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.
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.
Listeria monocytogenes was isolated from critical control points in a Danish turkey processing plant, from turkey products and from cases of human listeriosis. During processing in the plant the prevalence of L. monocytogenes ranged from 25.9 to 41.4%. Cleaning and disinfection decreased the prevalence to 6.4%. Isolates of L. monocytogenes were characterized by pulsed-field gel electrophoresis (PFGE) using restriction endonuclease ApaI. Identical DNA types were obtained from turkey products and the processing line even after cleaning and disinfection. Two identical DNA types were demonstrated among isolates from turkey products and human cases of listeriosis. The prevalence of L. monocytogenes in turkey products ranged from 7.3 to 17.4% for ready-to-eat products and raw products, respectively. Since none of the 27 flocks examined before slaughter sampled positive for L. monocytogenes and the prevalence increased during processing, the potential risk from turkey meat was apparently due to factory hygiene rather than intrinsic contamination of the turkeys.
Forty Listeria monocytogenes isolates obtained in European and Far East regions of Russia were differentiated on the basis of polymorphism of 5 markergenes. Total length of concatemers obtained after sequencing of internal fragments of genes inlA, inlB, inlC, inlE and prs was 3029 b.p. Comparative analysis of concatemers' sequences revealed 237 variable nucleotides. Totally, 25 sequence types were revealed, and isolates from European and Far East regions belonged to different types. On the dendrogram isolates split on 2 clusters, which correspond to early described phylogenetic lines of L. monocytogenes specie. Isolates obtained in European and Far East regions formed independent subclusters within main clusters. Fifteen clinical isolates of L. monocytogenes belonged to 7 different types. Analysis of epidemiologic data on time and place of isolates obtaining suggested that isolates of the same sequence type are epidemiologically related and might represent one strain; index of discrimination for proposed typing method was calculated as 0.982.
Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. We investigated the prevalence and characteristics of L. monocytogenes isolates from surface waters derived from catchments within the South Nation River watershed (Ontario, Canada). This watershed is dominated by urban and rural development, livestock and crop production, and wildlife habitats. From June to November 2005, a total of 314 surface water samples were collected biweekly from 22 discrete sampling sites characterized by various upstream land uses. Presumptive Listeria spp. were isolated using a selective enrichment and isolation procedure, and 75 L. monocytogenes isolates were identified based on colony morphology, hemolytic activity, and amplification of three pathogenicity genes: iap, inlA, and hlyA. Thirty-two of 314 (10%) surface water samples were positive for the presence of L. monocytogenes, but detection ranged between 0 and 27% depending on the sampling date. Isolates belonging to serovar group 1/2a, 3a (50%) and group 4b, 4d, 4e (32%) were dominant. L. monocytogenes populations were resolved into 13 EcoRI ribotypes and 21 ApaI and 21 AscI pulsotypes. These had Simpson indexes of discrimination of up to 0.885. Lineage I-related isolates were dominant (61%) during the summer, whereas lineage II isolates were dominant (77%) in the fall. Isolates were, on average, resistant to 6.1 +/- 2.1 antibiotics out of 17 tested. Half of the L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, and some isolates were found to be moderately to highly virulent by in vitro Caco-2 plaque formation assay (up to 28% of entry). There was a statistically significant link between the occurrence of L. monocytogenes and proximity to an upstream dairy farm and degree of cropped land. Our data indicate that L. monocytogenes is widespread in the studied catchments, where it could represent a public health issue related to agricultural land use.
Cites: J Food Prot. 2006 Jan;69(1):93-10516416906
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Automated ribotyping, pulsed-field gel electrophoresis (PFGE) and serotyping were evaluated for the epidemiological study of isolates of Listeria monocytogenes collected in Finland in 1997-1999 from human blood (n = 116) and the food industry (n = 72). The isolates divided into six serotypes, 23 EcoRI ribotypes, 54 AscI PFGE types, and 57 final subtypes if all results were combined. The discrimination index of ribotyping was lower (0.873) than that of PFGE (0.946). Two final subtypes dominated among human isolates, and identical subtypes were also found among food industry isolates. All PFGE types were serotype-specific, whereas two ribotypes included isolates of two serotypes. Isolates of serotype 3a, involved in an outbreak in Finland in 1999, matched one of these ribotypes, which also included some food industry isolates of serotype 1/2a. Ribotyping with EcoRI would not have been sufficient to define the outbreak in Finland caused by serotype 3a isolates. Although ribotyping is applicable as the first method in outbreak situations, human and food isolates with identical ribotypes should be investigated further by PFGE.
Listeria monocytogenes is a food-borne human-pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities. Here we report the genome sequences for two L. monocytogenes strains (N53-1 and La111) that were isolated 6 years apart from two different Danish fish processers. Both strains are of serotype 1/2a and belong to a highly persistent DNA subtype (random amplified polymorphic DNA [RAPD] type 9). We demonstrate using in silico analyses that both strains belong to the multilocus sequence typing (MLST) type ST121 that has been isolated as a persistent subtype in several European countries. The purpose of this study was to use genome analyses to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical, with a predicted protein homology of 99.94%, differing at only 2 proteins. No single-nucleotide polymorphism (SNP) differences were seen between the two strains; in contrast, N53-1 and La111 differed from the EGD-e reference strain by 3,942 and 3,471 SNPs, respectively. We included a persistent L. monocytogenes strain from the United States (F6854) in our comparisons. Compared to nonpersistent strains, all three persistent strains were distinguished by two genome deletions: one, of 2,472 bp, typically contains the gene for inlF, and the other, of 3,017 bp, includes three genes potentially related to bacteriocin production and transport (lmo2774, lmo2775, and the 3'-terminal part of lmo2776). Further studies of highly persistent strains are required to determine if the absence of these genes promotes persistence. While the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicates that subtypes with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype.
Listeria monocytogenes strains that were isolated from 314 human listeriosis cases in Finland during an 11-year period were analyzed by O:H serotyping and pulsed-field gel electrophoresis (PFGE). Serotyping divided the isolates into five serotypes, the most common being 1/2a (53%) and 4b (27%). During the study period, the number of cases caused by serotype 1/2a increased from 22% in 1990 to 67% in 2001, and those caused by serotype 4b decreased from 61 to 27%, respectively. PFGE with restriction enzyme AscI divided the strains into 81 PFGE genotypes; among strains of serotypes 1/2a and 4b, 49 and 18 PFGE types were seen, respectively. PFGE type 1 (serotype 1/2a) was the most prevalent single type (37 strains). Together with six other, closely related PFGE types, PFGE type 1 formed a group of 71 strains, representing 23% of all 314 strains. Strains of PFGE type 1 have also been isolated from cold smoked fish, suggesting a source of human infections caused by this type. Moreover, PFGE type 24 (serotype 1/2c) was significantly associated with gender: 5% of 180 male subjects but none of 132 female subjects (P = 0.012). An electronic database library was created from the PFGE profiles to make possible the prompt detection of new emerging profiles and the tracing of potential infection clusters in the future.
To investigate the prevalence of Listeria monocytogenes in poultry products, and to elucidate whether poultry products may be linked to listeriosis cases. A further goal was to identify contamination routes for L. monocytogenes to broiler carcasses.
Poultry products (385 samples) were screened for L. monocytogenes. The recovered isolates and 19 patient isolates were characterized by multilocus enzyme electrophoresis and restriction enzyme analysis. The poultry isolates showed great genetic diversity, but no identical subclones were identified from poultry sources and patients. One slaughterhouse was examined in detail during a 16-month period. The contamination rates increased along the processing line, and one subclone was found during the whole period. Only low prevalence of the bacteria was revealed from broiler faeces.
The prevalence of L. monocytogenes in poultry products was high, but no listeriosis cases was linked to poultry products. Broilers seem to be contaminated during the slaughter process, and specific strains may persist in the processing environment. Broiler faeces does not seem to be an important source of L. monocytogenes in poultry products.
Preventive measures to avoid contamination of poultry products by L. monocytogenes must be taken in the processing plants.
Febrile gastroenteritis in five healthy persons was associated with the consumption of vacuum-packed cold-smoked rainbow trout containing Listeria monocytogenes. L. monocytogenes isolates from the incriminated fish product lot and the stool samples were all of serotype 1/2a and were indistinguishable by pulsed-field gel electrophoresis employing AscI and SmaI.
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