Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis

This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.     

Comparison of ribotyping,randomly amplified polymorphic DNA,and pulsed-field gel electrophoresis for molecular typing of Vibrio tapetis

Brown ring disease, caused by Vibrio tapetis, is an important pathological problem in different species of cultured clams. In order to evaluate the genetic diversity of the pathogen, twenty-seven isolates of V tapetis with different origin were screened by ribotyping (RT), pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA PCR (RAPD). Restriction with PvuII, SalI, and SmaI gave 2 RT patterns, differentiating in all cases the strain 0202RD isolated from carpet-shell clams (Ruditapes decussatus) from the other strains tested. The use of NotI generated strain specific PFGE profiles, which could be grouped in two main clusters. Cluster 1 grouped all but one strain and was subdivided into six PFGE subtypes (1a to 1f) which joined at a similarity level of 75.6%. Cluster 2 included again only the isolate 0202RD. RAPD analysis yielded the same results with three different primers, this method being able to differentiate the isolates from R. decussatus from those isolated from other clam species. Of the three techniques evaluated, PFGE was the most discriminating of the three techniques evaluated, followed in discriminating power by RAPD and RT tests. On the basis of the results obtained, we conclude that the RAPD procedure, which is more rapid and easier to perform than the other techniques, shows to be very useful to analyze large amounts of strain collections from an epidemiological monitoring stanpoint. In addition, PFGE is of great utility to evaluate the genetic diversity of strains involved in an outbreak and to study the spreading of a specific clone.     

Genotyping of Mycoplasma bovis isolates using multiple-locus variable-number tandem-repeat analysis

Mycoplasma bovis has been considered an important cause of mastitis, pneumonia, and arthritis in bovines with a highly detrimental economic impact in the livestock industry. Previous epidemiological studies, using different typing techniques showed that the isolates were usually heterogeneous and results were difficult to compare between different laboratories. Reliable and comparable molecular typing techniques using geographically and temporal distinct isolates have never been used. The objective of this study was to use multiple-locus variable-number tandem-repeat analysis (MLVA) for the differentiation of M. bovis isolates. This typing scheme was developed using the sequenced genome of the M. bovis PG45 type strain. Nine tandem-repeat sequences were selected and the genetic diversity of 37 isolates of wide geographic and temporal origins was analyzed. The results were compared to those obtained with random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) for the same isolates. Cluster concordance between techniques was evaluated using Adjusted Rand and Wallace coefficients, and different cutoff levels of similarity were tested. Acceptable values of ≥0.5 for all techniques for the Wallace coefficient were only observed at the most relaxed cutoff level, i.e., 52% for MLVA, 29% for PFGE and 18% for RAPD. The Simpson's index of diversity was 0.91 for MLVA, 0.99 for RAPD analysis and 0.99 for PFGE. This MLVA assay is compatible with simple PCR and agarose gel-based electrophoresis steps as well as with high-throughput automated methods. The molecular typing scheme presented in this study provides a fast, reliable, and cost-effective typing method for surveillance of M. bovis epidemiology.     

Electrophoretic karyotypes of <Emphasis Type="Italic">C. neoformans</Emphasis> serotype A recovered from Thai Patients with AIDS

Thirty-seven clinical isolates of C. neoformans were recovered from AIDS patients and all were serotype A according to standard typing tests. They were further analyzed using RAPD, PCR fingerprinting, and PFGE along with 2 additional reference isolates ATCC 34871 (serotype A) and RV 45981 (serotype D). Using 2 different RAPD primers, all of the clinical isolates and the reference serotype A (ATCC 34871) gave similar RAPD patterns while serotype D (RV 45981) gave distinctive pattern. Corresponding result was also obtained upon PCR by using a primer for microsatellite (GACA)₄. However, using a primer specific to minisatellite M13 + 1, all PCR fingerprinting gave similar gel patterns (M1) for 35/37 of the clinical isolates and the reference serotype A while two clinical isolates generated different patterns called M2 and M3. The reference serotype D gave distinctive pattern called M4. PFGE gave 17 different karyotypes that could be categorized into 4 groups named EKA (1–6), EKB (1–5), EKC (1– 5) and EKD (1). The reference serotype A fell into group EKA as EKA6 while the reference serotype D fell into group EKC as EKC5. Among the clinical isolates, EKA group (20/37 isolates) and type EKA1 (16/20) dominated with only one isolate each for types EKA2 to EKA5. The next most prevalent was group EKB (12/37 isolates) which dominately fell in type EKB1 (8/12) and only one isolate each for types EKB2 to EKB5. Group EKC (4/37 isolates) and group EKD (1/37) had only one isolate for each type (EKC1 to EKC 4 and EKD1). The 2 predominant karyotypes (EKA1, 16/37 and EKB1, 8/37) may represent two originally common clones of C. neoformans expose among the patients. The high discriminatory power of PFGE infers the benefit of subtyping which lead to better understanding on the epidemiology and pathogenic potential of C. neoformans subtypes. Moreover, PCR fingerprinting and RAPD infer the feasibility of detail analysis between serotypes A and D for unencapsulated C. neoformans.     

A comparison of molecular typing methods for Moraxella catarrhalis

AIMS: Three molecular typing techniques were examined to determine which method was the most discriminatory in order to perform epidemiological typing of Moraxella catarrhalis. METHODS AND RESULTS: Twenty-five Mor. catarrhalis isolates obtained from nasopharyngeal aspirates collected from Aboriginal and non-Aboriginal children were subjected to random amplified polymorphic DNA (RAPD) analysis, automated ribotyping and pulsed field gel electrophoresis (PFGE). RAPD analysis determined two Mor. catarrhalis types, automated ribotyping with PstI determined four Mor. catarrhalis ribogroups and PFGE analysis with NotI determined 21 pulse field groups within the 25 isolates examined. CONCLUSIONS: Analysis of discrimination index and typeability demonstrated that PFGE is the most discriminatory method for typing Mor. catarrhalis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that PFGE is the most appropriate molecular tool for the epidemiological study of Mor. catarrhalis.     

Use of molecular typing methods to trace the dissemination of Listeria monocytogenes in a shrimp processing plant.

Molecular typing of bacteria has been widely used in epidemiological studies but not as extensively for tracing the transmission of pathogenic bacteria in food plants. This study was conducted to examine the potential use of two molecular typing methods, random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE), to trace Listeria monocytogenes contamination in a shrimp processing plant. Ribotyping and phase typing were also performed on a select number of strains. One hundred fifteen strains of L. monocytogenes collected in different areas of a shrimp processing plant were first serotyped and then subtyped by molecular typing. RAPD and PFGE showed great promise for typing L. monocytogenes isolates since distinguishable and reproducible DNA polymorphisms were obtained. When the composite profile from both (RAPD and PFGE) methods was generated, there was an increase in the discriminatory power to discern differences between strains of L. monocytogenes. The results indicated that environmental strains all fell into composite profile groupings unique to the environment, while strains from both water and utensils shared another composite profile group. L. monocytogenes fresh shrimp isolates belonging to one profile group were found in different areas of the processing line. This same profile group was also present in food handlers from the processing and packaging areas of the plant.     

Comparison of five molecular subtyping methods for differentiation of Salmonella Kentucky isolates in Tunisia

Salmonellosis is one of the most common causes of food-borne infection worldwide. In the last decade, Salmonella enterica serovar Kentucky has shown an increase in different parts of the world with the concurrent emergence of multidrug-resistant isolates. These drug-resistant types spread from Africa and the Middle East to Europe and Asia. Although S. Kentucky serovar is of potential human relevance, there is currently no standardized fingerprinting method for it, in Tunisia. In the present study, a collection of 57 Salmonella Kentucky isolates were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), ribotyping, enterobacterial repetitive intergenic consensus (ERIC) fingerprinting, and Random Amplification of Polymorphic DNA. Plasmid profiling showed a discriminatory index (D) of 0.290, and only 9 out of 57 (16 %) isolates carried plasmids, which represents a limitation of this technique. Fingerprinting of genomic DNA by PFGE and ribotyping produced 4 and 5 patterns, respectively. Distinct PFGE patterns (SX1, SX2, SX3, and SX4) were generated for only 28 strains out of 57 (49.1 %) with a D value of 0.647. RAPD fingerprinting with primers RAPD1 and RAPD2 produced 4 and 20 patterns, respectively. ERIC fingerprinting revealed 14 different patterns with a high discriminatory index (D) of 0.903. When the methods were combined, the best combination of two methods was ERIC-2 with RAPD2. These results indicates that a single method cannot be relied upon for discriminating between S. Kentucky strains, and a combination of typing methods such ERIC2 and RAPD2 allows further discrimination.     

PFGE and RAPD profiling differentiates closely related isolates of Ancylobacter aquaticus

1,2-dichloroethane (DCA) is a toxic synthetic haloalkane produced annually in excess of 20 billion tons. Five bacterial isolates capable of complete mineralization of DCA have recently been isolated from wastewater treatment facilities in South Africa. Pulsed field gel electrophoresis (PFGE) and random amplification of polymorphic DNA (RAPD) analysis were employed in this study to identify phylogenetic differences between these closely-related bacteria. Analysis of the 16S rDNA sequences of the selected isolates revealed similarities to previously characterised isolates of Ancylobacter aquaticus. It has been previously shown that all isolates follow the same catabolic pathway and possess an identical hydrolytic dehalogenase (DhlA) involved in the initial carbonchlorine bond cleavage. Analysis of homology matrices deduced from RAPD and restriction profiles, constructed using the GelCompar software package, revealed that although some of the isolates possessed identical profiles using one primer or restriction endonuclease, differences were observed when a different primer was used. Furthermore, the results obtained indicate that the previously characterised isolate A. aquaticus AD25 is significantly different from the isolates used in this study. PFGE was also able to show that isolates of A. aquaticus do not possess the 200 kb plasmid containing the hydrolytic dehalogenase gene previously identified in the DCA-degrading bacterium Xanthobacter autotrophicus GJ10. This study has been able to demonstrate that RAPD and PFGE analysis are suitable molecular tools for the differentiation of closely-related A. aquaticus isolates and may be routinely used in the differentiation of environmentally important bacteria.     

Polyphasic study of the genetic diversity of lactobacilli associated with 'Almagro' eggplants spontaneous fermentation, based on combined numerical analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoresis patterns

AIMS: The goal of this study was to assess the genetic diversity of lactic acid bacteria (LAB) from the complex natural ecosystem present in the spontaneous fermentation of 'Almagro' eggplants by a polyphasic approach based on molecular techniques. METHODS AND RESULTS: Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) were applied to 149 Lactobacillus isolates obtained from that fermentation process. Two random primers, OPL-05 and ArgDei-For, and two rare-cutting enzymes, SfiI and SmaI, chosen after preliminary testing on the basis of band intensity and distribution, were used. RAPD and PFGE generated electrophoretic patterns suitable for strain discrimination, but further discrimination was achieved when combined numerical analysis of the results from both methods and the results previously obtained by SDS-PAGE whole cell protein analysis, was carried out. The findings indicated a considerable degree of genomic diversity in the LAB microbiota studied and especially in the Lactobacillus plantarum isolates. In terms of species assignment, the polyphasic study allowed a definite and well-founded identification of 98.7% of the isolates. CONCLUSIONS: The combined numerical analysis of RAPD and PFGE patterns represented a useful tool to discriminate the diversity of the Lactobacillus strains responsible for the spontaneous fermentation of this pickle. The species identification and strain typing results from the polyphasic study were regarded as the most exact compromise yielding the fewest contradictions based on the available data. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined numerical analysis of RAPD-PCR and PFGE patterns has not yet been employed to study the genetic diversity of LAB from an ecosystem like that found in fermenting vegetables.     

Identification and genetic fingerprinting of Brachyspira species

Six Brachyspira type and reference strains, and 14 well characterized porcine field isolates representing all recognised porcine Brachyspira spp. were compared by different molecular methods. Sequence analysis of the 16S rRNA and the nox genes, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) were used in the study. In addition the isolates were analysed by five species-specific PCR systems. The topologies of the dendrograms obtained from each of the four typing systems were different. The B. pilosicoli isolates formed monophyletic clusters in all dendrograms, but with different sister lines. All five porcine Brachyspira species formed monophyletic clusters in the nox gene-based dendrogram only. All five PCR systems accurately identified their targets, except for the nox gene-based B. intermedia-specific system, by which it was not possible to identify one of the presumed B. intermedia isolates, and the other B. intermedia-specific system, based on the 23S rRNA gene, gave a positive reaction for one B. innocens isolate. In an extended study, 46 additional isolates and the original eight isolates with the phenotypes of B. hyodysenteriae or B. intermedia were compared by PFGE and PCR. The PFGE results indicated a high genetic diversity of isolates with the phenotype of B. intermedia. Thirty-three of 34 tested isolates could be identified by one or both of the two B. intermedia-specific PCR systems used, however, only 19 of the 34 isolates were positive in both systems.     

Persistence of a Salmonella enterica serotype typhimurium clone in Danish pig production units and farmhouse environment studied by pulsed field gel electrophoresis (PFGE)

The clonal relationship among Salmonella enterica serotype Typhimurium isolates from selected pig production units in Denmark was investigated by the pulsed field gel electrophoresis (PFGE) typing method to determine environmental survival and spread of Salmonella in different herds. Thirty-four Typhimurium isolated during 1996-1998 from porcine faeces and environmental samples from three pig farms designated 1, 3 and 5 were characterised by PFGE using two restriction enzymes. Farm 5 supplied piglets to farm 1 and the herds were located close to each other. Results of PFGE analysis showed both intra- and inter-relationships, i.e. identical PFGE patterns among the faecal and environmental isolates from farm 1 and farm 5. All the isolates from farm 3 irrespective of the source showed identical PFGE patterns, but were different from samples from farms 1 and 5. This study indicates spread between farms and survival of a farm-specific clone. Furthermore, identical PFGE patterns of isolates from piglet supplier and finisher herds indicate that the farrow-to-grower herd of farm 5 was sub-clinically infected prior to delivery to farm 1 and thereby caused the transmission of Salmonella.     

Diverse geno- and phenotypes of persistent Listeria monocytogenes isolates from fermented meat sausage production facilities in Portugal

The persistence of Listeria monocytogenes in food-associated environments represents a key factor in transmission of this pathogen. To identify persistent and transient strains associated with production of fermented meat sausages in northern Portugal, 1,723 L. monocytogenes isolates from raw material and finished products from 11 processors were initially characterized by random amplification of polymorphic DNA (RAPD), PCR-based molecular serotyping, and epidemic clone characterization, as well as cadmium, arsenic, and tetracycline resistance typing. Pulsed-field gel electrophoresis (PFGE) typing of 240 representative isolates provided evidence for persistence of L. monocytogenes for periods of time ranging from 10 to 32 months for all seven processors for which isolates from different production dates were available. Among 50 L. monocytogenes isolates that included one representative for each PFGE pattern obtained from a given sample, 12 isolates showed reduced invasion efficiency in Caco-2 cells, including 8 isolates with premature stop codons in inlA. Among 41 isolates representing sporadic and persistent PFGE types, 22 isolates represented lysogens. Neither strains with reduced invasion nor lysogens were overrepresented among persistent isolates. While the susceptibility of isolates to lysogenic phages also did not correlate with persistence, it appeared to be associated with molecular serotype. Our data show the following. (i) RAPD may not be suitable for analysis of large sets of L. monocytogenes isolates. (ii) While a large diversity of L. monocytogenes subtypes is found in Portuguese fermented meat sausages, persistence of L. monocytogenes in this food chain is common. (iii) Persistent L. monocytogenes strains are diverse and do not appear to be characterized by unique genetic or phenotypic characteristics.     

Biodiversity of Clostridium botulinum type E strains isolated from fish and fishery products

The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (SmaI-XmaI and XhoI) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.     

Biodiversity of Clostridium botulinum Type E Strains Isolated from Fish and Fishery Products

The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (SmaI-XmaI and XhoI) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.     

Comparison of traditional and molecular typing methods of Escherichia coli O157

An account is given using typing methods and detection of virulence genes of different serotypes of Escherichia coli isolated in Hungary. By hybridization using SLT-I and SLT-II probes and PCR method using stx1-2, eae and ehx primers we could differentiate O157 strains of different serotypes into eight (stx, eae, ehxA positive; stx, eae positive; stx, ehxA positive; stx positive; eae, ehxA positive; eae positive; ehxA positive; stx, eae, ehxA negative) types. The discriminatory power of phage typing proves to be much higher than that of the plasmid profile. RAPD typing with different primers could confirm or exclude the subtypes identity of the isolated E. coli O157 serotypes. Escherichia coli O157:HNM isolates could be sorted in six different phage types and six different RAPD types with ERIC-1, in five RAPD types with ERIC-2 and in seven types with M13 primers. Escherichia coli O157:H7 showed six different phage types and three RAPD types with ERIC-1 and ERIC-2 and five types with M13 primers. According to our results the standard PFGE protocol [32] gives the opportunity to differentiate epidemiologically independent but evolutionary related or unrelated isolates, but the practical value of PFGE method for epidemiological purposes must be confirmed by other or more restriction enzymes or using an other protocol. Summarizing our results we suggest the use of phage and RAPD typing and in doubtful cases the PFGE method.     

Characterization of halotolerant rhizobia isolated from root nodules of Canavalia rosea from seaside areas

Twelve nodule isolates from Canavalia rosea, an indigenous leguminous halophyte growing in the seaside areas of southern Taiwan, were effective symbionts for the original host and able to grow at NaCl concentrations up to 3-3.5% (w/v). The taxonomy of these isolates was investigated using a polyphasic approach, including phenotypic characteristics, banding patterns of total proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), genomic fingerprint patterns from random amplified polymorphic DNA (RAPD) analysis, pulsed-field gel electrophoresis (PFGE) analysis, amplified 16S rDNA restriction analysis (ARDRA), 16S rRNA gene sequencing, and nifH gene sequencing. Based on the SDS-PAGE, RAPD, PFGE and ARDRA results, the 12 isolates are highly diverse. The 16S rRNA and nifH gene sequences were determined for isolates with distinct ARDRA patterns and compared with other members of the rhizobial species. We propose these isolates should be classified into the genus Sinorhizobium and distinguished from the current species of this genus.     

Heterogeneity in expression of lipopolysaccharide by strains of Salmonella enterica serotype Typhimurium definitive phage type 104 and related phage types

AIMS: To investigate lipopolysaccharide (LPS) expression in Salmonella enterica serotype Typhimurium definitive phage type 104 (Salmonella Typhimurium DT104) and related phage types. METHODS AND RESULTS: Isolates were examined for the expression of LPS by SDS-PAGE and silver staining and subtyped by Pulsed Field Gel Electrophoresis (PFGE). The 100 isolates expressed one of two LPS profiles designated A (72%) and B (28%). LPS profiling was able to discriminate between isolates of identical PFGE type. Among 10 groups of outbreak isolates examined, each group was of a single LPS profile: A, 8/10 and B, 2/10. All 10 outbreaks were identical by PFGE analysis. CONCLUSIONS: Isolates of Salmonella Typhimurium DT104 and related phage types expressed one of two distinct LPS profiles. The two LPS profiles appear similar but shifted and in phase with one another, suggesting that the heterogeneity is due to changes in the LPS core region rather than among the repeating oligosaccharide units of the long-chain LPS. SIGNIFICANCE AND IMPACT OF THE SUTDY: LPS profiling provides a useful adjunct to PFGE and other molecular methods for the subtyping of this group of bacteria in epidemiological investigations.     

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.     

Genotypic analysis of Escherichia coli recovered from product and equipment at a beef-packing plant

AIMS: To identify sources of Escherichia coli on beef by characterizing strains of the organism on animals, equipment and product at beef-packing plant. METHODS AND RESULTS: Generic E. coli were recovered from hides, carcasses, beef trimmings, conveyers and ground beef during the summer of 2001 (750 isolates) and winter of 2002 (500 isolates). The isolates were characterized by Random Amplification of Polymorphic DNA (RAPD). The numbers of E. coli recovered from dressed carcasses were less than the numbers recovered from hides. The numbers recovered from chilled carcasses were too few for meaningful analysis of the strains present on them but the numbers recovered from trimmings and ground beef were larger. The RAPD patterns showed that the majority of isolates from hides, carcasses, beef trimmings, conveyers and ground beef were of similar RAPD types, but a few unique RAPD types were recovered from only one of those sources. The E. coli populations present on the hides of incoming animals and in the beef-processing environment were highly diverse. Randomly selected E. coli isolates from each of the five sources were further characterized by pulsed-field gel electrophoresis (PFGE). Most genotypes of E. coli defined by PFGE corresponded to the E. coli types defined by RAPD. CONCLUSIONS: The hides of the incoming animals appeared to be only one of the sources of the E. coli on trimmings and in ground beef, as additional sources were apparently present in equipment used for carcass breaking. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that hazardous microbiological contamination of meat may occur after the dressing of carcasses at commercial beef-packing plants, which suggests that attention should be given to the control of the contamination of meat during carcass breaking as well as during the dressing of carcasses.     

Comparison of molecular techniques for the typing of Mycoplasma hyopneumoniae isolates

In this study, we compared the potential of amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) analysis, restriction fragment length polymorphism (RFLP) of the gene encoding lipoprotein P146, and the variable number of tandem repeats (VNTR) of the P97 encoding gene, as possible methods for typing an international collection of Mycoplasma hyopneumoniae isolates. All techniques showed a typeability of 100% and high intraspecific diversity. However, the discriminatory power of the different techniques varied considerably. AFLP (>0.99) and PCR-RFLP of the P146 encoding gene (>0.98) were more discriminatory than RAPD (0.95) and estimation of the VNTR of P97 (<0.92). Other, preferentially well spread, tandem repeat regions should be included in order for this latter technique to become valuable for typing purposes. RAPD was also found to be a less interesting typing technique because of its low reproducibility between different runs. Nevertheless, all molecular techniques showed overall more resemblance between strains isolated from different pigs from the same herd. On the other hand, none of the techniques was able to show a clear relationship between the country of origin and the fingerprints obtained. We conclude that AFLP and an earlier described PFGE technique are highly reliable and discriminatory typing techniques for outlining the genomic diversity of M. hyopneumoniae isolates. Our data also show that RFLP of a highly variable gene encoding P146 may be an equally useful alternative for demonstrating intraspecific variability, although the generation of sequence variability of the gene remains unclear and must be further examined.