Random amplified polymorphic DNA fingerprinting of Campylobacter jejuni and C. coli isolated from human faeces, seawater and poultry products

~) INSTITOT PASTEUR]ELs~-VIER Res. MicrobioL Paris 1995 1995, 146. 685-696

Random amplified polymorphic DNA f'mgerprinthlg of Campylobacterjejuni and C. coli isolated

from human faeces, seawater and poultry products

J. Hernandez (t)(*), A. Fayos (i) M.A. Ferrus o) and R J . Owen (2)

tP; Departamento de Biotecnologia, Universidad Polit6cnica, Camino de Vera 14, 46022 Valencia (Spain), and

(2) National Collection o f Type Cultures, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT

SUMMARY

The polymerase chain reaction (PCR) technique was used to obtain randomly amplified polymorphic DNA (RAPID) profiles from 64 type and serotype reference strains and 114 isolates of Campylobacterjejuniand C. colifrom food, seawater and human faeces, Genetic diversity was detected among the strains as a total of 118 different RAPID profiles were obtained, each one containing from 4 to 11 bands between 0.30 and 1.50 ld). The discriminatory power of a random 10-mer primer (sequence 5'-CAATCGCCGT~3") was assessed. In general, no profiles were common to strains of the same Penner sero- group, but occasional strains from different Penner serotypes shared identical band profiles, RAPID analysis also differentiated between the species, and after numerical analysis, five main clusters were defined at the 40 % similarity level, corresponding to C. jejuni, C. co/i and C. /at /wi th some exceptions. RAPD profiling of Campy/obeyer is highly discriminatory and is a valuable new alternative to traditional typing in epidem- i o k ~ s t u d , s.

Key-words: Enteritis, RAPD, Campylobacter jejuni, Campyiob ~cter coli, Campylo- bacter lari; Genetic diversity, Typing, Epidemiology.

INTRODUCTION

Therrnophilic campylobacters are of world- wide significance in human and animal disease. Campy. obac ter j e j u n i in particular, is recog- rdzed as a major cause of acute bacterial enteritis in man in most developed countries (Grif- f i ths and Park, 19913; Heal ing et al . , 1992;

Tauxe, 1992). Sources of human in fec t ion remain largely undetermined, but contaminated foods, notably poultry and raw milk, are widely regarded as important vehic les of ;,nfection (Bu tz l e r and Oos te rom, 1991 ; Hernandez , 1993).

A c e t a t e methods of strain identification are essential for epidemiological purposes. Con-

Submitted January 25, 1995, accepted May 3 I, 1995.

(*) C~','esp~nd; lg author.

686 J. HERNANDEZ ET AL.

ventionaI phenotypic methods based on biotyping (Lior, 1984), serotyping (Penner and Hen- nessy, 1980) or phage typing (Khakhria and Lior, 1992) have been applied to campylobacters for over a decade. New molecular genotyp- ing methods based on cla-omosom~,l DNA analysis are more stable and avoid dependence on expressed and poss ibly var iable pheno typ ic features. Plasmid profiling has not been developed to any extent as a typing method, because fewer than 50 % of campylobacters carry plas- mids (Owen and Hernandez, 1990). The two molecular methods that provide precise and stable strain markers applicable to pure cultures of campylobacters are r ibosomal D N A ( r D N A ) gene p ro f i l e s f rom S o u t h e r n b lo t h y b r i d i z a t i o n ( r ibo typ ing) and p o l y m e r a s e chain reaction (PCR) -generated f ingerprints f rom random pr imer sequences (Owen and Hernandez, 1993).

Ribotyping is of value in typing most bacterial pathogens including Campylobacter species (Fayos et al., 1992; Gr imont and Gri- mont, 1986; Hernandez et aL, 1991a), but is t ime-consuming and not well suited to routi,~e use. The PCR has revolu t ionized molecular b i o l o g y t h r o u g h tbe i n t r o d u c t i o n of new g e n e t i c a s s a y s b a s e d c n s e l e c t i v e D N A amplif icat ion (Saiki et al., 1988). In 1990, a PCR method based on the t :mpl i f icat ion of random DNA fragments using a single primer of arbi trary sequence was descr ibed (Welsh and McClelland, 1990; Williams et al., 1990). RAPD analysis has since been applied to The typing of different pathogenic microorganisms including isolates of Campylobacter (Mazurier et aL, 1992), Listeria monocytogenes (Farber and Addison, 1994; Mazur ier and Wernars, 1992), Borre l ia burgdor fer i ~.Welsh et al. , 1992) and Proteus mirabi l is (Bingen et al., 1993).

In the present study, we evaluated the application of RAPD fingerprinting with a 10-mer

pr imer of a~ ~ ~trary sequence, for de tec t ing DNA polyrnc:phisms in C. jejuni and C. coii strains. The aim was to determine the genetic diversity among the strains and to assess its discriminatory power for typing in epidemiological studies.

M A T E R I A L S A N D M E T H O D S

Microorganisms and growth n,edia

The 178 strains of Campylobacter used are listed in table I with sources, strain numbers and Penner serotype if available ; 114 strains of C. jejuni and C. cell were isolated in V~lencia, Spain (1990-1993) from human faeces, seawater and poultry products. Also, 64 reference strains representing different Campylobacter sr~ecies, subspe- cies and Penner serotypes were included.

The primary isolation media were "Blood-free modif ied C C D A - P r e s t o n medium" (Oxoid SRCM739) with cefoperazone, and blood agar base no. 2 (Oxoid CM271) supple,q:ented with 5 % (v/v) defibrinated sheep blood (Oxoid SR51) and Butzler selective supplement (Oxoid SR85).

All bacteria were cultivated at 37°C for 48 h on 5 % (v/v) defibrinated sheep blood agar under microaerophilic conditions in an anaerobic jar (catalyst removed) that was evacuated to a pres- sure of 560mm of Hg (ca. 74.7 kPa) ond filled with 10% CO 2, 5% 0 2 and 85% N 2. Strains were preserved at - 7 0 ° C on glass beads on nutrient broth no. 2 (Oxoid CM67) containing 10% (v/v) glycerol, and were also lyophilized in 5 % (w/v) inositol serum.

Conventional bacteriological tests

The isolates were examined using the following conventional bacteriological tests: Gram stain, growth at 42°C• catalase production• nitrate reduc- tion hippurate hydrolysis HaS rapid test and DNA hydrolyms as described previously (Benjamin et al., 1983; Hernandez et al,, 1991b). Isolates were biotyped according to the extended scheme of Lior (1984).

C r A B -- cetyltrimethylammonium bromid... I RAPD = random amplified polymorphic DNA. PCR = polymorase chain reaction. 1 Taq ffi Thermus aquaticus.

RAPD OF CAMPYLOBACTER 687

RAPD analysis Computation of strain similarities

A 200-B1 suspension of bacterial cells was boiled for l0 min and centrifuged for 5 rain. The OOze ~ of the supernatant was measured and dilutions were prepared to an OD26o=0.15 according to Mazurier et al. (i992); 10 B1 of each dilution were used in the amplification reaction. Also, purified genomic DNA for RAPD analysis was obtained by a miniprep pro- tocol (Wilson, 1987) using cetyltrimethylammonium bromide (CTAB). The primer OPA-11 (Operon Technologies Inc., Alameda, CA) with sequence 5"- CAATCGCCGT-3" was used according to Fayos et ai. (1993).

A reaction volume of 100 BI was made up by addition of 2.5 U of Taq polymerase (DynaZyme TM

Thermostable DNA Polymerase, Finnzymes Oy, Finland), 10 mM Tris-HCl pH 8.8 at 25°C, 50 mM KCI, 1.5 mM MgCl 2, 0.1% Triton-Xl00, 200 BM of each deoxynucleotide triphosphate, 0.3 BM of the primer, 10 lal of the cell suspension (OD2~=0.15) and sterile distilled water. A negative control in which DNA was replaced with sterile distilled water was also included. The solutions were overlaid with 100 ptl of paraffin oil and cycled through the following temperature prof'fle: an initial denaturation step at 94°C for 1 min, 45 cycles of 94°C for 1 min (denaturation), 36°C for 1 rain (annealing) and 72°C for 2 rain (DNA chain extension), and a final elon- gation step at 72°(2 for 5 rain. Incubation was performed in a thermocycler (PHC-3 Thermal Cycler, Techne Corporation, Cambridge, UK). Standard procedures were adopted to eliminate contamination (Kwok and Higuchi, 1989). Duplicated RAPD analysis from two different DNA extractions was performed for each strain to assess reproducibility. Also, strain NCTC 11168 was included in each experimental run. The amplified DNA products were electrophoresed on 1.2% (w/v) agarose gels (ultrapure electrophoresis grade, Gibco-BRL) and stained in ethidium bromide solution, Either a 123- bp DNA ladder (Gibco-BRL) or bacteriophage ~, DNA digested with HindlII (Gibco-BRL) was used as a size marker lbr the PCR products.

Fragment size estimation

Gels were photographed and interpreted visually by three different observers. Molecular size of fragments was estimated by comparison with the molecular size marker using the computer program HOW- BIG (Grabner and Hofbauer, 1991), based on the reciprocal ec:relation of size to migration distance of DNA. To perform the calculations, the program automatically chose the three marker bands migrat- ing closest to the test band, thereby reducing the error to 0.5 % or less.

To compare RAPD patterns from different gels, the bands were coded according to size to minimize errors when determining similarities by computer- assisted methods of analysis. The amplification patterns were screened for bands within 17 different size ranges from 0.30 to 1.6 kb, and positive (pres- ence) or negative (absence) results were recorded. Bands of faint intensity were excluded. Double bands falling within a given range were scored as a single band. Computed similarities among strains were estimated by means of the Dice coefficient (negative matches excluded), and clustering of strains was based on the unweighted average pair group method (UPGMA) to facilitate the plotting of a dendrogram (Sneath and Sokal, 1973). All com- putations were performed using the NTSYS-PC program of Rohlf (1987).

Discrimination index

The discrimination index of the RAPD technique was calculated by application of the Simpson numerical index of diversity (Hunter and Gaston, 1988).

RESULTS

RAPD fingerprhtts

RAPD analysis of 208 strains of Campylo- bacter yielded multiple amplification products in 178 of the isolates. The other 30 strmns failed to give a PCR product, probably due to DNase activity by those particular isolates. The effect could not be eliminated even when DNA purified by the CTAB method was used, and these isolates were considered as non-typable by this method.

The DN A fragments resulting from the PCR amplification were electrophoretically s ep~- ated. The resul tant f ingerpr in t s compr i sed between 4 and 11 bands for each strain, witla sizes between 0.33 and 1.51 kb. Representa- t ive examples are shown in f igure 1. The results were similar for duplicate cultures of the same isolate, al though in some cases, a variation was observed in the relative amounts of amplification of certain DNA fragments, or


XABCDEFGHIJKXLMNOPQRSTUVX

: :!i

g

w ~=, ¢41r q l l - w ,qllt w ,q ..=,

kb

- 4.36

- 2.04

- 0.56

Fig. 1. Arbitrarily primed PCR (RAPD) patterns of representative strains of Campylobacter after electrophoresis

on a 1.2 % agarose gel. Genomic DNA was amplified using a 10-mer primer

with sequence 5'-CAATCGCCGT-3'. Lanes contained the following strains (see table I for strain numbers): X-- bacteriophage ~ DNA HindlII digest fragments, A =sterile distilled water (negative control), B = 107, C=I64, D= 137, E=13, F=72, (3=89, H=167, 1=112, J=73, K=15, L=I14, M=160, N=168, O=1($9, P=38, Q=97, R=161, 5=98, T=I4, U=I8, V=I6 (C. jejuni NCTC 11168T). Sizes (in kilobases) are indicated on the right.

in the lack of a band faintly represented in the alternative analysis. Only bands that appeared in both analyses were scored as part of the characteristic profile in the numerical study. Control assays in which cell suspensions were replaced by plain distil led water yielded no detectable amplified product.

A h igh degree of genet ic d ivers i ty was detected among the strains, which represented 118 different RAPD types (table I). The Simp- son numerical index gave a value of 0.990 showing a high discrimination ability for the method.

The inc lus ion in the s tudy of re ference strains representing a broad range of different Penner serotypes revealed no re la t ionsh ip between RAPD type and serotype. In some cases, strains with identical RAPD profiles had di f ferent serotypes, Converse ly , some strains with the same serotyp¢ had different RAPD profiles.

A n a l y s i s o f f i n g e r p r i n t s : d e t a i l s o f m a i n c lusters

Numerical analysis of the 178 RAPD patterns enabled the plot t ing of a dendrogram showing the level o f s imi lar i ty among the strains included in the study (fig. 2). Five main clusters (I-V) were defined at the 40 % similarity level, mainly corresponding to C. jejuni, C. coli and C. lari. These clusters were subdivided into subgroups at the 45 % similari ty level. Table II summarizes the different clusters and subgroups with details on species, RAPD types, and most predominant bands for each ciuster.

Cluster I contained 124 strains (99 %) phe- notypically characterized as C. jejuni subsp. jejuni, but with one hippurate-negative strain (1%) identified as C. coll. A total of 71 RAPD p r o f i l e s (P I - P 7 1 ) f r o m 125 s t r a ins were defined. All C. jejuni strains isolated from foods were in this cluster, but there were also representative strains belonging to all the other isolation origins. The majority of strains had charac ter is t ic ampl i f ica t ion DNA bands of 1.51, 0.79, 0.62 and 0.36 kb.

Cluster I was subdivided into subgroups la and Ib (fig. 2). Subgroup la was defined at the 57% similarity level and contained 116 strains from all isolation origins (RAPD prof'fles P l - P65). Subgroup Ib, defined at the 70 % simi- laxity level, contained only 9 C. jejuni strains (RAPD pl'ofiles P66-P71) isolated from foods (2 strains) and from human faeces (7 strains). Amplification DNA bands of 1.51 and 0.36 kb were found in all s t rains c lus tered in subgroup Ib.

Cluster II comprised a total of 33 strains: 29 strains (88 %) were ident if ied as C. coil and the remaining 4 strains (12%) were C. jejuni subsp, doylei including the type strain NCTC 11950 T. Thir ty different RAPD profiles (P72-P101) were defined. A major pro- por t ion of the strains (55 %) were isola ted from seawater, and they represented 67 % of all the seawater isolates. There were no spe- cially characteristic single bands for strains in this cluster.


No.

Table I. Strains of Campylobacter used in the study.

Source Species RAPD type Serotype

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 2O 21 22 23 24 25 26 27 28 29 3O 31 32 33 34 35 36 37 38 39 4O 41 42 43 44 45 46 47 48 49 5O 51 52 53

RAPD group Ia Faeces Cjj PI Faeces Cjj PI Faeces Cjj PI Faeces Cc P2 Seawater Cjj P2 Faeces Cjj P3 NCTC A709/92 Cii P3 NCTC A705/92 Cjj P3 NCTC A715/92 Cjj P3 NCTC A707/92 Cjj P3 NCTC 11322 Cjj P3 Poultry Cjj P3 NCTC A 198/81 Cjj P3 NCTC A176/82 Cjj P3 NCTC A178/81 Cjj P4 NCTC 11168 Cjj P4 NCTC A7 !6/92 Cjj P5 NCTC A714/92 Cjj P5 NCTC A703/92 Cjj P5 NCTC 10983 Cjj P5 NCTC 12500 Cjj P6 NCTC A 185/81 Cjj P7 Poultry Cjj P8 Faeces Cjj IX) Faeces Cjj P9 Faeces Cjj P9 Faeces Cii P9 NCTC A174181 Cjj P9 NCTC A704/92 Cjj P9 NCTC A711/92 Cjj P9 Seawater Cjj P l0 Faeces Cjj P 11 Faeces Cjj P12 Seawater Cjj P12 Faeces Cjj PI3 NCTC A191/81 Cjj PI4 Faeces Cjj Pl5 NCTC A170/82 Cjj Pl5 NCTC A622/89 Cjj Pl6 NCTC A627/89 Cjj PI6 NCTC A625/89 Cjj P16 NCTC A623/89 Cjj Pl6 NLYFC A628/89 Cjj PI6 NCTC A624/89 Cjj P16 NCTC A626/89 Cjj PI6 NCTC A174/82 Cjj PIT Faeces Cjj P18 Faeces Cjj PI9 Faeces Cjj P19 Poultry Cjj P20 Faeces Cjj P21 Faeces Cjj P21 Poultry Cjj P21

01 02 01 02 01

31 43 08 O2 01 01 02 02 01 17

O3 02 01

23

05 01 0I 01 01 01 01 01 29

690 .L H E R N A N D E Z E T AL.

No. Source Species RAPD type Serotype

54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99

100 101 102 103 104 105 106 107 108 109

Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces Faeces NCTC A180/81 NCTC A199/81 NCTC A203/81 Faeces NCTCA700/92 Faeces Faeces Faeces Poultry Poultry Faeces Faeces Faeces Faeces Faeces Faeces Faeces NCTCA189/81 NCTCA200/81 Faeces Faeces Faeces Faeces Faeces Faeces NCTC A177/81 NCTC A172/82 NCTC A175/82 NCTC A701/92 Faeces Faeces Faeces Poultry Faeces Faeces NCTCA710/92 NCTCA195/81 Faeces Faeces

c~ c~ c~ c~ c~ c~ c~ c~ c~ c~ c~ c~ c~ c~ c~ c~ c~ c~

c~ c~ c~ c~ c~ c~ c~ c~ c~ c~ c~ c~

c~ c~ c~ c~ c~ c~ c~ c~ c~

c~ c~ c~

c~ ch c~ c~ ch c~

c~ cjj

P22 P22 P23 P24 P24 P24 P25 P25 P25 P25 P25 P25 P25 P'26 P26 P27 P27 P'28 P28 P28 P29 P30 P31 P32 P33 P34 P35 P36 P36 P37 P38 P39 P40 P41 P42 P43 P44 P44 P44 P45 P46 P47 P48 P49 P49 P50 P51 P52 P53 P54 P55 P56 P57 P58 P59 P60

10 32 36

02

21 33

07 12 42 Ol

Ol 27



!10 111 112 113 114 115 116

117 118 119 120 I21 I22 123 124 125

126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143

144 145 146 147 148 149 150 151 152 153 154 155

156

Faeces NC'I C A 187/81 NCTC A202/81 NCTC A179/81 NCTC A205/81 NCTC A 173/82 NCTC A186/81

RAPDgrot,p Ib Faeces Faeces Faeces Faeces Poultry Faeces Faeces Faeces Poultry

RAPD group l la Faeces Faeces Faeces Poultry Seawater Seawater Faeces Faeces NCTC A 185/82 Seawater NCTC A193/81 NCTC A 197/81 NCTC A 180/82 NCTC A 183/82 Seawater NCTC A602/89 NCTC A607/89 NCTC A611/89

RAPD group Hb Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater Seawater NCTC 11950

RAPD group l lc Seawater

Cjj C~ C~ C~ C~ C~ C~

C~ C~ C~ Cjj C~ C~ C~ C~ C~

Cc Cc Cc Cc Cc Cc Cc Cc Cc Cc Cc Cc Cc Cc Cc Cjd Cjd Cjd

Cc Cc Cc Cc Cc Cc Cc Cc Cc Cc Cc Cjd

Cc

P61 P61 P62 P63 P64 P64 P65

P66 P67 P68 P68 P68 P69 P70 PTI P7I

P72 P73 P74 P75 P76 P77 P78 P79 P80 P81 P82 P83 P84 P85 P86 P87 P88 P89

I:'90 I:'90 P91 P92 P93 P94 P95 P96 P96 P96 P97 P98

P99

19 35 09 38 45 18

54

25 30 47 51

63 NT 35



157 158

159 160 161 162 163 164 165 166 167

168 169

170 171 172 173 174 175

176 177

17~

RAPD group l id Seawa~r Cc Seawa~r Cc

PIO0 PI01

RAPD group Hla Faeces Cc P102 NCTC A206/81 Cc P103 39 NCTC A173/82 Ce P104 14 Faeces Cc PI05 Faeces Cc P106 NCTC A196/81 Cc PI07 28 NCTC A181/82 Ce P108 48 Seawater Cc Pl09 NCTC A201/81 Cc P110 34

RAPD group m b NCTC A207/81 Cjj NCTC A208/81 Cjj

P l l l Pl12

RAPD group l l lc Seawater Cc P113 Seawater Cc P113 Seawater Ce P 114 Seawater Cc P114 Seawater Cc P ! 14 NCTC 12143 Cc P115

RAPD group IV Faeces Cjj Faeces Cjj

RAPD group V NCTC 11352 CI

PII6 PII7

PI18

40 41

Cjj=C. jejuni subsp, jejuni; Cjd=C. jejuni -ubsp. doylei: C¢=C. coli: CIffiC lari.

Clus te r III c o n t a i n e d 15 (88 %) C. coli strains and 2 (12 %) C. jejuni subsp, je juni strains. Fourteen RAPD profiles were defined (P102-P115). Amplification bands of 0.79 and 0.62 kb were f r equen t ly a feature of these strains. No food isolate was included in this cluster.

Cluster IV comprised two C. jejuni subsp. je juni s t ra ins i so la ted f rom h u m a n faeces (RAPD profiles Pl16-Pl17), with tile common amplification bands of 1.29, 0.36 and 0.33 kb.

Cluster V contained C. lari NCTC 11352 T

(RAPD profile P118) with amplification bands of 1.51, 0.88 and 0.55 kb (table II).

Analysis of fingerprints: details of subgroups

Figure 3 shows the subdivis ion of strains into a number of subgroups at the 45 % similarity level. For the human and poultry isolates, the RAPD types belor:ging to cluster la p redomina ted over the o ther types. A m o n g seawater isolates, the predominant RAPD type


'Fable H. Campylobacter strains, species and DNA band composition cf RAPD fingerprint clusters.

Most predominant DNA Cluster Species Total RAPD types bands (kb)

Ia P1-P65 1.51, 0.79, 0.62, 0.36 Cjj {*) 115 Cc 1

lb Cjj 9 P66-P71 1.5 l, 0.36 lla P72-P89 0.88, 0.40, 0.36

Cc 15 Cjd 3

lib tagO-P98 0.67, 0.55, 0.5t Cc 11 Cjd 1

IIc Cc 1 P99 1.11, 0.74, 0.67, 0.55, 0.47, 0.36 lid Ce 2 P100-PI01 0.88, 0.51, 0.36 IHa Ce 9 PI02-P110 0.79, 0.67, 0.62, 0.33 mb Cjj 2 Pl I 1-P112 1.00, 0.79, 0.62, 0.51 l i l t Cc 6 P113-P115 1.00, 0.79, 0.40 IV Cji 2 P116-P117 1.29, 0-36, 0.33 V CI I P118 1.51, 0.88, 0.55

(*) See footnote for table !.

was in subgroup l ib , whereas a number of subgroups compris ing seawater isolates (IIb, IIc, l id and IIIe) did not contain human or food isolates. In general, poultry and human isolates exhibited a similar distribution of RAPD pro-- files, but the seawater isolates appeared as a

more genomically distinctive group.

DISCUSSION

R A P D ana lys i s was success fu l ly appl ied p r ev ious ly to the d i f f e ren t i a t ion of l imi ted numbers of C. jejuni and C. coli strains (Mazu- t ier et al., 1992 ; Giesendorf et aL, 1994). In this study, we have examined a large selection of about 200 Campylobacter isolates of diverse origins as well as reference strains representing different Campylobacter species and Penner serotypes.

In a previous study of C. jejuni Penner sere-

groups 01 and 02 (Fayos et al. , 1993), we found that RAPD analysis provided a better discrimination than serotyping, as each of these two a n t i g e n i c g roups c o m p r i s e d d i f f e r en t R A P D types . T h a t was c o n f i r m e d in the present study of a broader range of serotypes, although in some cases serotyping was able to d iscr iminate between the same RAPD type. Nevertheless, RAPD analysis proved to have an excel lent discr iminat ion abil i ty (Simpson index=0.990) and reproducibility. RAPE} fingerprinting confu'ms the high degree of varia- t ion among Campylobacter strains that was previously detected by other molecular typing methods (Owen et al. , 1990 ; Owen et aL, 1993; Yan et al., 1991). In addition, this technique provides the advantages of other geno- typic methods such as the greater amount of information to be analysed and the stability of these characters. All RAPD procedures were r igorous ly cont ro l led in this study, but the question of good interlaboratory reproducibil- i ty r ema ins to be ach ieved . Some au thors


PERCENTAGE SIMILARITY 0 10 20 3 0 4 0 50 60 70 80 t . _ . , , , • | , , ,

9O IO0 | ,

C l u s t e r

I a

~b

I I a

i I b

I I C

I I d

m I I I a

- - I I I b

I I I c

I V

Fig. 2. Dendrogram of the numerical analysis based on the RAPD patterns of strains listed in table I.

The numbers on the horizontal axis indicate the percentage similarities as determined by the Dice coefficient. The vertical axis shows the main clusters defined at the 40 % similarity level.

obtained different results when using different thermal cyclers under identical reaction conditions (Ellsworth et al., 1993; MacPherson et al., 1993; Penner et al., 1993), and other fac-

tors such as the make of Taq and DNA purifi- cation procedures need to be critically assessed b e f o r e R A P D p r o f i l i n g can be g e n e r a l l y adopted as a standard typing procedure.

The numerical analysis of the amplification band patterns reveals the existence of an asso- ciation between RAPD groups and Campylo- bacter species, showing the possibi l i t ies of this method as a taxonomic tool. Analogous results have been achieved in other microbial genera (Van Belkum, 1994). The similar distribution of RAPD pattern clusters among faecal and pou l t r y i so la t e s c o n f i r m s the ro le o f ch.;c'ken meat in the transmission of the cam- pylobacteriosis. Surprisingly, most of the seawater isolates belonged to clusters which were not represented by faecal or poultry isolates. That fact suggests the existence of Campylo- bacter strains apparently not pathogenic and not transmitted through chicken meat.

In conclusion, RAPD f ingerpr int ing pro- v ides a d i sc r imina to ry and rapid means of comparing Campylobacter isolates, at least in intralaboratory studies. Such data are valuable in the ep idemioiogica i survei l lance and for investigating the distribution of types in different environments. Further invest igat ions are needed to find appropriate conditions for good interlaboratory reproducibility.

Acknowledgements

Part of this work was supported by the Direcci6n General de lnvestigaei6n Cientlfica y T~enica (DGICYT PB91-0887). A.F. is the recipient of a research grant from the Consejeda de Cultura, Education y Ciencia de la Generalidad de Valencia (Spain).

Profils RAPD de Campylobacterjejuni et C. coil isol~s de selles hnmaines, d'eau de met

et de produits de volaille

La PCR a 6t6 utilisre pour l'obtention de profils RAPD de 64 types et srrotypes de rrfrrence et 114 souches de Campylobacter jejuni et C. coli isolres d 'al iments , d 'eau de mer et de selles humaines. La diversit6 grnrtique est exprimre par 118 profils distincts, avec 4 ~ 11 bandes allant de


Percent of isolates

8 0 4

la Ib Ila lib IIc lid Ilia IIIb IIIc IVa V AFLPCR dusters

1 F a e c e s [ ] S e a water [~Poul t r / [ ]Reference

Fig. 3. Two-dimensional histogram showing the distribution of Campylobacter strains in tLAPD clusters and subgroups, defined at the 45 % similarity level, according to their sources.

0.30/t 1.50 kb, profils g6n6ralement bien distincts du s6rogroupage de Penner, alors que certaines souches de s6rotypes de Penner diff6rents ont des profils RAPD ident iques . L ' a n a l y s e R A P D assure la diff6renciadon des esp~ces. Une analyse num6rique d6gage cinq regroupements principaux (niveau de s imilar i t6 ~ 40 %) c o r r e s p o n d a n t aux esp~ces C. jejuni, C. coli et C. lari ~ de rares exceptions pr~s. La d~termination des proffls RAPD du genre Campy- lobacter s'av/~re tr~s diff6rentielle et offre ainsi une solution de relais dans le typage ~pid~miologique.

Mots-c lds: Ent6rite, RAPD, C a m p y l o b a c t e r jejuni, Campylobacter coil, Campylobacter lari ; Divcrsit6 g~n6tique, Typage, Epid6miologie.

References

Benjamin, J., Leaper, S., Owen, RJ. & Skirrow, M.B. (1983), Description of Campylobacter laridis, a new species comprising the Nalidixic Acid Resistant

Thermophilic Campylobacter (NARTC group). Curt. MicrobioL, 8, 23 !-238.

Bingen, E., Boissinot, C., Dcsjardins, P., Cave, H., Bra- himi, N., LamberbZechovsky, N., Denamur, E, Blot, P. & Ellen, J. (1993), Arbitrarily primed polymerase chain reaction provides rapid differentiation of Pro- teus mirabilis isolates from a pediatric hospital. J. Clin. Microbiol., 31, 1055-1059.

Butzler, J.P. & Oosterom, J. (1991), Campylobacter: pathogenicity and significance in foods. Int. J. Food Microbiol.. 12, 1-8.

Ellsworth, D.L., Rittenhouse, K.D. & Honeycutt, R.L. (1993), Artifactual variation in randomly amplified polymorphic DNA banding patterns. Biotechniques, 14, 214-217.

Father, J.M. & Addison, CJ. (1994), RAPD typing for dis- tinguishing species and strains in the genus Listeria. J. AppL BacterioL. 77, 242-250.

Fayos, A., Owen. RJ., Desai, M. & Hemandez, J. (1992), Ribosomal RNA gene restriction fragment diversity ,amongst Lior biotypes and Penner serotypes of Cam- pylobacter jejuni and Campylobacter coil FEMS MicrobioL Left., 95, 87-94.

Fayos, A., Owen, R.J., Hemandez, J., Jones, C. & Las- tovica, A. (1993), Molecular sabtyping by genome and plasmid analysis of Campylobacterjejuni sere- groups 01 and 02 (Penner) from sporadic and out-

696 J. H E R N A N D E Z ET AL.

break cases of human diarrhoea. EpidemioL blfect., 111, 415-427.

Giesendorf, B.A.J., Goossens, H., Niesters, H.G.M., Van Belkum, A., Koeken, A., Endtz, H.P., Stegeman, H. & Quint, W.G.V. (1994), Polymerase chain reaction- mediated DNA fingerprinting for epidemiological studies on Campylobacter spp. J. Meal. MicrobioL, 40, 141-147.

Grabner, M. & Hofbauer, R. (1991), A computer program for molecular weight determination of DNA fragments (HOWBIG). CABIOS, 7, 317-319.

Griffiths, P.L. & Park, R.W.A. (1990), Campylobacters associated with human diarrhoeal disease. J. AppL BacterioL, 69, 281-301.

Grimont, F. & Grimont, P.A.D. (1986), Ribosomal ribonu- cleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur/MicrobioL, 137B, 165-175.

Healing, T.D, Greenwood, M.H. & Pearson, A.D. (1992), Campylobacters and enteritis. Rev. Med. MicrobioL, 3, I59-167.

Hemandez, J. (1993), Incidence and control of Campylo- bacter in foods. Microbiologia (SEM), 9, 57-65.

Hernandez, J., Fayos, A. & Owen, R.J. (1991a/, Biotypes and DNA ribopatterns of thermophilie campylobacters from faeces and seawater in Eastern Spain. Lett. AppL MicrobioL, 13, 207-211.

Hernandez, J., Owen, R.J., Costas, M. & Lastovica, A. (1991 b), DNA-DNA hybridization and analysis of restriction endonuclease and rRNA gene patterns of atypical (catalase-weak/negative) Campylobacter jejuni from paediatric blood and faecal cultures. J. AppL BacterioL, 70, 71-80.

Hunter, P.R. & Gaston, M.A. (19881, Numerical index of the discrimination ability of typing systems: an application of Simpson's index of diversity. J. Clin. MicrobioL, 26, 2465-2466.

Khakhria, R. & Lior, H. (19921, Extended phage-typing scheme for Campylobacter jejuni and Campylobacter coli. EpidemioL Infect., 108, 403-414.

Kwok, S. & Higuchi, R. (1989), Avoiding false positives with PCR. Nature (Lend.), 339, 237-238.

Lior, H. (1984), New extended biotyping scheme for Cam- pylobacter jejuni, Carnpylobacter coli, and Campylo- bacter laridis. J. Clin. Microbiol., 20, 636-640.

MacPherson, J.M., Eckstein, P.E., Scoles, GJ. & Gajad- har, A.A. (1993), Variability of the random amplified polymorphic DlqA assay among thermal eyelets, and effects of primer and DNA concentration. MoL Cell. Probes, 7, 293-299.

Mazurier, S. & Wernars, K. (19921, Typing of Listeria strains by random amplification of polymorphie DNA. Res. Microbiol., 143, 499-505.

Mazurier, S., van de Giessen, A., Heuvelman, CJ. & Wer- nars, K. (1992), RAPD analysis of Campylobacter isolates: DNA fingerprinting without the need to pur- ify DNA. Lett. AppL Microbiol., 14, 260-262.

Owcn, R.J. & Hernandez, J. (19901, Occurence of plas- mids in "Carnpylobacter upsaliensis" (catalase negative or weak group) from geographically diverse patients with gastroenteritis or bacteraemia. Eur. J. EpidemioL, 6, 1 i 1-I 17.

Owen, R.J. & Hemandez, J. (1993), Ribotyping and arbi-

trary-primer PCR fingerprinting of campytobacters, in "New techniques in food and beverage microbiology", SAB Technical Series 31 (R.G. Krotl, A. Gil- mour & M. Sussman) (pp. 265-285). l:;luckwell Sci- entific Publ., Oxford.

Owen, R.J., Hernandez, J. & BoRon, F. (1990), DNA restriction digest and ribosomal RN& gene patterns of Carapylobacter jejuni: a comparison with bio-, sere-, and bacteriophage-types of United Kingdom outbreak strains. Epidemiol. Infect., 105, P65-275.

Owen, RJ., Desai, M. & Garcia, S. (1993), Molecular typing of thermotolerant species of Campylobacter with ribosomal RNA gene patterns. Res. MicrobioL, 144, 709-720.

Penner, J.L. & Hennessy, J.N. (1980), Passive haemagglu- tination technique for serotyping Campylobacter fetus subsp, jejuni on the basis of soluble heat stable antigens. J. Ciin. Microbiol., 12, 732-737.

Penner, G,A,, Bush, A,, Wise, R,, Kim, W., Domier, L., Kasha, K., Laroche, A., Stoles, G., Molnar, S.J. & Fedak, G. (1993), Reproducibility of random amplified polymorphic DNA (RAPD) analysis among laboratories. PCR Methods Applications, 2, 341-345.

Rohlf, J. (1987), Numerical Taxonomy and Multivariate Analysis System for the IBM PC Microcomputer (pp. 1-36). Applied Biostatistics Ine, Setauket, New York.

Saiki, R.K., Gelfand, D.H., Stoffel S., Scharf, S.J., Higu- chi, R., Horn, G.T., Mullis, K.B. & Ehrlieh, H.A. (1988), Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487-491.

Sneath, P.H.A. & Sokal, R.R. (1973), Numerical taxonomy. W.H. Freeman Co, San Francisco.

Tauxe, R.V. (1992), Epidemiology of Campylobacter jejuni infections in the United States and other indus- trialized nations, in "Campylobacter jejuni: current status and future trends" (I. Nachamkin, M.J. Blaser & L.S. Tompkins) (pp. 9-19). American Society for Microbiology, Washington, DC.

Van Belkum, A. (19941, DNA fingerprinting of medically important microorganisms by use of PCR. Clin. Microbiol. Rev., 7, 174-184.

Welsh, J. & McClelland, M. (1990), Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acid Res., 18, 7213-7218.

Welsh, J., Pretzman, C., Pestle, D., Saint Girons, I,, Baran- ton, (3. & McClelland, M. (1992), Genomic fingerprinting by arbitrarily primed polymerase chain re.ac- tion resolves Borrelia burgdorferi in three distinct phyletic groups. Int. J. Syst. Bacteriol., 42, 370-377.

Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. & Tingey, S.V. (1990), DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res., 18, 6531-6535.

Wilson, K. (1987), Preparation of genomic DNA from bacteria, in "Current protocols in molecular biology" (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.A. Smith, J.G. Seidman & K. Struhl). John Wiley & Sons, New York.

Yah, W., Chang, N. & Taylor, D.E. (1991), Pulsed-field electrophoresis of Campylobacter jejuni and Campy- lobacter coil genomic DNA and its epidemiologic application. J. Infect. Dis., 163, 1068-1072.

Random amplified polymorphic DNA fingerprinting of Campylobacter jejuni and C. coli isolated from human faeces, seawater and poultry products

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