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.
We here report on the development of a novel multiplex PCR with product detection in a Luminex 100 suspension array system. The assay covers the nine most important bacterial and viral pathogens found in Danish meningitis patients. The microorganisms include Neisseria meningitidis, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Streptococcus agalactiae, herpes simplex virus types 1 and 2, and varicella-zoster virus. The study was based on 1,187 samples, of which 55 were found to be positive by PCR. The assay was found to have an excellent sensitivity and an excellent specificity compared to the results of a "gold standard," defined by routine laboratory tests, for the two most important pathogens, S. pneumoniae (95 and 99.1%, respectively) and N. meningitidis (100 and 99.7%, respectively). The method provides a valuable supplement to the traditional microscopy and culture of cerebrospinal fluid (CSF) samples in a routine diagnostic setting, and results can be available within 1 workday. The method is suitable for use for the initial screening and identification of nine important microorganisms in CSF samples from patients with suspected meningitis. Compared to microscopy and culture of CSF, this rapid and sensitive method will support physicians with the selection of the appropriate antimicrobial agents and the initiation of timely treatment in the absence of live microorganisms in the CSF.
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.
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.
The causative agent of listeriosis, a serious disease of humans and animals, Listeria monocytogenes is a ubiquitous bacterium that inhabits both anthropogenic and pristine environments. We report L. monocytogenes isolation from wild animals, humans, food and the environment of a far eastern region of Russia. In total, 654 samples of internal organs of small rodents belonging to the Muridae and Cricetidae families, and 986 samples of the liver and muscles of mollusks and fish were examined to obtain 7 and 14 independent L. monocytogenes isolates, respectively. The wild animal isolates were compared with human (n=9), food (n=8) and environmental (n=3) isolates obtained in the same region. Twenty of the 21 wild animal isolates belonged to the serovar 4b. The serovars 4b, 1/2a, 1/2b, and 4b, 1/2a, 1/2b, 1/2c were found between human and food isolates, respectively. All isolates were characterized into molecular subtypes by DNA sequencing of the 618 bp internal fragment of the house keeping gene prs and 621 bp internal fragment of the virulence gene inlB. Sequence analysis revealed 4 and 13 alleles for prs and inlB fragments, respectively. Distinct prs and inlB alleles clustered into two groups consistently with established phylogenetic lineages. Among isolates of every lineage, the nucleotide diversity of the prs fragment was low; the nucleotide diversity of the inlB fragment was low among wild animal isolates and higher among human isolates. All rodent isolates and 10 of 14 marine organism isolates carried the same allele of the inlB fragment, which was also found among environmental (two of three), food (two of eight) and human (two of nine) isolates.
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.
'Gravad' rainbow trout artificially contaminated with Listeria monocytogenes was analyzed by use of a 4 h enrichment period followed by extraction of DNA and PCR amplification. This procedure made it possible to detect 10-100 cfu L. monocytogenes per gram 'gravad' rainbow trout, within 12 h. After a prolonged enrichment period of 24 h, numbers as low as 1-10 cfu L. monocytogenes per gram could be detected. The method described may be a useful tool for screening samples of 'gravad' rainbow trout for the presence of L. monocytogenes, since it is sensitive, rapid and simple.