Comparison of 23S polymerase chain reaction^restriction fragmentlength polymorphism and ampli¢ed fragment length polymorphismtechniques 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) andamplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter speciesisolated from clinical and poultry samples. 23S PCR^RFLP analysis performed to fingerprint 69 strains exhibited an excellent level oftypability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation ofCampylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed betweenthe 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 a69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observedbetween the AFLP profiles and the origin of the strains. When used in combination, 23S PCR^RFLP and single-enzyme AFLP methodscan be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters. > 2002 Federation ofEuropean 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 havebeen recognised as important agents of acute bacterialgastrointestinal infections, with Campylobacter jejuni themost common species associated with diarrhoeal illnessin man in both developed and developing countriesaround the world [1,2]. The major infection route for hu-mans is supposed to be the consumption of contaminatedpoultry products [3]. Although some others sources havebeen involved, the current epidemiology of infection re-mains undetermined [4].Accurate and reproducible methods of strain identi¢ca-

tion are essential for epidemiological purposes. Conven-tional phenotypic methods based on biotyping, serotyping

or phage-typing have been widely applied to the genusCampylobacter [5]. However, they usually lack discrimina-tory power or typability, and can fail to di¡erentiate atyp-ical strains [6,7].Molecular methods with high discriminatory power are

greatly required for a reliable identi¢cation of main Cam-pylobacter species specially in epidemiological studies toassess the transmission routes in epidemic outbreaks [8].An identi¢cation scheme for Campylobacter species us-

ing restriction fragment length polymorphism of PCR-am-pli¢ed 23S rRNA genes (PCR^RFLP) has been describedand showed to be able to identify Campylobacter and Ar-cobacter species, being more discriminatory, faster andmore cost e¡ective than phenotypic tests [9].More recently, the ampli¢ed fragment length polymor-

phism technique (AFLP), has been applied to several bac-terial species, including Helicobacter [10] and Campylo-bacter [11,12], showing great stability and discriminatorypower [13]. However, single-enzyme AFLP has not yetbeen 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

* Corresponding author. : Tel. : +34 (96) 387 7423;Fax: +34 (96) 387 9429.

E-mail address: jhernand@btc.upv.es (J. Herna¤ndez).

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Table 1Strains of Campylobacter used in the study and typing results

Straina Speciesb Source Specimen PCR^RFLP typec AFLP typec

R1 Cj NCTC 11168 Faeces r-2 a-4R2 Cj NCTC 11322 Faeces r-2 a-6R3 Cc NCTC 11366 Faeces r-10 a-8C01 Cj Hospital A Faeces r-2 a-1C02 Cj Hospital B Faeces r-2 a-8C03 Cj Hospital B Faeces r-2 a-1C04 Cj Hospital B Faeces r-4 a-4C05 Cj Hospital B Faeces r-2 a-5C06 Cj Hospital B Faeces r-4 a-4C07 Cj Hospital B Faeces r-4 a-4C08 Cj Hospital B Faeces r-2 a-2C09 Cj Hospital C Faeces r-2 a-4C10 Cj Hospital C Faeces r-2 a-2C11 Cc Hospital D Faeces r-10 a-4C12 Cc Hospital D Faeces r-10 a-5C13 Cj Hospital D Faeces r-4 a-2C14 Cj Hospital D Faeces r-11 a-4C15 Cj Hospital D Faeces r-2 a-1C16 Cj Hospital D Faeces r-5 a-1C17 Cj Hospital D Faeces r-8 a-3C18 Cj Hospital D Faeces r-4 a-1C19 Cj Hospital D Faeces r-4 a-3C20 Cj Hospital D Faeces r-1 a-1C21 Cj Hospital D Faeces r-2 a-3C22 Cj Hospital D Faeces r-7 a-1C23 Cc Hospital D Faeces r-10 a-1P01 Cj Market A Skin r-1 a-6P02Ad Cj Market A Liver r-6 a-5P02B Cj Market A Liver r-6 a-6P03A Cj Market A Liver r-6 a-6P03B Cj Market A Liver r-6 a-6P04 Cj Market A Liver r-4 a-6P05 Cj Market A Liver r-4 a-6P06 Cj Market A Liver r-1 a-6P07 Cj Market A Skin r-2 a-7P08 Cj Market A Skin r-2 a-6P09 Cj Market A Hamburger r-2 a-6P10 Cj Market A Hamburger r-2 a-6P11 Cj Market A Skin r-4 a-6P12 Cc Market A Skin r-3 a-6P13A Cj Market A Skin r-2 a-6P13B Cj Market A Skin r-2 a-6P14 Cj Market A Skin r-2 a-6P15A Cj Market A Carcass r-11 a-6P15B Cj Market A Carcass r-2 a-6P15C Cj Market A Carcass r-2 a-6P16A Cj Market B Carcass r-2 a-6P16B Cj Market B Carcass r-2 a-6P17 Cc Market B Carcass r-10 a-6P18 Cc Market B Carcass r-10 a-6P19 Cj Market B Carcass r-2 a-5P20 Cj Market B Carcass r-2 a-6P21 Cj Market B Carcass r-2 a-5P22 Cj Market C Carcass r-6 a-6P23 Cj Market C Liver r-2 a-6P24 Cj Market C Liver r-2 a-5P25 Cj Market C Liver r-2 a-5P26A Cj Market C Liver r-1 a-5P26B Cj Market C Liver r-1 a-6P26C Cj Market C Liver r-1 a-5P26D Cj Market C Liver r-1 a-4P27 Cj Market C Carcass r-2 a-5P28 Cc Market C Carcass r-10 a-6

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In this study, we have evaluated the combination of 23SPCR^RFLP and single-enzyme AFLP (sAFLP) ¢nger-printing methods for identi¢cation and typing of thermo-philic Campylobacter.

2. Materials and methods

2.1. Bacterial strains and growth conditions

A total of 23 clinical and 43 chicken isolates were usedin this study. Three NCTC reference Campylobacterstrains were also included for comparative purposes. Table1 provides information about their strain number, bio-chemical identi¢cation, sources, specimens, and typingpro¢les. Occasionally, several colonies showing morpho-logical di¡erences were selected from the same sampleand, consequently, they were considered as di¡erent iso-lates for typing purposes (Table 1).Clinical isolates were obtained in faeces of di¡erent pa-

tients su¡ering from diarrhoeic syndromes, within a periodof 5 years at four di¡erent hospitals. Chicken sampleswere collected from four di¡erent local markets.The primary isolation media were modi¢ed CCDA-

Preston selective agar (Oxoid CM739) with cefoperazone(16 g l31), and Campylobacter selective agar (Merck) sup-plemented with 5% de¢brinated sheep blood and Butzlerselective supplement (Oxoid SR085E).Once isolated, Campylobacter strains were maintained on

Blood agar base No. 2 (Oxoid CM67) supplemented with5% of sheep blood and incubated for 24 h at 37‡C undermicroaerophilic conditions (5% O2, 10% CO2, 85% N2).All strains were identi¢ed, prior to genotypic analysis,

by the API-Campy system (Biome'rieux). When doubtfulor unacceptable identi¢cation pro¢les were obtained, Liorbiotype 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 usingthe cetyltrimethylammonium bromide method [15].

2.3. PCR^RFLP analysis

A 2.6-kb 23S rRNA fragment from Campylobacterstrains was ampli¢ed using the LS1 and LS2 primerswith sequences 5P-GGATTTCCGAATGGGGCAACCC-3P and 5P-GTTTCGTGCTTAGATGTTTC-3P respectively[16]. Both primers were synthesised commercially (MWG-Biotech, Germany).Ampli¢cation was performed in a ¢nal volume of 100

Wl, containing 200 ng of genomic DNA, 2.5 U of Taqpolymerase (New England BioLabs, UK), 1.5 mM MgCl2,0.1 mM of each deoxynucleotide triphosphate (dNTP), 1.5WM of each primer in a reaction bu¡er (75 mM Tris^HClpH 8.0, 50 mM KCl, 20 mM ammonium sulfate). Thereaction mixture was overlaid with 50 Wl of mineral oilto prevent evaporation [9].An initial denaturation step at 95‡C for 5 min was fol-

lowed by 28 cycles of denaturation at 94‡C for 1 min,annealing at 58‡C for 1 min, and extension at 72‡C for2 min. Ampli¢ed products were visualised by 0.8% (w/v)NuSieve agarose gel electrophoresis and stained withethidium bromide (0.5 Wg ml31).Ampli¢ed DNA was concentrated by adding ethanol

Table 1. (Continued).

Straina Speciesb Source Specimen PCR^RFLP typec AFLP typec

P29 Cj Market C Carcass r-1 a-5P30 Cj Market C Liver r-9 a-5P31 Cj Market C Liver r-2 a-5P32 Cj Market C Carcass r-2 a-6P33 Cj Market D Carcass r-2 a-6P34 Cj Market D Carcass r-2 a-6

aR, reference strains; C, clinical isolates; P, poultry isolates.bCj, C. jejuni ; Cc, C. coli.cPCR^RFLP and AFLP types were de¢ned according to the results of the numerical analysis.dA, B, C and D represent di¡erent strains isolated from the same sample.

Fig. 1. 23S PCR^RFLP patterns of representative strains of Campylo-bacter after ampli¢cation of a 2.6-kb 23S rRNA fragment with primersLS1 and LS2, digestion with endonuclease HpaII, and electrophoresison a 3% agarose gel. Lane M, 100-bp DNA ladder used as molecularmarker.

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and resuspended in 15 Wl of TE bu¡er. DNA was thendigested 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% bromo-phenol blue) and fragments were visualised by 3% agarosegel electrophoresis in TAE bu¡er (40 mM Tris^acetate,2 mM EDTA, pH 8.3).

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 indi-cate the percentage similarities as determined by the Dice coe⁄cient. Vertical axis shows the main groups de¢ned at the 54% similarity level.

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2.4. AFLP analysis

A 10-Wg aliquot of genomic DNA was digested with 20U of restriction enzyme HindIII (New England BioLabs,UK) at 37‡C overnight in a ¢nal volume of 20 Wl contain-ing 5 mM spermidine trihydrochloride (Sigma).A 2.5-Wg aliquot of restriction fragments was then li-

gated 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 (NewEngland 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 inactivateT4 ligase.A 5-Wl aliquot of ligated DNA was then used as a tem-

plate for DNA ampli¢cation in 50 Wl of reaction mixturecontaining 2.5 mM of MgCl2, dNTP solution (200 mMeach) 200 ng of HIG primer (5P-GGTATGCGAACA-GAGCTTG-3P), and 1 U of Taq polymerase (GibcoBRL, Paisley, UK) in PCR bu¡er provided by the manu-facturer.The mixture was subjected to an initial denaturing step

at 94‡C for 5 min, and 33 ampli¢cation cycles consistingof 94‡C for 1 min, 60‡C for 1 min and 72‡C for 2 min.Ampli¢ed DNA fragments were separated electrophor-

etically in agarose gels (2.5% w/v) run in TAE bu¡er at70 V for 3 h. Following ethidium bromide stainings, thesizes of the visualised restricted fragments on the gels werecalculated from migration distances using UPGMA algo-rithms. Cluster analyses were performed using the Ntsysprogram, version 2.0 (Exeter Software, New York, USA).

3. Results

3.1. PCR^RFLP results

Stable and reproducible PCR^RFLP patterns formedby ¢ve to nine bands ranging from 230 to 1700 bp weredetected (Fig. 1). A common band of 525 bp was presentin all strains. Two bands of 600 bp and 315 bp were seenin 98.3% and 87% of the strains respectively.Numerical analysis of band patterns produced a den-

drogram in which all strains were grouped at 54% similar-ity level (Fig. 2), and a total of 11 di¡erent types werede¢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 chick-en-speci¢c strains was present, so no relationship betweenthe 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 referencestrain NCTC 11366, while all the rest of the types wereformed exclusively by C. jejuni isolates (30 strains).

3.2. AFLP results

Analysis of the 69 isolates resulted in 66 di¡erent band-ing patterns, with three to 22 ampli¢ed DNA fragments of235^1430 bp (Fig. 3). Although no common bands weredetected, 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 eightmain types could be di¡erentiated at 69% homology de-gree.Types a-1, a-2 and a-3 were composed exclusively of

strains of clinical origin. Type a-4 was formed by sevenclinical isolates, one poultry isolate, and one referencestrain of C. jejuni. Type a-5 was formed mainly by chickenisolates (12 out of 14 strains). No human-speci¢c strainwas 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. colireference strain.When PCR^RFLP and AFLP data were directly com-

pared by numerical analysis, no relationship could bedemonstrated.

4. Discussion

By 23S PCR^RFLP analysis all strains studied were¢ngerprinted using HpaII to digest the DNA, which indi-cates an excellent level of typability. Conserved restrictionpro¢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 belong-ing to the same species produced similar patterns and weregrouped together on the dendrogram. However, no signi¢-

Fig. 3. AFLP patterns of representative strains of Campylobacter aftergenomic DNA digestion with endonuclease HindIII, ligation with adapt-ers ADH1 and ADH2, ampli¢cation with HIG primer, and electropho-resis on a 2.5% agarose gel. Lane M, 100-bp DNA ladder used as mo-lecular marker.

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cant relationship could be observed between the pro¢lesand the origin of the strains; this supports the hypothesisthat con¢rms that poultry products are a likely source ofhuman infections.AFLP genotyping has proved to be a highly sensitive

method for subtyping and discriminating Campylobacterstrains. The high degree of variation obtained amongCampylobacter strains has been previously detected byother typing methods [17].Strains with a homology level higher than 95% were

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 thepercentage similarities as determined by the Dice coe⁄cient. The vertical axis shows the main groups de¢ned at the 51% similarity level.

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epidemiologically closely related as they had been isolatedfrom the same sample (P02A-P02B, P03A-P03B, P13A-P13B). So, AFLP patterns linked at 95% homology degreeor more were coded as AFLP subtypes within the sametype, and these strains were considered subclonal variantsfrom one original strain.In conclusion, 23S PCR^RFLP showed to be of special

usefulness for species identi¢cation, while AFLP was avaluable strain typing tool. Both methods indicated ahigh degree of genomic diversity within thermophilic cam-pylobacters, irrespective of the host and geographical ori-gin of the strains. When used in combination, they mayprovide accurate information of strain relationships atgenotypic level allowing epidemiological surveillance ofstrains.

Acknowledgements

This work was supported by the CICYT ProjectALI1999-0539. Y.M. was the recipient of a research grantfrom the Ministerio de Ciencia y Tecnolog|¤a.

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