing disorders, such as leukemia and alcoholism. Sixteen patients had been treated with corticosteroids, which were combined with cytostatic drugs in nine. Meningoencephalitis was diagnosed in 52 patients and was fatal in 16. The clinical symptoms did not differ from those in purulent meningitis caused by other bacteria. In the cerebrospinal fluid the cellular response was dominated by polymorphonuclear cells in 29 patients and by mononuclear cells in 20. Ten patients had septicemia, which was fatal in four. Clinical symptoms were dominated by chills, high fever and general prostration. One patient had pleurisy and one an abscess of the neck; both recovered. Serotypes 1 and 4b prevailed and were equally common. Many patients developed raised antibody titers in both the O-agglutination test and the complement fixation test. The titers were often not positive until after a month. Moderate granulocytosis was the rule and monocytosis was rarely seen. Ampicillin alone or combined with an aminoglycoside seemed to be the drug of choice in the treatment of listeriosis. An alternative drug was tetracycline. Most deaths occurred within six days of onset of the illness. Early diagnosis and treatment were imperative. Most patients recovered and serious sequelae were rare.
Sixty-two strains of Listeria monocytogenes isolated in Canada and Switzerland were investigated. Comparison based on molecular genotypes confirmed that strains in these two countries are genetically diverse. Interestingly strains from both countries displayed similar range of cold growth phenotypic profiles. Based on cold growth lag phase duration periods displayed in BHI at 4??C, the strains were similarly divided into groups of fast, intermediate and slow cold adaptors. Overall Swiss strains had faster exponential cold growth rates compared to Canadian strains. However gene expression analysis revealed no significant differences between fast and slow cold adapting strains in the ability to induce nine cold adaptation genes (lmo0501, cspA, cspD, gbuA, lmo0688, pgpH, sigB, sigH and sigL) in response to cold stress exposure. Neither was the presence of Stress survival islet 1 (SSI-1) analysed by PCR associated with enhanced cold adaptation. Phylogeny based on the sigL gene subdivided strains from these two countries into two major and one minor cluster. Fast cold adaptors were more frequently in one of the major clusters (cluster A), whereas slow cold adaptors were mainly in the other (cluster B). Genetic differences between these two major clusters are associated with various amino acid substitutions in the predicted SigL proteins. Compared to the EGDe type strain and most slow cold adaptors, most fast cold adaptors exhibited five identical amino acid substitutions (M90L, S203A/S203T, S304N, S315N, and I383T) in their SigL proteins. We hypothesize that these amino acid changes might be associated with SigL protein structural and functional changes that may promote differences in cold growth behaviour between L.?monocytogenes strains.