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Comparison of 23S polymerase chain reaction–restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters
FEMS Microbiology Letters 211 (2002) 97^103
www.fems-microbiology.org
Comparison of 23S polymerase chain reaction^restriction fragment length polymorphism and ampli¢ed fragment length polymorphism techniques as typing systems for thermophilic campylobacters Yolanda Moreno, Mar|¤a A. Ferru¤s, Alicia Vanoostende, Manuel Herna¤ndez, Rosa M. Montes, Javier Herna¤ndez Departamento de Biotecnolog|¤a, Universidad Polite¤cnica, Camino de Vera 14, 46022 Valencia, Spain Received 12 February 2002; received in revised form 27 March 2002; accepted 5 April 2002 First published online 2 May 2002
Abstract In this study, we evaluated the combination of polymerase chain reaction^restriction fragment length polymorphism (PCR^RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR^RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR^RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters. > 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Typing; Polymerase chain reaction^restriction fragment length polymorphism; Ampli¢ed fragment length polymorphism; Campylobacter
1. Introduction Since the late 1970s, thermophilic campylobacters have been recognised as important agents of acute bacterial gastrointestinal infections, with Campylobacter jejuni the most common species associated with diarrhoeal illness in man in both developed and developing countries around the world [1,2]. The major infection route for humans is supposed to be the consumption of contaminated poultry products [3]. Although some others sources have been involved, the current epidemiology of infection remains undetermined [4]. Accurate and reproducible methods of strain identi¢cation are essential for epidemiological purposes. Conventional phenotypic methods based on biotyping, serotyping
* Corresponding author.: Tel.: +34 (96) 387 7423; Fax : +34 (96) 387 9429. E-mail address : [email protected] (J. Herna¤ndez).
or phage-typing have been widely applied to the genus Campylobacter [5]. However, they usually lack discriminatory power or typability, and can fail to di¡erentiate atypical strains [6,7]. Molecular methods with high discriminatory power are greatly required for a reliable identi¢cation of main Campylobacter species specially in epidemiological studies to assess the transmission routes in epidemic outbreaks [8]. An identi¢cation scheme for Campylobacter species using restriction fragment length polymorphism of PCR-ampli¢ed 23S rRNA genes (PCR^RFLP) has been described and showed to be able to identify Campylobacter and Arcobacter species, being more discriminatory, faster and more cost e¡ective than phenotypic tests [9]. More recently, the ampli¢ed fragment length polymorphism technique (AFLP), has been applied to several bacterial species, including Helicobacter [10] and Campylobacter [11,12], showing great stability and discriminatory power [13]. However, single-enzyme AFLP has not yet been applied to thermophilic campylobacters.
0378-1097 / 02 / $22.00 > 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 6 7 0 - 5
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Table 1 Strains of Campylobacter used in the study and typing results Straina
Speciesb
Source
Specimen
PCR^RFLP typec
AFLP typec
R1 R2 R3 C01 C02 C03 C04 C05 C06 C07 C08 C09 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 P01 P02Ad P02B P03A P03B P04 P05 P06 P07 P08 P09 P10 P11 P12 P13A P13B P14 P15A P15B P15C P16A P16B P17 P18 P19 P20 P21 P22 P23 P24 P25 P26A P26B P26C P26D P27 P28
Cj Cj Cc Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cc Cc Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cc Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cc Cj Cj Cj Cj Cj Cj Cj Cj Cc Cc Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cc
NCTC 11168 NCTC 11322 NCTC 11366 Hospital A Hospital B Hospital B Hospital B Hospital B Hospital B Hospital B Hospital B Hospital C Hospital C Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market B Market B Market B Market B Market B Market B Market B Market C Market C Market C Market C Market C Market C Market C Market C Market C Market C
Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Skin Liver Liver Liver Liver Liver Liver Liver Skin Skin Hamburger Hamburger Skin Skin Skin Skin Skin Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Liver Liver Liver Liver Liver Liver Liver Carcass Carcass
r-2 r-2 r-10 r-2 r-2 r-2 r-4 r-2 r-4 r-4 r-2 r-2 r-2 r-10 r-10 r-4 r-11 r-2 r-5 r-8 r-4 r-4 r-1 r-2 r-7 r-10 r-1 r-6 r-6 r-6 r-6 r-4 r-4 r-1 r-2 r-2 r-2 r-2 r-4 r-3 r-2 r-2 r-2 r-11 r-2 r-2 r-2 r-2 r-10 r-10 r-2 r-2 r-2 r-6 r-2 r-2 r-2 r-1 r-1 r-1 r-1 r-2 r-10
a-4 a-6 a-8 a-1 a-8 a-1 a-4 a-5 a-4 a-4 a-2 a-4 a-2 a-4 a-5 a-2 a-4 a-1 a-1 a-3 a-1 a-3 a-1 a-3 a-1 a-1 a-6 a-5 a-6 a-6 a-6 a-6 a-6 a-6 a-7 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-5 a-6 a-5 a-6 a-6 a-5 a-5 a-5 a-6 a-5 a-4 a-5 a-6
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Table 1. (Continued). Straina
Speciesb
Source
P29 P30 P31 P32 P33 P34
Cj Cj Cj Cj Cj Cj
Market Market Market Market Market Market
C C C C D D
Specimen
PCR^RFLP typec
AFLP typec
Carcass Liver Liver Carcass Carcass Carcass
r-1 r-9 r-2 r-2 r-2 r-2
a-5 a-5 a-5 a-6 a-6 a-6
a
R, reference strains ; C, clinical isolates; P, poultry isolates. Cj, C. jejuni; Cc, C. coli. c PCR^RFLP and AFLP types were de¢ned according to the results of the numerical analysis. d A, B, C and D represent di¡erent strains isolated from the same sample. b
In this study, we have evaluated the combination of 23S PCR^RFLP and single-enzyme AFLP (sAFLP) ¢ngerprinting methods for identi¢cation and typing of thermophilic Campylobacter.
2. Materials and methods 2.1. Bacterial strains and growth conditions A total of 23 clinical and 43 chicken isolates were used in this study. Three NCTC reference Campylobacter strains were also included for comparative purposes. Table 1 provides information about their strain number, biochemical identi¢cation, sources, specimens, and typing pro¢les. Occasionally, several colonies showing morphological di¡erences were selected from the same sample and, consequently, they were considered as di¡erent isolates for typing purposes (Table 1). Clinical isolates were obtained in faeces of di¡erent patients su¡ering from diarrhoeic syndromes, within a period of 5 years at four di¡erent hospitals. Chicken samples were collected from four di¡erent local markets. The primary isolation media were modi¢ed CCDAPreston selective agar (Oxoid CM739) with cefoperazone (16 g l31 ), and Campylobacter selective agar (Merck) supplemented with 5% de¢brinated sheep blood and Butzler selective supplement (Oxoid SR085E). Once isolated, Campylobacter strains were maintained on Blood agar base No. 2 (Oxoid CM67) supplemented with 5% of sheep blood and incubated for 24 h at 37‡C under microaerophilic conditions (5% O2 , 10% CO2 , 85% N2 ). All strains were identi¢ed, prior to genotypic analysis, by the API-Campy system (Biome'rieux). When doubtful or unacceptable identi¢cation pro¢les were obtained, Lior biotype was determined [14]. Bacterial strains were preserved in 20% (v/v) glycerol at 380‡C. 2.2. DNA extraction Chromosomal DNA was extracted and puri¢ed using the cetyltrimethylammonium bromide method [15].
2.3. PCR^RFLP analysis A 2.6-kb 23S rRNA fragment from Campylobacter strains was ampli¢ed using the LS1 and LS2 primers with sequences 5P-GGATTTCCGAATGGGGCAACCC3P and 5P-GTTTCGTGCTTAGATGTTTC-3P respectively [16]. Both primers were synthesised commercially (MWGBiotech, Germany). Ampli¢cation was performed in a ¢nal volume of 100 Wl, containing 200 ng of genomic DNA, 2.5 U of Taq polymerase (New England BioLabs, UK), 1.5 mM MgCl2 , 0.1 mM of each deoxynucleotide triphosphate (dNTP), 1.5 WM of each primer in a reaction bu¡er (75 mM Tris^HCl pH 8.0, 50 mM KCl, 20 mM ammonium sulfate). The reaction mixture was overlaid with 50 Wl of mineral oil to prevent evaporation [9]. An initial denaturation step at 95‡C for 5 min was followed by 28 cycles of denaturation at 94‡C for 1 min, annealing at 58‡C for 1 min, and extension at 72‡C for 2 min. Ampli¢ed products were visualised by 0.8% (w/v) NuSieve agarose gel electrophoresis and stained with ethidium bromide (0.5 Wg ml31 ). Ampli¢ed DNA was concentrated by adding ethanol
Fig. 1. 23S PCR^RFLP patterns of representative strains of Campylobacter after ampli¢cation of a 2.6-kb 23S rRNA fragment with primers LS1 and LS2, digestion with endonuclease HpaII, and electrophoresis on a 3% agarose gel. Lane M, 100-bp DNA ladder used as molecular marker.
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Fig. 2. Dendrogram of the numerical analysis based on the 23S PCR^RFLP patterns of strains listed in Table 1. Numbers on the horizontal axis indicate the percentage similarities as determined by the Dice coe⁄cient. Vertical axis shows the main groups de¢ned at the 54% similarity level.
and resuspended in 15 Wl of TE bu¡er. DNA was then digested with 10 U of HpaII (New England BioLabs, UK) in a ¢nal reaction volume of 20 Wl at 37‡C for 2 h. Restriction reaction was stopped by adding 3 Wl of stop-
mix solution (50 mM EDTA, 0.3% Ficoll, 0.3% bromophenol blue) and fragments were visualised by 3% agarose gel electrophoresis in TAE bu¡er (40 mM Tris^acetate, 2 mM EDTA, pH 8.3).
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2.4. AFLP analysis A 10-Wg aliquot of genomic DNA was digested with 20 U of restriction enzyme HindIII (New England BioLabs, UK) at 37‡C overnight in a ¢nal volume of 20 Wl containing 5 mM spermidine trihydrochloride (Sigma). A 2.5-Wg aliquot of restriction fragments was then ligated with the ADH1 (5P-ACGGTATGCGACAG-3P) and ADH2 (5P-AGCTCTGTCGCATACCGTGAG-3P) adapters in a reaction volume of 20 Wl at 37‡C for 3^4 h. The ligation mixture consisted of 2.5 Wl of digested DNA, 0.2 Wg of each adapter, and 1 U of T4 DNA ligase (New England BioLabs, UK) in ligase bu¡er. Prior to the PCR, ligated DNA was diluted 50/50 in distilled water and heated at 80‡C for 10 min to inactivate T4 ligase. A 5-Wl aliquot of ligated DNA was then used as a template for DNA ampli¢cation in 50 Wl of reaction mixture containing 2.5 mM of MgCl2 , dNTP solution (200 mM each) 200 ng of HIG primer (5P-GGTATGCGAACAGAGCTTG-3P), and 1 U of Taq polymerase (Gibco BRL, Paisley, UK) in PCR bu¡er provided by the manufacturer. The mixture was subjected to an initial denaturing step at 94‡C for 5 min, and 33 ampli¢cation cycles consisting of 94‡C for 1 min, 60‡C for 1 min and 72‡C for 2 min. Ampli¢ed DNA fragments were separated electrophoretically in agarose gels (2.5% w/v) run in TAE bu¡er at 70 V for 3 h. Following ethidium bromide stainings, the sizes of the visualised restricted fragments on the gels were calculated from migration distances using UPGMA algorithms. Cluster analyses were performed using the Ntsys program, version 2.0 (Exeter Software, New York, USA).
3. Results 3.1. PCR^RFLP results Stable and reproducible PCR^RFLP patterns formed by ¢ve to nine bands ranging from 230 to 1700 bp were detected (Fig. 1). A common band of 525 bp was present in all strains. Two bands of 600 bp and 315 bp were seen in 98.3% and 87% of the strains respectively. Numerical analysis of band patterns produced a dendrogram in which all strains were grouped at 54% similarity level (Fig. 2), and a total of 11 di¡erent types were de¢ned at 100% linkage level. Five types (r-3, r-5, r-7, r-8, r-9) were represented by a single isolate. No separate type of human-speci¢c or chicken-speci¢c strains was present, so no relationship between the type and the origin of the strains could be detected. Types r-3 (one strain) and r-10 (seven strains) contained only Campylobacter coli isolates, including the reference strain NCTC 11366, while all the rest of the types were formed exclusively by C. jejuni isolates (30 strains).
Fig. 3. AFLP patterns of representative strains of Campylobacter after genomic DNA digestion with endonuclease HindIII, ligation with adapters ADH1 and ADH2, ampli¢cation with HIG primer, and electrophoresis on a 2.5% agarose gel. Lane M, 100-bp DNA ladder used as molecular marker.
3.2. AFLP results Analysis of the 69 isolates resulted in 66 di¡erent banding patterns, with three to 22 ampli¢ed DNA fragments of 235^1430 bp (Fig. 3). Although no common bands were detected, 92% of the strains shared a 600-bp size band. Following numerical analysis, the resulting dendrogram (Fig. 4) showed a high diversity among the strains studied. All the isolates joined at 51% similarity level, and eight main types could be di¡erentiated at 69% homology degree. Types a-1, a-2 and a-3 were composed exclusively of strains of clinical origin. Type a-4 was formed by seven clinical isolates, one poultry isolate, and one reference strain of C. jejuni. Type a-5 was formed mainly by chicken isolates (12 out of 14 strains). No human-speci¢c strain was included amongst the 30 isolates constituting type a-6. Only type a-7 was composed of a single isolate. Finally, type a-8 comprised one clinical isolate and the C. coli reference strain. When PCR^RFLP and AFLP data were directly compared by numerical analysis, no relationship could be demonstrated.
4. Discussion By 23S PCR^RFLP analysis all strains studied were ¢ngerprinted using HpaII to digest the DNA, which indicates an excellent level of typability. Conserved restriction pro¢les were generated for all the strains tested. This method did allow for the di¡erentiation at species level between C. jejuni and C. coli because strains belonging to the same species produced similar patterns and were grouped together on the dendrogram. However, no signi¢-
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Fig. 4. Dendrogram of the numerical analysis based on the AFLP patterns of strains listed in Table 1. The numbers on the horizontal axis indicate the percentage similarities as determined by the Dice coe⁄cient. The vertical axis shows the main groups de¢ned at the 51% similarity level.
cant relationship could be observed between the pro¢les and the origin of the strains; this supports the hypothesis that con¢rms that poultry products are a likely source of human infections. AFLP genotyping has proved to be a highly sensitive
method for subtyping and discriminating Campylobacter strains. The high degree of variation obtained among Campylobacter strains has been previously detected by other typing methods [17]. Strains with a homology level higher than 95% were
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epidemiologically closely related as they had been isolated from the same sample (P02A-P02B, P03A-P03B, P13AP13B). So, AFLP patterns linked at 95% homology degree or more were coded as AFLP subtypes within the same type, and these strains were considered subclonal variants from one original strain. In conclusion, 23S PCR^RFLP showed to be of special usefulness for species identi¢cation, while AFLP was a valuable strain typing tool. Both methods indicated a high degree of genomic diversity within thermophilic campylobacters, irrespective of the host and geographical origin of the strains. When used in combination, they may provide accurate information of strain relationships at genotypic level allowing epidemiological surveillance of strains.
Acknowledgements This work was supported by the CICYT Project ALI1999-0539. Y.M. was the recipient of a research grant from the Ministerio de Ciencia y Tecnolog|¤a.
References [1] Friedman, C.R., Neimann, J., Wegener, H.C. and Tauxe, R.V. (2000) Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations. In: Campylobacter, 2nd edn. (Nachamkin, I. and Blaser, M.J., Eds.), pp. 121^138. ASM Press, Washington, DC. [2] Richard, A., Oberhelman, D. and Taylor, D.N. (2000) Campylobacter infections in developing countries. In: Campylobacter, 2nd edn. (Nachamkin, I. and Blaser, M.J., Eds.), pp. 139^154. ASM Press, Washington, DC. [3] Nachamkin, I. (1997) Campylobacter jejuni. In: Food Microbiology. Fundamentals and Frontiers (Doyle, M.P., Beuchat, L.R. and Montville, T.J., Eds.), pp. 159^170. ASM Press, Washington, DC. [4] Thomas, C., Gibson, H., Hill, D.J. and Mabey, M. (1999) Campylobacter epidemiology : an aquatic perspective. J. Appl. Microbiol. Symp. 85 (Suppl.), 168S^177S.
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[5] Patton, C.M., Wachsmuth, I.K., Evins, G.M., Kiehlbauch, J.A., Plikaytis, B.D., Troup, N., Tompkins, L. and Lior, H. (1991) Evaluation of 10 methods to distinguish epidemic-associated Campylobacter strains. J. Clin. Microbiol. 29, 680^688. [6] Patton, C.M. and Wachsmuth, I.K. (1992) Typing schemes: are current methods useful ? In: Campylobacter jejuni: Current Status and Future Trends (Nachamkin, I., Blaser, M.J. and Tompkins, L.S., Eds.), pp. 110^128. ASM Press, Washington, DC. [7] Owen, R.J. and Gibson, J.R. (1995) Update on epidemiological typing of Campylobacter. PHLS Microbiol. Dig. 12, 2^6. [8] Meinersmann, R.J. (1998) Criteria for molecular typing methods. In: Campylobacter, Helicobacter and Related Organisms (Lastovica, A.J., Newell, D.G. and Lastovica, E.E., Eds.), pp. 582^583. Institute of Child Health, University of Cape Town, Cape Town. [9] Hurtado, A. and Owen, R.J. (1997) A molecular scheme based on 23S rRNA gene polymorphisms for rapid identi¢cation of Campylobacter and Arcobacter species. J. Clin. Microbiol. 35, 2401^2404. [10] Gibson, J.R., Slater, E., Xerry, J., Tompkins, D.S. and Owen, R.J. (1998) Use of an ampli¢ed-fragment length polymorphism technique to ¢ngerprint and di¡erentiate isolates of Helicobacter pylori. J. Clin. Microbiol. 36, 2580^2585. [11] Duim, B., Wassenaar, T.M., Rigter, A. and Wagenaar, J. (1999) High resolution genotyping of Campylobacter strains isolated from poultry and humans with ampli¢ed-fragment length polymorphism ¢ngerprinting. Appl. Environ. Microbiol. 65, 2369^2375. [12] Kokotovic, B. and On, S.L.W. (1999) High-resolution genomic ¢ngerprinting of Campylobacter jejuni and Campylobacter coli by analysis of ampli¢ed fragment length polymorphisms. FEMS Microbiol. Lett. 173, 77^84. [13] Lindstedt, B.A., Heir, E., Vardunt, T., Melby, K.K. and Kapperud, G. (2000) Comparative ¢ngerprinting analysis of Campylobacter jejuni subsp. jejuni strains by ampli¢ed fragment length polymorphism genotyping. J. Clin. Microbiol. 38, 3379^3387. [14] Lior, H. (1984) New extended biotyping scheme for Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis. J. Clin. Microbiol. 20, 636^640. [15] Wilson, K. (1987) Preparation of genomic DNA from bacteria. In: Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A., Seidman, J.G. and Struhl, K., Eds.), unit 2.4.1. John Wiley and Sons, New York. [16] Hurtado, A. and Owen, R.J. (1997) A rapid identi¢cation scheme for Helicobacter pylori and other species of Helicobacter based on 23S rRNA gene polymorphisms. Syst. Appl. Microbiol. 20, 222^231. [17] Hernandez, J., Fayos, A., Ferrus, M.A. and Owen, R.J. (1995) Random ampli¢ed polymorphic DNA ¢ngerprinting of Campylobacter jejuni and C. coli isolated from human faeces, seawater and poultry products. Res. Microbiol. 146, 685^696.
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Comparison of 23S polymerase chain reaction^restriction fragment length polymorphism and ampli¢ed fragment length polymorphism techniques as typing systems for thermophilic campylobacters Yolanda Moreno, Mar|¤a A. Ferru¤s, Alicia Vanoostende, Manuel Herna¤ndez, Rosa M. Montes, Javier Herna¤ndez Departamento de Biotecnolog|¤a, Universidad Polite¤cnica, Camino de Vera 14, 46022 Valencia, Spain Received 12 February 2002; received in revised form 27 March 2002; accepted 5 April 2002 First published online 2 May 2002
Abstract In this study, we evaluated the combination of polymerase chain reaction^restriction fragment length polymorphism (PCR^RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR^RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR^RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters. > 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Typing; Polymerase chain reaction^restriction fragment length polymorphism; Ampli¢ed fragment length polymorphism; Campylobacter
1. Introduction Since the late 1970s, thermophilic campylobacters have been recognised as important agents of acute bacterial gastrointestinal infections, with Campylobacter jejuni the most common species associated with diarrhoeal illness in man in both developed and developing countries around the world [1,2]. The major infection route for humans is supposed to be the consumption of contaminated poultry products [3]. Although some others sources have been involved, the current epidemiology of infection remains undetermined [4]. Accurate and reproducible methods of strain identi¢cation are essential for epidemiological purposes. Conventional phenotypic methods based on biotyping, serotyping
* Corresponding author.: Tel.: +34 (96) 387 7423; Fax : +34 (96) 387 9429. E-mail address : [email protected] (J. Herna¤ndez).
or phage-typing have been widely applied to the genus Campylobacter [5]. However, they usually lack discriminatory power or typability, and can fail to di¡erentiate atypical strains [6,7]. Molecular methods with high discriminatory power are greatly required for a reliable identi¢cation of main Campylobacter species specially in epidemiological studies to assess the transmission routes in epidemic outbreaks [8]. An identi¢cation scheme for Campylobacter species using restriction fragment length polymorphism of PCR-ampli¢ed 23S rRNA genes (PCR^RFLP) has been described and showed to be able to identify Campylobacter and Arcobacter species, being more discriminatory, faster and more cost e¡ective than phenotypic tests [9]. More recently, the ampli¢ed fragment length polymorphism technique (AFLP), has been applied to several bacterial species, including Helicobacter [10] and Campylobacter [11,12], showing great stability and discriminatory power [13]. However, single-enzyme AFLP has not yet been applied to thermophilic campylobacters.
0378-1097 / 02 / $22.00 > 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 6 7 0 - 5
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Table 1 Strains of Campylobacter used in the study and typing results Straina
Speciesb
Source
Specimen
PCR^RFLP typec
AFLP typec
R1 R2 R3 C01 C02 C03 C04 C05 C06 C07 C08 C09 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 P01 P02Ad P02B P03A P03B P04 P05 P06 P07 P08 P09 P10 P11 P12 P13A P13B P14 P15A P15B P15C P16A P16B P17 P18 P19 P20 P21 P22 P23 P24 P25 P26A P26B P26C P26D P27 P28
Cj Cj Cc Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cc Cc Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cc Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cc Cj Cj Cj Cj Cj Cj Cj Cj Cc Cc Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cj Cc
NCTC 11168 NCTC 11322 NCTC 11366 Hospital A Hospital B Hospital B Hospital B Hospital B Hospital B Hospital B Hospital B Hospital C Hospital C Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Hospital D Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market A Market B Market B Market B Market B Market B Market B Market B Market C Market C Market C Market C Market C Market C Market C Market C Market C Market C
Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Skin Liver Liver Liver Liver Liver Liver Liver Skin Skin Hamburger Hamburger Skin Skin Skin Skin Skin Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Liver Liver Liver Liver Liver Liver Liver Carcass Carcass
r-2 r-2 r-10 r-2 r-2 r-2 r-4 r-2 r-4 r-4 r-2 r-2 r-2 r-10 r-10 r-4 r-11 r-2 r-5 r-8 r-4 r-4 r-1 r-2 r-7 r-10 r-1 r-6 r-6 r-6 r-6 r-4 r-4 r-1 r-2 r-2 r-2 r-2 r-4 r-3 r-2 r-2 r-2 r-11 r-2 r-2 r-2 r-2 r-10 r-10 r-2 r-2 r-2 r-6 r-2 r-2 r-2 r-1 r-1 r-1 r-1 r-2 r-10
a-4 a-6 a-8 a-1 a-8 a-1 a-4 a-5 a-4 a-4 a-2 a-4 a-2 a-4 a-5 a-2 a-4 a-1 a-1 a-3 a-1 a-3 a-1 a-3 a-1 a-1 a-6 a-5 a-6 a-6 a-6 a-6 a-6 a-6 a-7 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-6 a-5 a-6 a-5 a-6 a-6 a-5 a-5 a-5 a-6 a-5 a-4 a-5 a-6
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Table 1. (Continued). Straina
Speciesb
Source
P29 P30 P31 P32 P33 P34
Cj Cj Cj Cj Cj Cj
Market Market Market Market Market Market
C C C C D D
Specimen
PCR^RFLP typec
AFLP typec
Carcass Liver Liver Carcass Carcass Carcass
r-1 r-9 r-2 r-2 r-2 r-2
a-5 a-5 a-5 a-6 a-6 a-6
a
R, reference strains ; C, clinical isolates; P, poultry isolates. Cj, C. jejuni; Cc, C. coli. c PCR^RFLP and AFLP types were de¢ned according to the results of the numerical analysis. d A, B, C and D represent di¡erent strains isolated from the same sample. b
In this study, we have evaluated the combination of 23S PCR^RFLP and single-enzyme AFLP (sAFLP) ¢ngerprinting methods for identi¢cation and typing of thermophilic Campylobacter.
2. Materials and methods 2.1. Bacterial strains and growth conditions A total of 23 clinical and 43 chicken isolates were used in this study. Three NCTC reference Campylobacter strains were also included for comparative purposes. Table 1 provides information about their strain number, biochemical identi¢cation, sources, specimens, and typing pro¢les. Occasionally, several colonies showing morphological di¡erences were selected from the same sample and, consequently, they were considered as di¡erent isolates for typing purposes (Table 1). Clinical isolates were obtained in faeces of di¡erent patients su¡ering from diarrhoeic syndromes, within a period of 5 years at four di¡erent hospitals. Chicken samples were collected from four di¡erent local markets. The primary isolation media were modi¢ed CCDAPreston selective agar (Oxoid CM739) with cefoperazone (16 g l31 ), and Campylobacter selective agar (Merck) supplemented with 5% de¢brinated sheep blood and Butzler selective supplement (Oxoid SR085E). Once isolated, Campylobacter strains were maintained on Blood agar base No. 2 (Oxoid CM67) supplemented with 5% of sheep blood and incubated for 24 h at 37‡C under microaerophilic conditions (5% O2 , 10% CO2 , 85% N2 ). All strains were identi¢ed, prior to genotypic analysis, by the API-Campy system (Biome'rieux). When doubtful or unacceptable identi¢cation pro¢les were obtained, Lior biotype was determined [14]. Bacterial strains were preserved in 20% (v/v) glycerol at 380‡C. 2.2. DNA extraction Chromosomal DNA was extracted and puri¢ed using the cetyltrimethylammonium bromide method [15].
2.3. PCR^RFLP analysis A 2.6-kb 23S rRNA fragment from Campylobacter strains was ampli¢ed using the LS1 and LS2 primers with sequences 5P-GGATTTCCGAATGGGGCAACCC3P and 5P-GTTTCGTGCTTAGATGTTTC-3P respectively [16]. Both primers were synthesised commercially (MWGBiotech, Germany). Ampli¢cation was performed in a ¢nal volume of 100 Wl, containing 200 ng of genomic DNA, 2.5 U of Taq polymerase (New England BioLabs, UK), 1.5 mM MgCl2 , 0.1 mM of each deoxynucleotide triphosphate (dNTP), 1.5 WM of each primer in a reaction bu¡er (75 mM Tris^HCl pH 8.0, 50 mM KCl, 20 mM ammonium sulfate). The reaction mixture was overlaid with 50 Wl of mineral oil to prevent evaporation [9]. An initial denaturation step at 95‡C for 5 min was followed by 28 cycles of denaturation at 94‡C for 1 min, annealing at 58‡C for 1 min, and extension at 72‡C for 2 min. Ampli¢ed products were visualised by 0.8% (w/v) NuSieve agarose gel electrophoresis and stained with ethidium bromide (0.5 Wg ml31 ). Ampli¢ed DNA was concentrated by adding ethanol
Fig. 1. 23S PCR^RFLP patterns of representative strains of Campylobacter after ampli¢cation of a 2.6-kb 23S rRNA fragment with primers LS1 and LS2, digestion with endonuclease HpaII, and electrophoresis on a 3% agarose gel. Lane M, 100-bp DNA ladder used as molecular marker.
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Fig. 2. Dendrogram of the numerical analysis based on the 23S PCR^RFLP patterns of strains listed in Table 1. Numbers on the horizontal axis indicate the percentage similarities as determined by the Dice coe⁄cient. Vertical axis shows the main groups de¢ned at the 54% similarity level.
and resuspended in 15 Wl of TE bu¡er. DNA was then digested with 10 U of HpaII (New England BioLabs, UK) in a ¢nal reaction volume of 20 Wl at 37‡C for 2 h. Restriction reaction was stopped by adding 3 Wl of stop-
mix solution (50 mM EDTA, 0.3% Ficoll, 0.3% bromophenol blue) and fragments were visualised by 3% agarose gel electrophoresis in TAE bu¡er (40 mM Tris^acetate, 2 mM EDTA, pH 8.3).
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2.4. AFLP analysis A 10-Wg aliquot of genomic DNA was digested with 20 U of restriction enzyme HindIII (New England BioLabs, UK) at 37‡C overnight in a ¢nal volume of 20 Wl containing 5 mM spermidine trihydrochloride (Sigma). A 2.5-Wg aliquot of restriction fragments was then ligated with the ADH1 (5P-ACGGTATGCGACAG-3P) and ADH2 (5P-AGCTCTGTCGCATACCGTGAG-3P) adapters in a reaction volume of 20 Wl at 37‡C for 3^4 h. The ligation mixture consisted of 2.5 Wl of digested DNA, 0.2 Wg of each adapter, and 1 U of T4 DNA ligase (New England BioLabs, UK) in ligase bu¡er. Prior to the PCR, ligated DNA was diluted 50/50 in distilled water and heated at 80‡C for 10 min to inactivate T4 ligase. A 5-Wl aliquot of ligated DNA was then used as a template for DNA ampli¢cation in 50 Wl of reaction mixture containing 2.5 mM of MgCl2 , dNTP solution (200 mM each) 200 ng of HIG primer (5P-GGTATGCGAACAGAGCTTG-3P), and 1 U of Taq polymerase (Gibco BRL, Paisley, UK) in PCR bu¡er provided by the manufacturer. The mixture was subjected to an initial denaturing step at 94‡C for 5 min, and 33 ampli¢cation cycles consisting of 94‡C for 1 min, 60‡C for 1 min and 72‡C for 2 min. Ampli¢ed DNA fragments were separated electrophoretically in agarose gels (2.5% w/v) run in TAE bu¡er at 70 V for 3 h. Following ethidium bromide stainings, the sizes of the visualised restricted fragments on the gels were calculated from migration distances using UPGMA algorithms. Cluster analyses were performed using the Ntsys program, version 2.0 (Exeter Software, New York, USA).
3. Results 3.1. PCR^RFLP results Stable and reproducible PCR^RFLP patterns formed by ¢ve to nine bands ranging from 230 to 1700 bp were detected (Fig. 1). A common band of 525 bp was present in all strains. Two bands of 600 bp and 315 bp were seen in 98.3% and 87% of the strains respectively. Numerical analysis of band patterns produced a dendrogram in which all strains were grouped at 54% similarity level (Fig. 2), and a total of 11 di¡erent types were de¢ned at 100% linkage level. Five types (r-3, r-5, r-7, r-8, r-9) were represented by a single isolate. No separate type of human-speci¢c or chicken-speci¢c strains was present, so no relationship between the type and the origin of the strains could be detected. Types r-3 (one strain) and r-10 (seven strains) contained only Campylobacter coli isolates, including the reference strain NCTC 11366, while all the rest of the types were formed exclusively by C. jejuni isolates (30 strains).
Fig. 3. AFLP patterns of representative strains of Campylobacter after genomic DNA digestion with endonuclease HindIII, ligation with adapters ADH1 and ADH2, ampli¢cation with HIG primer, and electrophoresis on a 2.5% agarose gel. Lane M, 100-bp DNA ladder used as molecular marker.
3.2. AFLP results Analysis of the 69 isolates resulted in 66 di¡erent banding patterns, with three to 22 ampli¢ed DNA fragments of 235^1430 bp (Fig. 3). Although no common bands were detected, 92% of the strains shared a 600-bp size band. Following numerical analysis, the resulting dendrogram (Fig. 4) showed a high diversity among the strains studied. All the isolates joined at 51% similarity level, and eight main types could be di¡erentiated at 69% homology degree. Types a-1, a-2 and a-3 were composed exclusively of strains of clinical origin. Type a-4 was formed by seven clinical isolates, one poultry isolate, and one reference strain of C. jejuni. Type a-5 was formed mainly by chicken isolates (12 out of 14 strains). No human-speci¢c strain was included amongst the 30 isolates constituting type a-6. Only type a-7 was composed of a single isolate. Finally, type a-8 comprised one clinical isolate and the C. coli reference strain. When PCR^RFLP and AFLP data were directly compared by numerical analysis, no relationship could be demonstrated.
4. Discussion By 23S PCR^RFLP analysis all strains studied were ¢ngerprinted using HpaII to digest the DNA, which indicates an excellent level of typability. Conserved restriction pro¢les were generated for all the strains tested. This method did allow for the di¡erentiation at species level between C. jejuni and C. coli because strains belonging to the same species produced similar patterns and were grouped together on the dendrogram. However, no signi¢-
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Fig. 4. Dendrogram of the numerical analysis based on the AFLP patterns of strains listed in Table 1. The numbers on the horizontal axis indicate the percentage similarities as determined by the Dice coe⁄cient. The vertical axis shows the main groups de¢ned at the 51% similarity level.
cant relationship could be observed between the pro¢les and the origin of the strains; this supports the hypothesis that con¢rms that poultry products are a likely source of human infections. AFLP genotyping has proved to be a highly sensitive
method for subtyping and discriminating Campylobacter strains. The high degree of variation obtained among Campylobacter strains has been previously detected by other typing methods [17]. Strains with a homology level higher than 95% were
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epidemiologically closely related as they had been isolated from the same sample (P02A-P02B, P03A-P03B, P13AP13B). So, AFLP patterns linked at 95% homology degree or more were coded as AFLP subtypes within the same type, and these strains were considered subclonal variants from one original strain. In conclusion, 23S PCR^RFLP showed to be of special usefulness for species identi¢cation, while AFLP was a valuable strain typing tool. Both methods indicated a high degree of genomic diversity within thermophilic campylobacters, irrespective of the host and geographical origin of the strains. When used in combination, they may provide accurate information of strain relationships at genotypic level allowing epidemiological surveillance of strains.
Acknowledgements This work was supported by the CICYT Project ALI1999-0539. Y.M. was the recipient of a research grant from the Ministerio de Ciencia y Tecnolog|¤a.
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