Evaluation of Phenotypic and Genotypic Methods for Subtyping ...

JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2000, p. 3800–3810 0095-1137/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 38, No. 10

Evaluation of Phenotypic and Genotypic Methods for Subtyping Campylobacter jejuni Isolates from Humans, Poultry, and Cattle EVA MØLLER NIELSEN,1* JØRGEN ENGBERG,2 VIVIAN FUSSING,2 LISE PETERSEN,1 CARL-HENRIK BROGREN,3 AND STEPHEN L. W. ON1 Danish Veterinary Laboratory, 1790 Copenhagen,1 Statens Serum Institut, 2300 Copenhagen,2 and Institute of Food Safety and Toxicology, 2860 Søborg,3 Denmark Received 2 March 2000/Returned for modification 11 June 2000/Accepted 28 July 2000

Six methods for subtyping of Campylobacter jejuni were compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (RiboPrinting); random amplified polymorphic DNA typing (RAPD); pulsed-field gel electrophoresis (PFGE); restriction fragment length polymorphisms of the flagellin gene, flaA (fla-RFLP); and denaturing gradient gel electrophoresis of flaA (flaDGGE). The methods were evaluated and compared on the basis of their abilities to identify isolates from one outbreak and discriminate between unrelated isolates and the agreement between methods in identifying clonal lines. All methods identified the outbreak strain. For a collection of 80 supposedly unrelated isolates, RAPD and PFGE were the most discriminatory methods, followed by fla-RFLP and RiboPrinting. fla-DGGE and serotyping were the least discriminative. All isolates included in this study were found to be typeable by each of the methods. Thirteen groups of potentially related isolates could be identified using a criterion that at least four of the methods agreed on clustering of isolates. None of the subtypes could be related to only one source; rather, these groups represented isolates from different sources. Furthermore, in two cases isolates from cattle and human patients were found to be identical according to all six methods. notypic 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

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 phe* Corresponding author. Mailing address: Department of Microbiology, Danish Veterinary Laboratory, 27 Bu ¨lowsvej, DK-1790 Copenhagen, Denmark. Phone: 45 3530 0100. Fax: 45 3530 0120. E-mail: emn @svs.dk. 3800

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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 IV—4th 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 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 formaldehydetreated 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-␮l PCR mixture contained 5.0 ␮l of Super Taq buffer (HT Biotechnology Ltd., Cambridge, United Kingdom); 5.0 ␮l of 100 mM Tris-HCl (pH 8.3); 3.0 ␮l 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 ␮l of heated bacterial lysate, or 2.5 ␮l of DNA purified by QIAamp. Computer-assisted analysis using GelCompar (Applied Maths, Kortrijk, Belgium) was used for identification of RFLP profiles. Starting with the more distinct DdeI profiles, profiles were compared with similar patterns derived from Danish C. jejuni strains contained in existing databases. In cases when a match

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could not be found, a new profile type was defined. One band difference or band shift sufficient to be reproducibly recorded distinguished one profile type from another. AluI profiles were identified subsequently and if possible given the same number as the DdeI profile of that strain. If more than one AluI profile were observed in combination with a DdeI profile, letters were used to indicate the relationship through the DdeI profile (fla-RFLP types 1/1 and 1/1a are distinguished by the AluI profile). When more than one DdeI profile were observed in combination with one AluI profile, the original AluI profile name was kept (fla-RFLP types 25/25 and 26/25 are distinguished by the DdeI profile). 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 ␮g using a 1-␮l inoculating loop. The colony material was mixed with 300 ␮l 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 ␮l of deionized formamide, 1 ␮l of size standard 2500-ROX, and 1 ␮l 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. The isolates were visually grouped according to combined profiles based on each of the three primers. A capital letter after the type number indicates that profiles were similar with only minor differences in intensity and position of banding profile. 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 ␮l of template DNA (25 to 100 pg) in a final volume of 25 ␮l 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 longterm 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. An estimate of melting behavior with or without the various GC clamps was made by Melt95 software (Ingeny, Leiden, The Netherlands) on the basis of DNA sequences of this flaA fragment (GenBank, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md.). The theoret-

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TABLE 1. Typing of 80 C. jejuni isolates with no known relationa Isolate no.

Source

Serotype

flaDGGE

RiboGroup

flaRFLPb

PFGE

913 943 5025 5130 5027 5048 680 5031 733 665 4006 5050 5037 5038 5033 5052 5044 669 4016 5042 5026 5019 690 4009 4022 4026 5029 5012 5015 5040 4025 5001 5007 4030 5024 5030 5032 5127b 5022 5010 5039 5023 5009 5110 4007 5041 5035 1006 5049 5018 5006 4037 657 659 4039 4005 5074 4027 5011 5051 936 5034 5126 4024 4042 1038 886 1040 4018 751 5043

Poultry Poultry Human Humanc Human Human Poultry Human Poultry Poultry Cattle Human Human Human Human Human Human Poultry Cattle Human Human Human Poultry Cattle Cattle Cattle Human Human Human Human Cattle Humand Human Cattle Human Human Human Human Human Human Human Human Human Humanc Cattle Human Human Poultry Human Human Human Cattle Poultry Poultry Cattle Cattle Human Cattle Human Human Poultry Human Humanc Cattle Cattle Poultry Poultry Poultry Cattle Poultry Human

1,44 1,44 1,44 1,44 1,44 3 1,44 1,44 1,44 1,44 1,44 1,44 1,44 1,44 1,44 1,44 1,44 1,44 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 complex 4 complex 4 complex 4 complex 4 complex 4 complex 4 complex 4 complex 4 complex 4 complex 5 6,7 6,7 6,7 11 11 11 12 12 15 18 19 19 19 19 23,36 23,36 29 37 37

12 12 9 6 7 7 7 7 8 9 6 9 9 9 2 3 3 3 3 3 3 4 4 4 4 4 6 4 4 5 5 5 5 5 3 6 7 7 7 3 6 9 5 7 13 10 3 8 3 5 5 6 2 5 1 3 3 9 5 1 4 10 7 7 8 2 8 8 6 4 6

23 23 23 23 23 25 25 25 25 25 25 25 37 37 18 10 2 25 1 1 23 1 23 23 19 23 23 23 23 23 24 24 24 24 24 24 23 20 35 6 29 24 21 28 30 38 39 30 13 23 7 9 33 33 32 11 11 10 17 26 8 4 27 27 31 42 27 27 27 16 14

2/2 2/2 28/28 2/2 20/1a 7/7 7/7 7/7 7/7 7/7 7/7 7/7a 20/20 20/20 21/21 26/25 27/27 35/35 1/1 1/1 1/1 1/1 1/1 1/1 16/16 16/16 14/14 1/1 1/1 1/1 1/1 1/1 1/1 12/12 1/1a 1/1a 20/20 34/34 14/14a 25/25 31/31 1/1 5/5 12/12 15/15 17/17 17/17 22/22 32/32 33/33 36/36 19/19 5/5 5/5 18/18 30/30 30/30 30/30 5/5 18/18 13/13 29/29 24/24 24/24 15/15a 37/37 15/15 15/15 31/31a 8/8 8/8

12 12 12 34 13 25 38 15 37 37 8 27 6 6 12 29 23 3 3 3 3 6 6 6 6 6 6 6 8 20 8 8 2 50 3 3 16 20 10 4 19 11 3 32 48 21 18 44 26 9 1 51 35 36 35 47 31 49 5 28 43 17 33 42 42 24 42 46 49 41 22

RAPD

27 27 27 27 3 51 5 5 5 12 17 11 3 3 4 48 43 5 2 2 10 9 2 2 2 2 2 41 37 52 1 1 1A 6 1 1 50 42 32 24 38 26 25 53 22 14 40 20 33 54 15 46 7 7 34 49 49 44 47 39 19 21 42 42A 56 45 31 31 23 18 18

TABLE 1—Continued Isolate no.

Source

Serotype

flaDGGE

RiboGroup

5056 726 786 5013 5045 827 684 5028 720

Human Poultry Poultry Human Human Poultry Poultry Human Poultry

37 42 42 53 53 55 55 55 57

4 11 11 4 4 5 5 6 5

12 34 34 3 3 40 15 43 36

8/8 6/6 6/6 23/23 23/23 6/6 11/11 38/38 11/11

PFGE

RAPD

30 35 35 7 7 35 39 14 40

13 29 29 8 8 16 55 35 36

a Boldface highlighting of source and typing data indicates that at least four typing methods agreed on the grouping. Boldface highlighting of the isolate number indicates that all six methods agreed on the grouping. b DdeI/AluI profiles. c Control isolate also included in Table 3. d Outbreak isolate also included in Table 3.

ical melting curves were compared with the curves experimentally made by perpendicular DGGE, and the denaturing gradient was designed according to the melting temperature of the main melting domain (18). A 20 to 40% (vol/vol) urea-formamide gradient gel was prepared from a 100% gel stock solution (7 M urea and 400 ml of formamide per liter) with a 6% (wt/vol) polyacrylamide gel (35.5:1 acrylamide/bisacrylamide ratio) prepared in 1⫻ Tris-acetate-EDTA buffer, pH 8.3. A 1-mm gel (20 by 30 cm) was made by mixing 35 ml of 20% (vol/vol) urea-formamide solution with 35 ml of 40% (vol/vol) urea-formamide denaturant solution in a linear gradient mixer and adding 382 ␮l of 10% (wt/vol) ammonium persulfate and 31 ␮l of N,N,N⬘,N⬘-tetramethylethylenediamine (TEMED) into each gel solution. Approximately 10 ␮l of samples was added to each of 32 wells. The PCR amplicons and DNA ladder were diluted according to DNA content before mixing 8 ␮l with 2 ␮l of 5⫻ gelloader containing bromophenol blue and xylene cyanol markers. The DGGE apparatus (2U-Phor; Ingeny) was run at 60°C for either 17 h at 76 V or 4 to 5 h at 200 V. The gel was stained with SYBR Green I for 30 min without destaining according to the manufacturer’s protocol (Molecular Probes, Eugene, Oreg.). The PCR-amplified homoduplexes revealed a single band pattern with migration positions corresponding to melting behavior (low-melting-point homoduplexes are positioned at a short migration distance, and high-melting-point homoduplexes are positioned at a longer migration distance). The band pattern was photographed and stored as a digital image (Kodak digital camera DC120). Kodak 1-D software was used to scan the lanes, locate the bands, and compare band positions with marker lanes.

RESULTS 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 TABLE 2. Performance of six typing methods tested on a collection of 80 C. jejuni isolates with no known relationship Typing method

No. of types

Prevalence (%) of dominant type

No. of unique typesa

D indexb

Serotyping fla-DGGE RiboPrinting fla-RFLP PFGE RAPD All 6 methods

18 13 40 40 50 56 74

21 (26.3) 14 (17.5) 15 (18.8) 13 (16.3) 9 (11.3) 7 (8.8) 2 (2.5)

5 1 28 23 39 44 68

0.868 0.896 0.945 0.960 0.974 0.984 0.998

a

Continued

flaRFLPb

b

Number of types represented by only one isolate in this collection. D index was calculated according to the work of Hunter and Gaston (15).

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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.

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

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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. 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.

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.

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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.

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. Clonal groups and typing system concordance. By use of the criterion that the agreement of strain groupings formed by four

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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). As a measure of typing system concordance, the boldface groups in Table 1 were also used as an indication of how often a given method disagreed with the other methods. Of the 38 isolates grouped in 13 types with at least four typing methods,

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.


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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.

serotyping agreed with the grouping in all cases, whereas flaDGGE disagreed in grouping 9 isolates, RiboPrinting disagreed for 3 isolates, fla-RFLP disagreed for 4 isolates, PFGE disagreed for 13 isolates, and RAPD disagreed for 8 isolates. The groups of isolates defined by each of the five genotypic methods are shown as bars in Fig. 8. In addition, the figure shows the occurrence of different serotypes within these groups and thereby gives an impression of the serotype variation within groups defined by the genotypic methods. DISCUSSION 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

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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. In the RAPD analysis, isolates were visually grouped according to profiles based on three primers. The G⫹C content of the 10-nucleotide primers, HLWL85, 1281, and 1254, was 50, 60, and 70%, respectively. For the closely related species Helicobacter pylori (G⫹C content similar to that of C. jejuni [30 to 33%]), it has been shown that 10-nucleotide primers with a 60 or 70% G⫹C content gave better results than those with 50% G⫹C (3). This was not the case in our study, as HLWL85 and 1254 most often produced more informative patterns than did 1281 (Fig. 4). A major drawback of RAPD has been reported to be its reproducibility (22). However, by using Ready-To-Go RAPD analysis beads followed by automated detection of fragments on a DNA sequencer, the number of susceptible steps has largely been reduced (E. M. Nielsen, J. Engberg, and V. Fussing, unpublished data). 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. Recently, the validity of fla-RFLP typing has been questioned, due to the potential of the fla genes to undergo recombination events, thereby greatly changing the RFLP profile (13). On the other hand, several recent studies conclude that fla-RFLP types can be linked to evolutionary genetic lineages of Campylobacter spp. (41–43). In this study, fla typing cor-

TABLE 3. Typing of C. jejuni isolates related to a waterborne outbreak Source

No. of strains

EIa

Serotype

fla-DGGE

Ribogroup

fla-RFLP

PFGE

RAPD

Patients Water Patient Patient Patient Patient

9 2 1 1 1 1

Yes Yes No No Unknown No

2 2 4 complex 19 2 1,44

5 5 7 7 7 6

24 24 28 27 20 23

1/1 1/1 12/12 24/24 34/34 2/2

8 8 32 33 20 34

1 1 53 42 42 27

a

EI, epidemiologically implicated.

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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.

rectly identified the epitype isolates from the waterborne outbreak and succeeded in grouping the strain collection in a way that seemed reasonable compared to the results from the remaining five typing tools, indicating that fla typing is overall a reliable epidemiological marker for these isolates. However, it was noted that two isolates (913 and 5025) that were grouped together by the other methods examined, and also by SalI-, KpnI-, and BamHI-based PFGE typing (30), were distinct by both fla-RFLP and fla-DGGE analyses. This suggests that these isolates represent a single clone in which the flaA gene

has undergone some spontaneous genetic change. It was evident that the combined use of DdeI and AluI enhanced the discriminatory power of fla-RFLP typing. In all but one case (AluI profile types 14 and 14a), the AluI profiles that were associated with the same DdeI profile were highly similar, distinguished by one or two band differences. As it is impossible to determine if one or two band differences are caused by major or minor sequence differences between the flaA genes in question, it is not meaningful to interpret similarity between

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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.

profiles as a close interstrain relationship but it is reasonable to regard each fla-RFLP type combination as a separate type. 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 system—i.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. fla-DGGE formed the lowest number of different types, but due to a more even distribution of types, the D index was slightly better than that for serotyping. Though fla-DGGE in some cases agreed on the grouping formed by the other methods (Table 1), most of the groups formed by fla-DGGE were subdivided by all other methods, including the less discriminative serotyping (Fig. 8). DGGE analysis can be sensitive down to single base mutations (8, 18), but the relatively low discriminatory power of the present fla-DGGE method may be the

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result of many different mutations in the flaA gene fragment counteracting each other in melting behavior. A gene with less polymorphism might be a better choice. Also, the large size of this fragment (747 bp) is not optimal for DGGE typing, which works best in the range of 200 to 400 bp. Further development of this new DGGE bacterial genotyping method will therefore involve selection of a smaller and less polymorphic DNA fragment. Furthermore, the heteroduplex analysis procedure has been shown in recent studies of Salmonella and Legionella typing to greatly enhance the precision and discriminatory power in nondenaturing assay systems (17, 39). 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. The most discriminatory methods, PFGE and RAPD, showed some level of agreement in terms of strain differentiation and grouping, but for about 40% of the isolates, the two methods disagreed. Both methods subdivided groups formed by the other method. Although both methods detect whole-genome polymorphisms, the principles underlying each method are quite different and different genetic variations may be detected. It is well established that PFGE profiles of related strains can be altered by a variety of genetic phenomena, including point mutations in restriction sites and genomic rearrangements (31, 48). Such phenomena may account for the differentiation of RAPD groups by PFGE profiling, especially where other markers are concordant with RAPD groupings (e.g., RAPD types 5, 7, 31, and 49). Furthermore, since the discriminatory potential of PFGE is dependent upon the restriction enzyme used, it is conceivable that the use of a morefrequent-cutting enzyme (e.g., KpnI) would further distinguish the SmaI-based PFGE types that were subdivided by RAPD, thereby yielding equivalent results. fla-DGGE is based on polymorphism on a smaller part of the flaA gene than the one used for fla-RFLP in this study. Though fla-DGGE and fla-RFLP are based on parts of the same gene, they measure different parameters (melting point of the whole amplicon versus position of restriction sites), and this is likely to be the reason for the lack of correlation between the two methods. 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.

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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.

The sporadic nature of human campylobacteriosis and the ubiquitous distribution of the bacteria have traditionally hindered the unequivocal identification of sources of infection. In our study, we found six groups each consisting of two supposedly unrelated strains where groupings from each of the typing

methods used were concordant. Three of the aforementioned groups contained isolates from more than one source: two groups comprised cattle and human isolates, and one group contained one isolate each from cattle and poultry (Table 1). The application of stringent criteria for strain identity has previously shown that isolates obtained from cattle and poultry are genetically similar to isolates from cases of human diarrhea (32). The agreement of six different phenotypic and genotypic markers, as described here, can similarly be said to be a stringent criterion for strain identity and thus provide further documentation of the presence of strains from cattle in human enteric disease. Although contaminated, undercooked poultry meat is believed to be a significant vector of sporadically detected human disease (47), these data show that the importance of other animal reservoirs such as cattle requires further study. 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


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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. Typing of bacterial isolates from different sources is a prerequisite for intervention and infection control and to contribute to risk assessment studies of sources of human campylobacteriosis. A comparison of Campylobacter types from food animals and foods of animal origin with isolates from humans makes it possible to produce estimates for the number of human cases attributable to certain animal sources. The more laboratories and countries involved in such surveillance, the better the knowledge of the global epidemiology of campylobacters that can be obtained. Standardization and harmonization of typing methods between laboratories involved in such studies are of utmost importance. Harmonization of the genotypic methods used in the present study (except fla-DGGE) has been initiated in a program of cooperation among several European laboratories (CAMPYNET; http://www.svs.dk /campynet). The applied method or methods must be definitive to render results comparable over time, area, etc. Serotyping is the only method that has been generally used for this purpose in typing of Campylobacter. However, by the construction of databases in a program suitable for the assimilation and analysis of molecular fingerprints, our results indicate that the molecular methods used in the present setup may be applicable as definitive typing tools. ACKNOWLEDGMENTS We gratefully thank all the persons at the participating laboratories who contributed with technical assistance in this study. REFERENCES 1. Akopianz, N., N. O. Bukanov, T. U. Westblom, S. Kresovich, and D. E. Berg. 1992. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res. 20:5137–5142. 2. Anonymous. 2000. Annual report on zoonoses in Denmark 1999, p. 1–28. Danish Zoonosis Centre, Danish Veterinary Laboratory, Copenhagen, Denmark.

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3. Berg, D., N. S. Akopianz, and D. Kersulyte. 1994. Fingerprinting microbial genomes using the RAPD or AP-PCR method. Methods Mol. Cell. Biol. 5: 13–24. 4. Bolton, F. J., A. V. Holt, and D. N. Hutchinson. 1984. Campylobacter biotyping scheme of epidemiological value. J. Clin. Pathol. 37:677–681. 5. Børresen-Dale, A. L., S. Lystad, and A. Langerød. 1998. Temporal temperature gradient gel electrophoresis (TTGE) compared with denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) in mutation screening. BioRadiation 99:12–13. 6. Brogren, C.-H., and L. Bergstro ¨m. 1999. New bacterial genotyping methods using mutation analysis in denaturing slap-gel and capillary electrophoresis. Jpn. J. Electrophor. 43:83. 7. Engberg, J., P. Gerner-Smidt, F. Scheutz, E. M. Nielsen, S. L. W. On, and K. Mølbak. 1998. Water-borne Campylobacter jejuni infection in a Danish town—a 6-week continuous source outbreak. Clin. Microbiol. Infect. 4:648– 656. 8. Fisher, S. 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