Salmonella enterica, Campylobacter and Yersinia species, Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes and Clostridium perfringens are the bacterial pathogens constituting the greatest burden of food-borne disease in Finland. Several molecular genetic methods have been applied to diagnose, discriminate and survey these bacteria. PCR, PCR-RFLP and PFGE are the most widely and successfully used. However, these methods are unable to replace conventional and internationally standardised phenotyping. Electronic database libraries of the different genomic profiles will enable continuous surveillance of infections and detection of possible infection clusters at an early stage. Furthermore, whole-genome sequence data have opened up new insights into epidemiological surveillance. Laboratory-based surveillance performed in a timely manner and exploiting adequate methods, and co-operation at local, national and international levels are among the key elements in preventing food-borne diseases. This paper reviews different applications of molecular genetic methods for investigating enteric bacterial pathogens and gives examples of the methods successfully used in diagnostics and epidemiological studies in Finland.
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
Cites: Appl Environ Microbiol. 2004 Aug;70(8):4458-6715294773
To evaluate the prevalence and genetic diversity of Listeria monocytogenes in wild birds and to compare the genotypes with isolates previously collected from foods and food processing environments.
Samples of wild birds' faeces (n = 212) were collected from a municipal landfill site and from urban areas in the Helsinki region and analysed by two-step enrichment and plating onto L. monocytogenes-selective agar. The overall prevalence of L. monocytogenes in bird faeces was 36% (95% CI 30-43%), and prevalence on the landfill site was significantly higher. All isolates were analysed with pulsed-field gel electrophoresis and compared with the L. monocytogenes profiles in an existing collection. Similar pulsotypes were found in birds and in isolates collected along the food chain.
Birds commonly carry L. monocytogenes, and strains are frequently similar with those detected in foods and food processing environments. Thus, birds may disseminate L. monocytogenes in nature and may also contaminate foods when entering the food processing environments and outdoor market places.
Populations of L. monocytogenes in wild birds and along the food processing chain overlap. Our findings add to the epidemiological data on this significant foodborne pathogen.
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.
The molecular epidemiology of Listeria monocytogenes was investigated in a longitudinal study of three Finnish dairy farms during 2013 to 2016. A total of 186 bulk tank milk (BTM), 224 milk filter sock (MFS), and 1,702 barn environment samples were analyzed, and isolates of L. monocytogenes were genotyped using pulsed-field gel electrophoresis. L. monocytogenes occurred throughout the year in all sample types, and the prevalence in MFS increased significantly during the indoor season. L. monocytogenes was more prevalent in MFS (29%) than in BTM (13%) samples. However, the prevalence of L. monocytogenes varied more between farms in samples of MFS (13 to 48%) than in BTM (10 to 16%). For each farm, the L. monocytogenes genotypes detected were classified by persistence (defined as persistent if isolated from =3 samples during =6 months) and predominance (defined as predominant if >5% prevalence on at least one farm visit). The prevalence of sporadic genotypes was 4 to 5% on all three farms. In contrast, the prevalence of persistent predominant genotypes varied between farms by 4% to 16%. The highest prevalence of persistent predominant genotypes was observed on the farm with the poorest production hygiene. Persistent predominant genotypes were most prevalent on feeding surfaces, water troughs, and floors. Genotypes isolated from the milking system or from cow udders had a greater relative risk of occurring in BTM and MFS than genotypes that only occurred elsewhere in the farm, supporting the hypothesis that L. monocytogenes is transmitted to milk from contamination on the udder surface or in the milking equipment.IMPORTANCEListeria monocytogenes is a ubiquitous environmental bacterium and the causative agent of a serious foodborne illness, listeriosis. Dairy products are common vehicles of listeriosis, and dairy cattle farms harbor L. monocytogenes genotypes associated with human listeriosis outbreaks. Indeed, dairy cattle farms act as a reservoir of L. monocytogenes, and the organism is frequently detected in bulk tank milk (BTM) and in the feces of clinically healthy cows. The ecology of L. monocytogenes in the farm environment is complex and poorly understood. Isolates of the same L. monocytogenes genotype can occur in the farm for years, but the factors contributing to the persistence of genotypes on dairy farms are unknown. Knowledge of the persistence patterns and contamination routes of L. monocytogenes on dairy farms can improve management of the contamination pressure in the farm environment and aid in the development of focused control strategies to reduce BTM contamination.
The investigation of the bacterial populations' heterogeneity contributes to the control of natural foci, causative agents of nosocomial infections, to the analysis of the microbial evolution. Multilocus sequence typing (MLST) was employed for the analysis of the diversity and features of the distribution of polyhostal ubiquitous microorganisms of the genera Burkholderia, Leptospira, and Listeria, which belong to three bacterial phyla: Proteobacteria, Spirochaetes, and Firmicutes. According to the bacterial samples analysis microbial genotypes prevalent and unique to Russia were identified; their occurrence in different Federal Regions was investigated; their similarity with global spread genotypes was appreciated. Obtained results allowed identifying common regularities of the selection of the microorganisms capable to cause the diseases of human and animals. The formation of genotypes that are most pathogenic for the host was demonstrated for all groups of bacteria. Leptospira spp. and Listeria monocytogenes strains with these genotypes have been circulating for a long time, being supported by natural foci. The formation of a wide variety of genotypes with different pathogenicity was demonstrated in the local geographic areas. In Russia, the zonal difference in all three groups of bacteria is most clearly traced to the Far Eastern Federal Region. The results are thought to contribute to analyzing the factors of selection and the phylogeny of the taxa under study.
Listeria monocytogenes is responsible for severe and often fatal food-borne infections in humans. A collection of 2,421 L. monocytogenes isolates originating from Ontario's food chain between 1993 and 2010, along with Ontario clinical isolates collected from 2004 to 2010, was characterized using an improved multilocus variable-number tandem-repeat analysis (MLVA). The MLVA method was established based on eight primer pairs targeting seven variable-number tandem-repeat (VNTR) loci in two 4-plex fluorescent PCRs. Diversity indices and amplification rates of the individual VNTR loci ranged from 0.38 to 0.92 and from 0.64 to 0.99, respectively. MLVA types and pulsed-field gel electrophoresis (PFGE) patterns were compared using Comparative Partitions analysis involving 336 clinical and 99 food and environmental isolates. The analysis yielded Simpson's diversity index values of 0.998 and 0.992 for MLVA and PFGE, respectively, and adjusted Wallace coefficients of 0.318 when MLVA was used as a primary subtyping method and 0.088 when PFGE was a primary typing method. Statistical data analysis using BioNumerics allowed for identification of at least 8 predominant and persistent L. monocytogenes MLVA types in Ontario's food chain. The MLVA method correctly clustered epidemiologically related outbreak strains and separated unrelated strains in a subset analysis. An MLVA database was established for the 2,421 L. monocytogenes isolates, which allows for comparison of data among historical and new isolates of different sources. The subtyping method coupled with the MLVA database will help in effective monitoring/prevention approaches to identify environmental contamination by pathogenic strains of L. monocytogenes and investigation of outbreaks.
Cites: Int J Food Microbiol. 2008 Jun 10;124(3):239-4918457891
Cites: Int J Food Microbiol. 1999 Aug 1;49(1-2):95-10210477075
Identification and prioritisation of food safety interventions requires an understanding of the relationship between food, pathogens and cases. Such understanding can be gained through different approaches, e.g. microbial subtyping to attribute cases of foodborne disease to food vehicles or other sources of illness. In this study, Listeria monocytogenes isolates (n=166) from (i) three categories of ready-to-eat (RTE) foods, (ii) food processing plant environments, and (iii) human listeriosis cases, all sampled during 2010 in Sweden, were subtyped. In addition, 121 isolates from human listeriosis cases, collected 2005-2009, were subtyped. Subtyping consisted of both serotyping (conventional method and PCR) and genotyping using pulsed-field gel electrophoresis (PFGE). Serotype 1/2a dominated in all three groups of isolates (range 73-96%). Eighteen percent of the human isolates (2010) belonged to serotype 4b, but only 1.4% of the food isolates. The food isolates differentiated into 19 pulsotypes (ID=0.843), the human isolates collected 2010 into 31 pulsotypes (ID=0.950) and the processing plant isolates into 22 pulsotypes (ID=0.991). Six of the pulsotypes were shared between the food and human isolates. These pulsotypes comprised 42% of the human isolates and 59% of the food isolates. For some processing plants, there was suggested persistence of one or more specific L. monocytogenes strains, as indicated by repetitive isolation of the same pulsotype from food. This study indicated the presence of L. monocytogenes in the processing plant environment as a likely source of contamination of gravad and cold-smoked fish, and this food category as an important source of human exposure to the pathogen.
Canadian cases and outbreaks of illness caused by Listeria monocytogenes between 1995 and 2004 were assessed. Isolates (722 total) were characterized by serotyping, and pulsed-field gel electrophoresis (PFGE) was performed to provide a means of detecting case clusters. Rates of listeriosis remained fairly consistent during the period of study, and patient characteristics were similar to those seen in studies of other populations. Most isolates were obtained from blood and cerebrospinal fluid, although during some outbreak investigations isolates were also obtained from stools. Serotype 1/2a predominated in isolates from patients in Canada, followed by serotypes 4b and 1/2b. Outbreaks caused by L. monocytogenes that occurred during the period of study were caused by isolates with serotypes 1/2a and 4b. A retrospective analysis of PFGE data uncovered several clusters that might have represented undetected outbreaks, suggesting that comprehensive prospective PFGE analysis coupled with prompt epidemiological investigations might lead to improved outbreak detection and control.
We analysed the surveillance data from listeriosis cases notified to the Finnish National Infectious Diseases Register between 1995 and 2004 and describe our recent experience in investigating clusters of listeriosis cases. The number of annual cases varied between 18 and 53 but no trends in incidence were identified (average annual incidence was 7 cases per million inhabitants). Only a few cases affected pregnant women or newborns. Most of the patients were elderly people with non-malignant underlying illnesses; 25% of them died from their infections. By routine sero- and genotyping of the listeria isolates, we detected several clusters; the vehicle for infection was only identified for two outbreaks. At least one quarter of listeriosis cases (78/315) was caused by a certain sero-genotype or closely related genotypes, which have also been found from vacuum-packed cold-smoked or cold-salted fish products. During 2000-2003, Finnish consumers were repeatedly informed about food precautions for risk groups. The information was also given to attending physicians and prenatal clinics.