A variation of the amplified-fragment length polymorphism (AFLP) technique using three restriction endonucleases, and assessment of the enzyme combination BglII-MfeI for AFLP analysis of Salmonella enterica subsp. enterica isolates.

We have performed amplified-fragment length polymorphism (AFLP) fingerprinting on a collection of Salmonella enterica subsp. enterica serovar typhimurium strains with a restriction endonuclease combination (BglII and MfeI) that has previously been used successfully for typing Campylobacter jejuni isolates with high resolution. Additionally, a variation of the AFLP assay in which two rare cutting restriction enzymes (XbaI and BsrGI) in combination with the frequent cutter (HinP1I) was examined. The BglII and MfeI enzyme combination offered low resolution for genotyping Salmonella typhimurium isolates and is not recommended for this common serovar. The three-enzyme combination gave a higher discrimination, and is thus a new alternate way of performing AFLP fingerprinting of S. typhimurium.

Harmonization of the multiple-locus variable-number tandem repeat analysis method between Denmark and Norway for typing Salmonella Typhimurium isolates and closer examination of the VNTR loci.

AIMS: Harmonization and evaluation of the multiple-locus variable-number tandem repeat analysis (MLVA) method for sub-typing Salmonella enterica ssp. enterica serovar Typhimurium (Salm. Typhimurium) in Denmark and Norway, and analysis of the typing data. METHODS AND RESULTS: The Salm. Typhimurium MLVA (STMLVA) method, which uses length polymorphisms in five tandem-repeated DNA loci to differentiate isolates, was harmonized between Denmark and Norway, using a common set of 14 isolates. The MLVA assay that is routinely used at the Norwegian Institute of Public Health was set up at the Statens Serums Institute. Both the institutes used an ABI-310 Genetic Analyzer for capillary separation of PCR products, and the same internal size standard. Running the same set of 14 test isolates in both countries and comparing the results showed an excellent typing match at all loci in all isolates. Subsequently, 461 isolates were genotyped in Norway and 454 isolates were genotyped in Denmark. The STMLVA assay displayed a large number of allelic profiles that were distinct for each country as well as shared profiles. Differences in variable number of tandem repeats allele frequencies and absence of amplification products were observed between Denmark and Norway. CONCLUSIONS: The MLVA method was set up in two different laboratories and produced completely matching typing data that could be shared rapidly by e-mail for comparison. Notably, differences in allele frequencies and absence of amplification were noted between the countries. SIGNIFICANCE AND IMPACT OF THE STUDY: The STMLVA method was shown to be easily implemented and to produce typing data, which were shared over the Internet. This enables increased speed of typing and comparison of data between countries, when compared with earlier typing methods. Information embedded in the allele frequencies might give clues to the origin and source of isolates.

Intestinal carriage of Campylobacter jejuni and Campylobacter coli among cattle from south-western Norway and comparative genotyping of bovine and human isolates by amplified-fragment length polymorphism.

In a survey conducted in 1999-2001, the carriage of thermotolerant Campylobacters in cattle was investigated, and the genetic diversity of C. jejuni within one herd was examined and compared with human isolates. C. jejuni, C. coli and other thermotolerant Campylobacter spp. were isolated from intestinal contents from 26%, 3% and 2% of 804 cattle, respectively. The carriage rate was higher in calves (46%) than in adults (29%). Twenty-nine C. jejuni isolates from one herd and 31 human isolates from the study area were genotyped with amplified-fragment length polymorphism (AFLP). Eighty-three % of the bovine isolates fell into three distinct clusters with 95-100% similarity, persistent in the herd for 5-10 months. Among human isolates, 58% showed >90% similarity with bovine isolates. The results show that cattle are a significant and stable reservoir for C. jejuni in the study area. Transmission between individuals within the herd may be sufficient to maintain a steady C. jejuni population independent of environmental influx. The results of this study have provided new information on C. jejuni and C. coli transmission, and also on the carriage in cattle, genotypes stability and similarity between bovine and human isolates.

Molecular epidemiology of Salmonella spp. isolates from gulls, fish-meal factories, feed factories, animals and humans in Norway based on pulsed-field gel electrophoresis.

The molecular epidemiology of 98 isolates of Salmonella serovar Agona (n = 27), S. Montevideo (n = 42) and S. Senftenberg (n = 29) from wild-living gulls, fish-meal factories, feed factories, humans and domestic animals was investigated using pulsed-field gel electrophoresis (PFGE) and computerized numerical analysis. Two of the S. Agona profiles were identified both in gulls and in two of the factories. In addition, one of these profiles was detected in two infected poultry farms. Two of the S. Montevideo profiles were also identified both in gulls and in two of the factories, and one of these profiles was observed in a human isolate. Four factories shared an identical S. Senftenberg profile. The S. Senftenberg profile found in gulls was not identified in any other source investigated. The presence of isolates with identical PFGE profiles indicates potential epidemiological links between different factories, as well as between gulls and factories.

Fluorescent amplified-fragment length polymorphism genotyping of Salmonella enterica subsp. enterica serovars and comparison with pulsed-field gel electrophoresis typing.

We have performed the fluorescently labeled amplified-fragment length polymorphism (FAFLP) method on 97 strains of the food-borne pathogen Salmonella enterica subsp. enterica comprising seven different serovars using the restriction enzymes EcoRI and MseI. From the total FAFLP fingerprinted strains, 81 were compared with pulsed-field gel electrophoresis (PFGE) typing of the same strains. The FAFLP method showed a discriminatory power equal to that of PFGE. We report a fast, robust, and high-resolution adaptation of the AFLP assay for fingerprinting S. enterica subsp. enterica serovars with capillary electrophoresis that can be scaled to high throughput on automated analysis instruments.

Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay.

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8, 700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as

Detection of low numbers of Salmonella in environmental water, sewage and food samples by a nested polymerase chain reaction assay.

A polymerase chain reaction (PCR) assay with two nested pairs of primers selected from conserved sequences within a 2.3 kb randomly cloned DNA fragment from the Salmonella typhimurium chromosome was developed. The nested PCR assay correctly identified 128 of a total of 129 Salmonella strains belonging to subspecies I, II, IIIb and IV. One strain of Salm. arizona (ssp. IIIa) tested negative. No PCR products were obtained from any of the 31 non-Salmonella strains examined. The sensitivity of the assay was 2 cfu, as determined by analysis of proteinase K-treated boiled lysates of Salm. typhimurium. The performance of the assay was evaluated for environmental water, sewage and food samples spiked with Salm. typhimurium. Water and sewage samples were filtered and filters were enriched overnight in a non-selective medium. Prior to PCR, the broth cultures were subjected to a rapid and simple preparation procedure consisting of centrifugation, proteinase K treatment and boiling. This assay enabled detection of 10 cfu 100 ml(-1) water with background levels of up to 8700 heterotrophic organisms ml(-1) and 10000 cfu of coliform organisms 100 ml(-1) water. Spiked food samples were analysed with and without overnight enrichment in a non-selective medium using the same assay as above. Nested PCR performed on enriched broths enabled detection of <10 cfu g(-1) food. Variable results were obtained for food samples examined without prior enrichment and most results were negative. This rapid and simple assay provides a sensitive and specific means of screening drinking water or environmental water samples, as well as food samples, for the presence of Salmonella spp.

Detection of low numbers of pathogenic Yersinia enterocolitica in environmental water and sewage samples by nested polymerase chain reaction.

Isolation of pathogenic Yersinia enterocolitica from water and sewage by traditional culture techniques is time-consuming and subsequent differentiation between pathogenic and non-pathogenic strains can be difficult and unreliable. A nested polymerase chain reaction (PCR) procedure was used for the detection of low numbers of Y. enterocolitica in spiked samples from natural surface sources with variable background flora ranging from oligotrophic water to sewage. Water and sewage samples were filtered and filters enriched overnight in a non-selective medium. Nested PCR conducted on enriched broth, prepared by use of a rapid and simple preparation step consisting of centrifugation, proteinase K treatment and boiling, enabled the detection of 8-17 cfu 100 ml-1 water with background levels of up to 8700 heterotrophic organisms ml-1 and 10,000 cfu coliform organisms 100 ml-1 water. The analysis can be completed within 2-3 d and should be a significant tool in monitoring environmental waters and drinking water sources for the presence of pathogenic Y. enterocolitica.

Molecular epidemiology of Salmonella typhimurium isolates from human sporadic and outbreak cases.

The molecular epidemiology of a representative collection of sporadic foreign and domestically acquired Salmonella Typhimurium (S. Typhimurium) isolates from Norwegian patients in 1996-9 was studied by numerical analysis of pulsed-field gel electrophoresis (PFGE) profiles. Three subclusters (E5, F1 and G1) comprised 47% of the 102 sporadic isolates investigated and 45% of the domestically acquired isolates fell in subclusters E5 and F1. Distinct seasonal and geographic variations were evident for these strains which have been responsible for both local outbreaks (E5) and a national epidemic (F1) where salmonella-infected hedgehogs and birds constituted the suggested primary source of infection. Subcluster G1 was dominated by imported multi-resistant definitive type (DT) 104 isolates. All multi-resistant isolates contained integron-associated gene cassette-structures. This study presents valuable information on the relative significance, geographic distribution and antibiotic resistance features of distinct S. Typhimurium clones causing human salmonellosis among Norwegians.

Molecular epidemiology and population genetics of Salmonella subspecies diarizonae in sheep in Norway and Sweden.

Fifty-four isolates of Salmonella enterica subsp. diarizonae (IIIb) in Norway, Sweden, England, the United States, France and Australia were characterized by pulsed-field gel electrophoresis (PFGE). This study focuses on serovar 61:k:1,5,(7) [S. IIIb 61:k:1,5,(7)] isolated from sheep. Digestion of the bacterial DNA with restriction enzyme XhaI yielded 15 distinct PFGE profiles comprising 12-16 fragments in the range 48.5-630.5 kbp. Four different profiles were identified in Norwegian sheep isolates and a single profile in Swedish isolates. The spatial and temporal distribution of profiles is discussed.

Genomic fingerprinting of shigatoxin-producing Escherichia coli (STEC) strains: comparison of pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment-length polymorphism (FAFLP).

For epidemiological studies of shigatoxin-producing Escherichia coli (STEC) infections, rapid, reproducible and highly discriminative methods are required. In this study, we examined the performance of the fluorescent amplified-fragment-length polymorphism (FAFLP) technique for epidemiological fingerprinting of STEC isolates and compared it to the acknowledged fingerprinting method pulsed-field gel electrophoresis (PFGE). A total of 88 STEC isolates, including 82 of serotype O157:H7 or O157:H-, were subjected to fingerprinting by both PFGE and FAFLP. The isolates included sporadic and epidemiologically related strains of both animal and human origin from widespread geographical locations. The FAFLP fingerprint patterns confirmed the clonal nature of STEC O157 strains. Among the 82 O157:H7/H- isolates belonging to 49 distinct groups of epidemiological unrelated isolates, 24 FAFLP profiles and 51 PFGE patterns were obtained. Thus, PFGE had a higher discriminatory power than FAFLP and overall correlated better to available epidemiological data. Consequently, the PFGE technique remains the method of choice in epidemiological investigations of STEC infections.

Detection of pathogenic Yersinia enterocolitica in foods and water by immunomagnetic separation, nested polymerase chain reactions, and colorimetric detection of amplified DNA.

A two-step polymerase chain reaction (PCR) procedure with two nested pairs of primers specific for the yadA gene of Yersinia enterocolitica was developed. The PCR assay identified all common pathogenic serogroups (O:3, O:5,27, O:8, O:9, O:13, and O:21) from three continents and differentiated pathogenic Y. enterocolitica from Y. pseudotuberculosis and from a variety of nonpathogenic yersiniae representing 25 serogroups and four species. The performance of the method was evaluated with seeded food and water samples. We compared two procedures for sample preparation prior to PCR: one was based on immunomagnetic separation of the target bacteria from the sample, using magnetic particles coated with immunoglobulin antibodies to Y. enterocolitica serogroup O:3, and the other method consisted of a series of centrifugation steps combined with proteinase treatment. Regardless of the method used, the PCR assay was capable of detecting 10 to 30 CFU/g of meat in 10(6)-fold excess of indigenous bacteria. When the samples were enriched overnight in a nonselective medium, the sensitivity was increased to approximately 2 CFU/g, except for samples with an extremely high background flora (> 10(7) CFU/g). We compared gel electrophoretic detection of PCR products with a colorimetric detection method designated DIANA (detection of immobilized amplified nucleic acids), which enabled easy visualization of amplified fragments in a microtiter plate format with an optical density reader. DIANA and gel electrophoresis showed complete concordance in their discrimination between positive and negative samples. The combination of immunomagnetic separation, nested PCR, and DIANA makes possible the development of a fully automated analytic process which requires a minimum of laboratory manipulations.

Comparative fingerprinting analysis of Campylobacter jejuni subsp. jejuni strains by amplified-fragment length polymorphism genotyping.

Amplified-fragment length polymorphism (AFLP) analysis with the endonucleases BglII and MfeI was used to genotype 91 Campylobacter jejuni subsp. jejuni strains from outbreaks and sporadic cases. AFLP-generated fragments were labeled with fluorescent dye and separated by capillary electrophoresis. The software packages GeneScan and GelCompar II were used to calculate AFLP pattern similarities and to investigate phylogenetic relationships among the genotyped strains. The AFLP method was compared with two additional DNA-based typing methods, pulsed-field gel electrophoresis (PFGE) using SmaI and restriction fragment length polymorphism analysis on PCR products (PCR-RFLP) of the flaA and flaB genes. We found that AFLP analysis of C. jejuni strains is a rapid method that offers better discriminatory power than do both PFGE and PCR-RFLP. AFLP and, to a lesser extent, PCR-RFLP could differentiate strains within the same PFGE profiles, which also makes PCR-RFLP an alternative to PFGE. We were able to clearly distinguish 9 of 10 recognized outbreaks by AFLP and to identify similarities among outbreak and sporadic strains. Therefore, AFLP is suitable for epidemiological surveillance of C. jejuni and will be an excellent tool for source identification in outbreak situations.