Multiple-locus variable-number tandem-repeats analysis of Listeria monocytogenes using multicolour capillary electrophoresis and comparison with pulsed-field gel electrophoresis typing.

The multiple-locus variable-number tandem-repeats analysis (MLVA) method for genotyping has proven to be a fast and reliable typing tool in several bacterial species. MLVA is in our laboratory the routine typing method for Salmonella enterica subsp. enterica serovar Typhimurium and Escherichia coli O157. The gram-positive bacteria Listeria monocytogenes, while not isolated as frequent as S. Typhimurium and E. coli, causes severe illness with an overall mortality rate of 30%. Thus, it is important that any outbreak of this pathogen is detected early and a fast trace to the source can be performed. In view of this, we have used the information provided by two fully sequenced L. monocytogenes strains to develop a MLVA assay coupled with high-resolution capillary electrophoresis and compared it to pulsed-field gel electrophoresis (PFGE) in two sets of isolates, one Norwegian (79 isolates) and one Swedish (61 isolates) set. The MLVA assay could resolve all of the L. monocytogenes serotypes tested, and was slightly more discriminatory than PFGE for the Norwegian isolates (28 MLVA profiles and 24 PFGE profiles) and opposite for the Swedish isolates (42 MLVA profiles and 43 PFGE profiles).

Study of polymorphic variable-number of tandem repeats loci in the ECOR collection and in a set of pathogenic Escherichia coli and Shigella isolates for use in a genotyping assay.

The Escherichia coli (E. coli) reference collection, ECOR, consists of 72 strains that are representative of the genotypic diversity, as indexed by multilocus enzyme electrophoresis (MLEE), in the species as a whole. MLEE revealed 4 main phylogenetic groups designated A, B1, B2 and D. We present a study of the relationship between the ECOR strains as determined by polymorphisms in seven variable-number of tandem repeats (VNTR) loci. Seven tandem repeats that were present in more than one of the fully sequenced E. coli strains were selected, and primers were constructed in order to amplify the targets in all species where the loci were present. The combined result for all VNTR loci was adapted as a multiple-locus variable-number tandem repeats analysis (MLVA) and showed that the ECOR collection was divided into 63 distinct genotypes. The ECOR phylogenetic groups defined by MLEE were not well conserved by MLVA. A set of 61 pathogenic isolates of both E. coli and Shigella spp. was then tested with the same set of VNTR loci, and revealed 54 distinct genotypes. In addition, the MLVA method was used to genotype isolates from patients and suspected sources in a recent outbreak of E. coli O103 in Norway. The pathogenic E. coli isolates contained the diarrhea causing categories EIEC, EAEC, STEC, ETEC and EPEC. Shigella isolates were of species S. flexneri, S. boydii, S. sonnei and S. dysenteriae. The MLVA method rapidly genotyped all isolates in the study at a Simpson's index of diversity of D=0.98.

Multiple-locus variable-number tandem-repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis.

The multiple-locus variable-number tandem-repeats analysis (MLVA) method is currently being used as the primary typing tool for Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) isolates in our laboratory. Our published initial MLVA was performed using a single fluorescent dye and the different patterns were assigned from gel images. Here we present a new and significantly improved assay using multiple dye colors, enhanced PCR multiplexing and the introduction of two new loci for better adaptation to capillary electrophoresis with increased speed. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from gel images. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. A total of 106 human, bird, animal and food isolates of S. Typhimurium, including 16 with definite type (DT) 104, were used for the development of the improved MLVA. The method is based on capillary separation of multiplexed PCR products from five VNTR loci in the S. Typhimurium genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned allele numbers, which were used for strain comparison.

Molecular epidemiology and disinfectant susceptibility of Listeria monocytogenes from meat processing plants and human infections.

We have investigated the molecular epidemiology of Listeria monocytogenes from the meat processing industry producing cold cuts and from cases of human listeriosis by discriminative pulsed-field gel electrophoresis (PFGE). A subset of the isolates was also investigated for susceptibility to a disinfectant based on quaternary ammonium compounds (QAC) frequently used in the meat processing industry. The purpose of this investigation was to obtain knowledge of sources, routes of contamination and genetic types of L. monocytogenes present along the production line in the meat processing industry, and to compare meat industry isolates and human isolates. Of the 222 isolates from four meat-processing plants, 200 were from two plants responsible for nearly 50% of the production of cold cuts in the Norwegian market. The strain collection included historical routinely sampled isolates (1989-2002) and isolates systematically sampled through a one year period (November 2001 to November 2002) from fresh meat and production environments in three plants. No isolates were obtained in samples from employees (throat, faeces). Human strains included all available reported isolates from Norwegian patients in selected time periods. The L. monocytogenes PFGE data showed a large genetic heterogeneity, with isolates separated into two genetic lineages and further subdivided into 56 different PFGE profiles. Certain profiles were observed on both sides of production (before and after heat treatment) indicating contamination of end products by fresh meat or fresh meat environments. While fresh meat isolates almost exclusively grouped within lineage I, isolates from end products showed a more balanced distribution between lineages I and II. Ten profiles were common among isolates from human and meat industry. Typing of human isolates identified a previously unrecognised outbreak. Generally, a higher QAC resistance incidence was observed among isolates from the meat processing industry than among human isolates although large plant to plant differences were indicated. No correlation between resistance and PFGE profile or resistance and persistence was observed.

Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis.

The Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method is currently being used as the primary typing tool for Shiga-toxin-producing Escherichia coli (STEC) O157 isolates in our laboratory. The initial assay was performed using a single fluorescent dye and the different patterns were assigned using a gel image. Here, we present a significantly improved assay using multiple dye colors and enhanced PCR multiplexing to increase speed, and ease the interpretation of the results. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. Seventy-two human and animal strains of Shiga-toxin-producing E. coli O157 were used for the development of the improved MLVA assay. The method is based on capillary separation of multiplexed PCR products of VNTR loci in the E. coli O157 genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison.

DNA fingerprinting of Shiga-toxin producing Escherichia coli O157 based on Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA).

BACKGROUND: The ability to react early to possible outbreaks of Escherichia coli O157:H7 and to trace possible sources relies on the availability of highly discriminatory and reliable techniques. The development of methods that are fast and has the potential for complete automation is needed for this important pathogen. METHODS: In all 73 isolates of shiga-toxin producing E. coli O157 (STEC) were used in this study. The two available fully sequenced STEC genomes were scanned for tandem repeated stretches of DNA, which were evaluated as polymorphic markers for isolate identification. RESULTS: The 73 E. coli isolates displayed 47 distinct patterns and the MLVA assay was capable of high discrimination between the E. coli O157 strains. The assay was fast and all the steps can be automated. CONCLUSION: The findings demonstrate a novel high discriminatory molecular typing method for the important pathogen E. coli O157 that is fast, robust and offers many advantages compared to current methods.

Molecular analyses of Salmonella enterica isolates from fish feed factories and fish feed ingredients.

Isolates of the most commonly observed salmonella serovars in Norwegian fish feed factories from 1998 to 2000 (Salmonella enterica serovar Agona, S. enterica serovar Montevideo, S. enterica serovar Senftenberg, and S. enterica serovar Kentucky) were studied by pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis and compared to isolates of the same serovars from fish feed ingredients, humans, and other sources (a total of 112 isolates). Within each serovar, a variety of distinct PFGE types (with similarity levels less than 90%) were observed in the feed ingredients and other sources, while only two distinct types of each serovar were identified in the factories. The combined results of PFGE and plasmid analyses showed that each factory harbored only a few S. enterica clones. Some of these clones persisted for at least 3 years in the factories, indicating that there was long-lasting contamination probably due to inadequate decontamination procedures.

Molecular epidemiology of Salmonella enterica serovar typhimurium isolates determined by pulsed-field gel electrophoresis: comparison of isolates from avian wildlife, domestic animals, and the environment in Norway.

The molecular epidemiology of 142 isolates of Salmonella enterica serovar Typhimurium from avian wildlife, domestic animals, and the environment in Norway was investigated using pulsed-field gel electrophoresis (PFGE) and computerized numerical analysis of the data. The bacterial isolates comprised 79 isolates from wild-living birds, including 46 small passerines and 26 gulls, and 63 isolates of nonavian origin, including 50 domestic animals and 13 environmental samples. Thirteen main clusters were discernible at the 90% similarity level. Most of the isolates (83%) were grouped into three main clusters. These were further divided into 20 subclusters at the 95% similarity level. Isolates from passerines, gulls, and pigeons dominated within five subclusters, whereas isolates from domestic animals and the environment belonged to many different subclusters with no predominance. The results support earlier results that passerines constitute an important source of infection to humans in Norway, whereas it is suggested that gulls and pigeons, based on PFGE analysis, represent only a minor source of human serovar Typhimurium infections. Passerines, gulls, and pigeons may also constitute a source of infection of domestic animals and feed plants or vice versa. Three isolates from cattle and a grain source, of which two were multiresistant, were confirmed as serovar Typhimurium phage type DT 104. These represent the first reported phage type DT 104 isolates from other sources than humans in Norway.