INTRODUCTION

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Campylobacter spp. are among the most frequently reported causes of bacterial enteritis in the developed countries. The number of human cases of campylobacteriosis has increased dramatically in recent years in many countries. In Denmark, the number of cases has more than tripled during the last 7 years from approximately 22 cases/100,000 inhabitants in the years 1980 to 1992 to 78/100,000 in 1999 (2). Approximately 95% of the Danish cases are caused by Campylobacter jejuni subsp. jejuni (hereafter C. jejuni).

A wide range of phenotypic and genotypic typing systems have been developed and used for epidemiological typing of Campylobacter spp. The phenotypic methods include serotyping with heat-stable (37) or heat-labile antigens (19), phage typing (40), and biotyping (4). The phenotypic methods, in particular the two serotyping systems, are used in laboratories worldwide, e.g., for surveillance of a large number of isolates. However, for improved discrimination of isolates one or more of the genotypic methods are usually selected. Some of the most commonly used genotypic methods for typing of Campylobacter are pulsed-field gel electrophoresis (PFGE), ribotyping, flagellin gene typing, random amplified polymorphic DNA typing (RAPD), and restriction endonuclease analysis (26, 35, 49). The genotypic methods have primarily been used for outbreaks and epidemiological studies of poultry flocks, etc., whereas less has been published regarding typing of sporadic human cases and surveillance of isolates from animal sources.

Despite the large number of different typing systems for Campylobacter, few studies comparing methods and their efficacies exist. Patton et al. (36) evaluated the ability of 10 phenotypic and genotypic methods to distinguish C. jejuni strains from animals and humans involved in four milk- and waterborne outbreaks. Three phenotypic methods (the two serotyping systems and multilocus enzyme electrophoresis) and three genotypic methods (PvuII/PstI ribotyping and two restriction endonuclease analysis methods) were able to correctly identify all epidemiologically implicated strains. In contrast, four other methods (biotyping, phage typing, plasmid profiling, and BglI/XhoI ribotyping) were not sufficiently discriminatory to make the correct groupings of strains. In another study, PFGE typing, heat-labile serotyping, biotyping, and fatty acid profile typing were compared for typing of C. jejuni and Campylobacter coli isolated from abattoirs (44). PFGE was found to be the most discriminatory method, and biotyping was found to be the least discriminatory. In two other studies, PFGE was also the most discriminatory method, whereas phage typing had low discrimination and typeability, and HaeIII/PstI ribotyping could differentiate between C. jejuni strains of different serotypes but differentiated only to a limited degree between strains of the same serotype (12, 34). In general, PFGE and ribotyping with some enzyme combinations showed good discriminatory power in the previous studies. In contrast, biotyping, phage typing, and plasmid profiling had low discriminatory power.

In the present study, we have evaluated one phenotypic method and five genotypic methods for subtyping of C. jejuni: heat-stable serotyping (Penner serotyping), PFGE, automated ribotyping (RiboPrinting), RAPD, PCR-restriction fragment length polymorphism (RFLP) on the flaA gene, and PCR-denaturing gradient gel electrophoresis (DGGE) on the flaA gene. Of these typing methods, PFGE, fla-RFLP, and serotyping have been used extensively for typing of C. jejuni by several laboratories including the laboratories participating in this study (25, 27, 28, 32). RAPD has also been used for typing of campylobacters previously (10, 14, 21). In the present study, the RAPD protocol included the use of standardized PCR analysis beads and fluorescence detection of the resulting profiles on an automated fragment analyzer. RiboPrinting is equivalent to ribotyping, the difference being that most of the process is automated. PCR-DGGE has been used for typing of human genes and mixed population fingerprinting (5, 23, 46) but has not previously been used for bacterial subtyping, although preliminary results have been presented (6; C.-H. Brogren and P. Venema, Abstr. IMBEM IV4th Int. Meet. Bacterial Epidemiol. Markers, abstr. S103, 1997).

The six methods were used for subtyping a collection of 90 C. jejuni isolates from animal sources, sporadic human cases, and a well-documented waterborne outbreak. The methods were evaluated and compared on the basis of their abilities to identify outbreak isolates and discriminate between unrelated isolates and the agreement between methods in identifying probable clones.

	MATERIALS AND METHODS

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Bacterial isolates. Ninety C. jejuni isolates obtained during an 11-month period in 1995-1996 were included in the study. Of these, 75 isolates were selected at random from strain collections at the Danish Veterinary Laboratory and the Statens Serum Institut: 40 human clinical isolates obtained from sporadic cases of campylobacteriosis with no known relation to each other, 20 isolates obtained from broiler chickens, and 15 isolates obtained from cattle. Fifteen isolates were related to a waterborne outbreak affecting a small Danish town during January to March 1996 (7). Of these, nine isolates were from patients with a confirmed relation to the outbreak, two were isolated from the suspected water, and four were clinical isolates from the same period and region but apparently epidemiologically unrelated to the outbreak (control isolates). Therefore, in total, 80 isolates were supposedly unrelated: the 75 randomly selected isolates, the four control isolates, and one representative of the outbreak isolates (strain 5001). All human strains were isolated from feces at the Statens Serum Institut using a standard procedure (7). All cattle and chicken strains were isolated at the Danish Veterinary Laboratory from fecal samples from healthy animals at slaughter according to a previously described procedure (27). Sets of identical cultures were prepared in 15% glycerol broth and stored at 80°C. One set was then distributed to each participating laboratory.

Serotyping. For antigen preparation, the bacteria were cultured on blood agar for 24 to 48 h at 42°C in a microaerobic atmosphere. Heat-stable serotyping (O serotyping) was performed according to the Penner serotyping scheme (37) with separate sets of sera for C. jejuni and C. coli (38). The C. jejuni strains were typed using all 47 C. jejuni antisera in the hemagglutination test. If a strain was nontypeable with these sera, the strain was also tested against the 19 C. coli antisera. The production of antisera has been described previously (27). If a strain reacted in more than one antiserum, it was designated a complex serotype, e.g., O:1,44, O:4,13,16,43,50,64 (=O:4 complex), O:6,7, or O:23,36. All complexes seen in this study were well-known and common C. jejuni serotype complexes (38). Different combinations of reactions with the O:4 complex were seen, but these were disregarded in the data analysis.

PFGE profiling. DNA-agarose samples were prepared from formaldehyde-treated bacterial cells using the protocol of Gibson et al. (11), modified as described previously (32, 33). DNA was digested with SmaI, and fragments were separated in a CHEF-DRIII PFGE system (Bio-Rad Laboratories, Hercules, Calif.), using parameters described previously (32). Any differences between PFGE profiles of strains were considered significant, and types were arbitrarily defined on that basis.

fla-RFLP typing. For fla-RFLP, a 1.7-kb fragment of the flaA gene was amplified and analyzed after digestion with two restriction enzymes, DdeI and AluI. Bacteria were grown on blood agar (5% cattle blood) overnight in a microaerobic atmosphere. Preparation of template for PCR and the PCRs were carried out largely as described by Nachamkin and colleagues (24) or, in a few cases, by using the commercial DNA isolation kit QIAamp tissue kit (Qiagen, Hilden, Germany) by use of the manufacturer's recommendations for gram-negative bacteria, without RNase treatment. The procedure was slightly modified so that each 50-µl PCR mixture contained 5.0 µl of Super Taq buffer (HT Biotechnology Ltd., Cambridge, United Kingdom); 5.0 µl of 100 mM Tris-HCl (pH 8.3); 3.0 µl of 25 mM MgCl₂, resulting in a total concentration of 3.0 mM Mg²⁺; 0.4 mM deoxynucleoside triphosphate (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom); 0.25 mM (each) primer; 2.5 U of Super Taq polymerase (HT Biotechnology Ltd.); and 5 µl of heated bacterial lysate, or 2.5 µl of DNA purified by QIAamp.

RiboPrinting. RiboPrinting was performed using the RiboPrinter, as recommended by the manufacturer (Qualicon, Wilmington, Del.). In brief, single colonies from a 24-h culture on a 5% yeast-enriched blood agar plate were suspended in a sample buffer and heated at 80°C for 15 min. After addition of lytic enzymes, samples were transferred to the RiboPrinter System. Further analysis, including HaeIII restriction of DNA, was carried out automatically. The RiboPrint profiles were aligned according to the position of a molecular size standard and compared with patterns obtained previously. Profiles were analyzed with the GelCompar software using the band matching coefficient of Dice and UPGMA (unweighted pair group method with averages) clustering to determine profile relatedness.

RAPD typing. Template DNA was extracted from a 24-h subculture on a yeast-enriched 5% blood agar plate by picking colony material corresponding to approximately 1 µg using a 1-µl inoculating loop. The colony material was mixed with 300 µl of a 20% slurry of Chelex-100 (Bio-Rad) in TE buffer (10 mM Tris [pH 8], 1 mM EDTA) and heated at 95°C for 10 min. The resin was pelleted by centrifugation at 10,000 rpm (Biofuge 13; Heraeus Sepatech) for 2 min. Two microliters of this suspension was used for subsequent amplifications. All PCR amplifications were performed using 25 pmol of primer with Ready-To-Go RAPD analysis beads (Amersham Pharmacia Biotech, Uppsala, Sweden), containing premixed, predispensed AmpliTaq DNA polymerase, as well as all necessary buffer ingredients and nucleotides. The cycling parameters were as follows: denaturing at 95°C for 30 s, annealing for 1 min at temperatures as stated below, and extension at 72°C for 2 min in a total of 31 cycles. Annealing started at 46°C with a 1°C decrease during 11 cycles until 36°C, followed by 20 cycles at 36°C. The ramping was done at 2.5°C/s and 1°C/s. Prior to cycling, samples were heated to 95°C for 5 min. Finally, an additional extension step of 72°C for 7 min was included. Amplifications were performed using a thermocycler, the PTC-200 Peltier Thermal cycler (MJ Research Inc., Watertown, Mass.), with a hot-start procedure. Fluorescently labeled primers 1281, 1254, and HLWL85 (1, 14, 21) were used in three independent amplifications, and the resultant PCR products were detected on an ABI PRISM 310 DNA Genetic Analyzer (Applied Biosystems, Naerum, Denmark) using the manufacturer's recommendations. In brief, 12 µl of deionized formamide, 1 µl of size standard 2500-ROX, and 1 µl of each of the three PCR amplifications were mixed and denatured at 95°C for 2 min and subsequently kept on ice until further processing. The ABI-310 instrument was prepared with a short capillary (47 cm) and POP4 polymer (4% performance optimized polymer; Applied Biosystems). Running conditions were as follows: injection time, 10 s; voltage, 15 kV; collection time, 45 min; electrophoresis voltage, 15 kV; and heat plate temperature, 60°C.

fla-DGGE typing. A 702-bp PCR fragment of the Campylobacter flaA gene (29) modified with a GC clamp attached to the 5' end of the reversed primer was used to optimize the DGGE point mutation analysis (42). The PCR amplicon was selected after testing 25 PCR systems for Campylobacter based on PCR fragments of 16S rRNA, 23S rRNA, flagellin genes flaA and flaB, and the VS1 gene (C.-H. Brogren, unpublished results). All four combinations of clamped systems (42) were tested. Based on band pattern and visual polymorphism, the chosen forward 20-mer primer (p1) was 5'-TAC TAC AGG AGT TCA AGC TT-3', and the 65-mer reversed primer (p4A) was 5'-GCG GGC GGG GCG GGG GCA CGG GGG GCG CGG CGG GCG GGG CGG GGG GTT GAT GTA ACT TGA TTT TG-3'. Template DNA was obtained by boiling a washed suspension of the isolate harvested from a brain heart infusion plate cultured under microaerobic conditions for 48 h. The DNA template samples were stored in small aliquots at 80°C. No improvement of the PCRs was observed after Qiagen DNA purification, which was therefore omitted. Ready-to-Go PCR beads (Amersham Pharmacia Biotech AB) were used for all PCR amplifications with addition of 5 pmol of each primer and 5 µl of template DNA (25 to 100 pg) in a final volume of 25 µl per reaction. PCR was performed within a Gene AMP System 2400 thermocycler (PE Biosystems, Foster City, Calif.) using the following protocol: 95°C for 5 min; cycling parameters at 95°C for 45 s, 60°C for 45 s, and 72°C for 45 s for 35 cycles; and final elongation at 72°C for 10 min. The final amplicon (747 bp) was stored at 4°C until analyzed or kept at 20°C for long-term storage. PCR amplicons were size controlled by 1.5% (wt/vol) Sepharide (Gibco-BRL, Glasgow, Scotland) agarose gel electrophorese. Purity and amount of DNA were evaluated visually.

	RESULTS

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Discriminatory power and typeability. The results of using six typing methods on a collection of 80 C. jejuni strains with no known relationships are presented in Table 1. Isolates grouped together by at least four of the six typing methods are marked with boldface in the table. With the exception of serotyping, all strains were assigned to types that had been defined arbitrarily according to the individual criteria described above. In Table 2, the methods are evaluated with respect to discriminatory index (D index), number of types obtained, number of unique types, and prevalence of dominant type. Serotyping and fla-DGGE typing were the least discriminatory methods, as they divided the 80 strains into 18 and 13 different types, respectively, with D indices of 0.868 and 0.896, respectively. Serotypes 2, 1/44, and 4 complex were the most common serotypes, a result which is in agreement with the serotype distribution generally seen for Danish isolates from these sources (27). PFGE and RAPD were the most discriminatory methods (D indices of 0.974 and 0.984, respectively), each dividing the 80 strains into more than 50 different types. RiboPrinting and fla-RFLP each divided the 80 strains into 40 types and resulted in D indices of 0.945 and 0.960, respectively. In this study, all strains were found to be typeable with each of the methods used. Representative pictures of profiles for each of the five genotypic methods are presented in Fig. 1 to 5.

View larger version (170K): [in this window] [in a new window]   FIG. 1.   PFGE profiles. Lane 1, isolate 5001; lane 2, isolate 4025; lane 3, isolate 5042; lane 4, isolate 5026; lane 5, isolate 733; lane 6, isolate 657; lane 7, isolate 4024. Lanes M, molecular size markers. The PFGE types are 8, 8, 3, 3, 37, 35, and 42 for lanes 1 to 7, respectively.

View larger version (111K): [in this window] [in a new window]   FIG. 2.   fla-RFLP profiles. (Left) DdeI restriction; (right) AluI restriction. Lane identities are as for Fig. 1. The fla-RFLP types are 1/1, 1/1, 1/1, 1/1, 7/7, 5/5, and 24/24 for lanes 1 to 7, respectively.

View larger version (59K): [in this window] [in a new window]   FIG. 3.   RiboPrinting. Lane identities are as for Fig. 1 (vertical lanes). The RiboGroups are 24, 24, 1, 23, 25, 33, and 27 for lanes 1 to 7, respectively.

View larger version (54K): [in this window] [in a new window]   FIG. 4.   RAPD profiles. Lane identities are as for Fig. 1 (vertical lanes). The RAPD types are 1, 1, 2, 10, 5, 7, and 42A for lanes 1 to 7, respectively.

View larger version (116K): [in this window] [in a new window]   FIG. 5.   fla-DGGE profiles. Lane identities are as for Fig. 1. The fla-DGGE types are, from left to right, 2, 3, 3, 5, 5, 7, and 8, respectively. The position of a single band is equal to a specific genotype. The DGGE type number is set low for the fastest-migrating and high for the shortest-migrating band.

Outbreak isolates. All methods assigned the 11 epidemiologically implicated isolates (nine clinical isolates and two water isolates) to the same type (Table 3). However, serotyping also assigned this type to an isolate from one of the control patients originating from the same area. Furthermore, all methods showed the existence of the epitype among unrelated human isolates from other areas and isolates from other sources (Table 1).

Sources. For each method, the origin of the most common types in the collection of 80 unrelated strains is shown in Fig. 6. The majority of these types were found in all three sources, though in different proportions. The dominant type in general was also the most common type among human and cattle isolates. Serotype 2 represented 29 and 40% of the human and cattle isolates, respectively, but only 5% (one isolate) of isolates from poultry. The same picture was seen for fla-DGGE types 3 and 6, RiboGroup 24, fla-RFLP type 1/1, and PFGE types 6 and 8. With the exception of the fla-DGGE types, these genotypes were mostly represented by isolates of serotype 2. Serotype 1,44 was dominant in poultry (30%) but was represented by only one cattle isolate (7%). Dominance in poultry was also seen for some of the common genotypes: RiboGroup 25, fla-RFLP type 7/7, PFGE type 12, and RAPD type 5. 

View larger version (35K): [in this window] [in a new window]   FIG. 6.   For each typing system, the most common types (four or more isolates) are presented as percentages of isolates from each source (human patient, poultry, and cattle). (a) Serotyping; (b) fla-DGGE; (c) RiboPrinting; (d) fla-RFLP; (e) PFGE; (f) RAPD.

Clonal groups and typing system concordance. By use of the criterion that the agreement of strain groupings formed by four or more of the methods indicated a close, probably clonal relationship, the grouping is indicated in Table 1 by boldface. Thirteen groups were formed in this way, accounting for two to eight isolates each, and in total 38 of the 80 strains were part of such a group. One of the groups consisted of strains from all three sources (Fig. 7). Isolates from both humans and cattle were represented in four groups, isolates from humans and poultry were in two groups, and the remaining six groups were represented by one source only (Fig. 7). With the criterion that all six methods should agree on grouping the isolates, seven groups of two isolates each were identified (parts of the groups in Fig. 7; indicated in Table 1 by boldface for isolate number). In four of these groups, both isolates originated from the same source (poultry or humans), but two pairs consisted of a cattle and a human isolate, and one pair consisted of a poultry and a cattle isolate. Interestingly, all methods recognized a cattle isolate (isolate 4025) as belonging to the epitype from the outbreak, and thus this isolate formed an identical pair with the outbreak strain that is included in Table 1 (human isolate 5001).

View larger version (25K): [in this window] [in a new window]   FIG. 7.   Groups of isolates formed by at least four typing methods (boldface groups in Table 1). Origins of isolates are indicated. The groups are numbered according to positions in Table 1, starting from the top, i.e., group 1 includes isolates 913, 943, 5025, and 5130.

View larger version (92K): [in this window] [in a new window]   FIG. 8.   Groups of isolates defined by each of the five genotypic methods. Numbers of isolates within each group are indicated on the y axis. Stacked bars show the serotype distribution within each bar group. For the four most common serotypes, the serotype is indicated by hatching of the bar, and the remaining serotypes are shown by serotype number in the bar.

	DISCUSSION

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Validation of typing methods includes evaluation of their performance. Several performance criteria are essential, in particular, typeability, reproducibility, stability, discriminatory power, and typing system concordance (45). In the present study, we have evaluated the typeability and discriminatory power of six methods for typing C. jejuni: Penner serotyping, fla-DGGE, RiboPrinting, fla-RFLP, PFGE, and RAPD. In addition, the concordance of these methods was evaluated. The test population consisted of 80 C. jejuni strains that were presumably unrelated epidemiologically and 11 strains related to an outbreak. The discriminatory power differed among the six marker systems with D indices in the range of 0.868 to 0.984. PFGE and RAPD were the most discriminatory methods followed by RiboPrinting and fla-RFLP. Serotyping and fla-DGGE typing were the least discriminatory methods in the study. All typing methods had a typeability of 100% on this collection of isolates. For serotyping, this typeability is higher than expected but not unusual, as we generally find more than 95% of Danish surveillance isolates from human patients, cattle, and broiler chickens typeable when using the full set of unabsorbed antisera (E. M. Nielsen, unpublished data). A higher proportion of nontypeable isolates has been reported in use of absorbed sera, e.g., 19% of Dutch poultry (16) and 21% of isolates from clinical cases in the United Kingdom using a modified scheme (9).

Performance of RAPD and PFGE. RAPD and PFGE profiling are well recognized as highly discriminatory tools for molecular typing of a wide range of bacteria, including C. jejuni (10, 20, 34, 49). This is reaffirmed in the present study, where PFGE and RAPD recognized 50 and 56 distinct profiles, respectively, among the 80 strains examined. The high discriminatory potential of PFGE and RAPD can be attributed to their ability to determine polymorphisms in the entire bacterial genome.

Performance of fla-RFLP and RiboPrinting. fla-RFLP and RiboPrinting were not as discriminatory as PFGE and RAPD but still identified 40 different types each. Both methods grouped the isolates in generally good accordance with the other methods (Table 1). However, several RiboGroups were subdivided by all other methods, e.g., the 15 isolates of RiboGroup 23, the most common group, were of three different serotypes (O:1,44, O:2, and O:4 complex) and eight different fla-RFLP types. Some of the fla-RFLP types were also subdivided by all other methods, but 12 of the 13 isolates of type 1/1, the most common type, were serotype 2. In general, typing based on the conserved ribosomal genes is considered a stable typing method. This could be the reason why other typing methods further divide some RiboGroups, e.g., 23 in this study.

Performance of serotyping and fla-DGGE. Serotyping was the least discriminatory method in this study, although a fairly high D index of 0.868 was still attained. Serotyping was the best primary method in the sense that the other methods could form the best hierarchic structure based on the serotyping, e.g., only one of the RAPD groups was subdivided by serotyping (Fig. 8). Serotyping never disagreed on the grouping identified by at least four of the methods (Table 1). Though serotyping was the least discriminatory method, this demonstrated the stability of the serotyping systemi.e., serotyping did not separate strains that the genotypic methods grouped together. In accordance with other studies (12, 34), strains of serotypes O:1,44 and O:2 were found to be more homologous than were strains of the O:4 complex, i.e., within serotypes O:1,44 and O:2 several large clonal groups of isolates were identified with the genotypic methods, whereas none were found in the O:4 complex. The use of absorbed antisera may have made it possible to separate isolates of the O:4 complex into more subtypes. The use of absorbed sera for Penner serotyping of poultry isolates revealed that only 4% of the isolates reacted with any of the antisera comprising the O:4 complex, and half of these reacted with O:13,50 (16). The other common complex, O:1,44, could not be separated in that study. A modified serotyping system based on absorbed antisera and direct agglutination was able to identify isolates with single reactions with the O:4 complex antisera (the majority of these were serotype 50) (9). However, as this modified scheme is not based on passive hemagglutination, the results are not in complete concordance with the traditional scheme (A. N. Oza et al., Abstr. 10th Int. Workshop CHRO, abstr. CE15, 1999). The value of using absorbed sera for the isolates in the present study is therefore difficult to estimate on the basis of these studies. In addition, it must be taken into account that the use of absorbed sera may reduce typeability.

Typing system concordance. The more typing systems showing the same pattern, the better the predictability of relationships between isolates. In this study, the six typing systems possess different discriminatory powers, which must be considered in the evaluation and comparison of methods. When the grouping of isolates formed by at least four typing systems was used for evaluation of concordance of methods, the highly discriminatory PFGE most often disagreed with the other methods, but also fla-DGGE had a high level of disagreement when its low discriminatory power was taken into account. Methods with a high level of agreement but different D indices show a hierarchic pattern, i.e., the highly discriminatory method split the types formed by the low-discriminatory method, but not vice versa. The most discriminatory typing system, RAPD, showed a hierarchic structure with serotyping as the primary system, as the majority of RAPD groups consisted of isolates of only one serotype (Fig. 8). Several of the groups formed by the other genotypic methods consisted of more than one serotype (Fig. 8), showing that the markers of these typing systems often are independent of the serotype. This is not surprising when a typing system is based on a single gene, e.g., the fla gene.

Identification of outbreak isolates and sources of sporadic human infection. The 11 isolates related to a waterborne outbreak were clearly identified by all six typing methods. The typing methods included in this study are thus sufficiently stable to correctly group isolates of clonal origin, even though the isolates were sampled over a period of 2 1/2 months from human diarrheal cases and from the contaminated water.

Applicability of typing systems. Depending on the nature of the bacterial species under investigation, more or less discriminatory methods are suitable for studying the epidemiology of the bacteria. Highly discriminatory methods or combinations of such are necessary for typing of clonal population, whereas stable and perhaps less discriminatory methods are necessary for typing panmictic populations in order to determine the correct relations between isolates. All typing systems evaluated in this study were able to identify the outbreak isolates, and none of the systems failed with respect to typeability and discriminatory power. However, the methods clearly showed different discriminatory powers and different levels of agreement in identifying clonal lines. The methods can be recommended for different uses on the basis of the results of this comparative study, combined with considerations of the costs and labor associated with the methods, as covered by recent reviews (30, 49). As a definitive typing system, Penner serotyping proved to be useful for typing of large numbers of isolates to obtain a coarse grouping of isolates and comparing the serotype distribution to other sources, other time periods, other countries and regions, etc. Serotyping can be supplemented with more discriminatory methods; e.g., serotyping can be used for an initial screening of isolates and then isolates for further typing can be selected. fla-DGGE showed a discriminatory power at the same level as that of serotyping, but the method was not useful as a primary selection method, as isolates in the groups formed by the more discriminatory methods were generally spread among several DGGE groups. The method needs to be further developed and evaluated. fla-RFLP and RiboPrinting are both fairly discriminative and can be used for screening high numbers of isolates. Furthermore, due to standardization and automation, RiboPrinting can be regarded as a definitive typing system. PFGE and RAPD are highly discriminatory methods, based on the whole genome, and these methods are therefore useful for ensuring genotypic similarity in cases of outbreaks.