Journal of Applied Bacteriology 1994, 77, 694-701

ldentif ication of Campylobacter jejuni, Campylobacter coli and Campylobacter lari by the nucleic acid amplification system NASBAR

M. Uyttendaele, R. Schukkink', B. van Gemen' and J. Debevere Department of Food Technology and Nutrition, Faculty of Agricultural and Applied Biological Sciences, UG, Ghent, Belgium and 'Organon Teknika Boseind 15, Boxtel, The Netherlands

4929/05/94. received 23 May 1994, revised 22 July 1994 and accepted 25 July 1994

M. UYTTENDAELE, R . SCHUKKINK, B . V A N GEMEN AND J. DEBEVERE. 1994. NASBA~, an isothermal amplification technique for nucleic acids, was evaluated for the specific identification of Campylobacter jejuni, Camp. coli and Camp. lari. A set of primers and a probe were chosen from the 16s rRNA sequence of Campylobacter. T h e probe was hybridized in solution with the amplified nucleic acids of 12 Campylobacter species and nine other Gram-negative bacteria. T h e probe was shown to hybridize specifically to the amplified single-stranded RNA of Camp. jejuni, Camp. coli and Camp. lari in an enzyme-linked gel assay (ELGA). In a C a m p . jejuni model system the combination of NASBAR and ELGA was able to detect ca 1000 rRNA molecules. The presence of an excess of Gram-negative bacteria did not influence the sensitivity of detection. A number of 6 cfu of Camp. jejuni, present in a total count of 4 x lo6 cfu of Gram-negative bacteria, resulted in a positive hybridization signal.

INTRODUCTION

Various studies have indicated that Campylobacter jejuni is the most common bacterial cause of acute gastro-intestinal infection in humans, exceeding rates of illness caused by both Salmonella and Shigella (Archer and Kvenberg 1985; Franco 1988). In addition to Camp. jejuni, the closely- related species, Camp. coli and Camp. lari, have also been implicated as agents of gastro-enteritis in humans. Campy- lobacter coli represented only approximately 3% of the Campylobacter isolates obtained from patients with campy- lobacter enteritis (Griffiths and Park 1990). Campylobaeter lari is a rather newly-described species that is very similar to Camp. jejuni (Stern and Karmi 1989). Unpasteurized milk has been implicated in a number of large outbreaks of campylobacter enteritis. Poultry is the premier vehicle in transmitting Camp. jejuni foodborne enteritis. Outbreaks associated with poultry rarely involve a large number of individuals. A few instances of campylobacter infection have been linked to the consumption of red meats (Stern and Karmi 1989; Griffiths and Park 1990). The fact that most of the reports are sporadic infections leads to an

Correspondence to Dr J Debevere, Department of Food Technology and Nutrttton, Faculty of Agrtcultural and Applred Biological Sciences, LJG, Coupure Ltnks 6i3, B-9000 Gent, Belgium

underestimation of the importance of this enteropathogen. Campylobacter jejuni, Camp. coli and Camp. lari require unusual growth conditions : reduced oxygen tension and a growth temperature of 42°C. This complicates the culture of these micro-organisms in a routine laboratory. In addi- tion, Campylobacter is a fastidious and slow-growing organism, easily suppressed by other enteropathogens. The selectivity of the isolation media is merely based on the presence of antibiotics. If resistance is acquired competing micro-organisms can easily interfere with the isolation pro- cedure. The traditional approach to the identification of the Campylobacter spp. is based on biochemical tests, resistance patterns and growth temperatures. Variation in a single test may result in misidentification of the strain or, at the least, confusion concerning the correct identity can easily occur. Such problems with isolation and identification often result in an inability to recover and identify the Campylobacter SPP.

DNA probes offer an attractive alternative for the detec- tion of pathogens (Walker and Dougan 1989). Specific seg- ments of the Campylobacter genome (Korolik et al. 1988) can be used as a probe or a synthetic oligonucleotide probe for the 16s rRNA gene (Rashtchian et al. 1986; Romaniuk et al. 1987) can be constructed. The polymerase chain reac- tion (PCR) offers an additional tool for the identification of micro-organisms, including Campylobacter (Giesendorf et

NASBAR IDENTIFICATION OF CAMPYLOBACTER 695

al. 1992; Oyofo et al. 1992). This technique facilitates exponential amplification of a preselected region of DNA and increases the sensitivity of detection. An alternative approach is NASBAR (Compton 1991 ; Kievits et al. 1991), a technique to selectively amplify RNA. Isothermal nucleic acid amplification of RNA in NASBAR is achieved through the concerted action of avian myeloblastosis virus reverse transcriptase (AMV-RT), T 7 RNA polymerase and RNase H (Fig. 1). The reaction starts with a non-cyclic phase, in which a downstream primer containing a tail-sequence of

+RNA 5' -3'

Primer I I 5' 3'

3' 5'

5' - 3' 3' - - - - - - - - 7

5l I RNaseH 1 Primer 2

5'-3' 5'- 3'

3'- 5' 3'- 5'

3' fvvvvw 5' 3'- 5'

3' w 5'

T7 RNA pol

T7 RNA POI

3' -5 ' --I r- Primer 2

I

3l 5 ' - - - - - 3' 3'fvvvyv 5' ___) 51 _ _ _ _ _ 3' __t 5'----.

Fig. 1 Schematic presentation of the NASBAR method applied to ssRNA. The method is based on extension of Primer 1 (T7-containing) by avian myeloblastosis virus reverse transcriptase (AMV-RT) on a RNA/DNA template, degradation of the RNA strand by RNase H, synthesis of a second DNA strand by AMV-RT and RNA synthesis by T7 RNA polymerase. With RNA synthesis the system enters the cyclic phase, based on the principles described above. RT, AMV-RT. Broken lines represent newly synthesized DNA and wavy lines represent RNA. For further explanation see text

RNase H Primer I 3'75,

the T7-promoter sequence anneals to the single-stranded target sequence. Through the action of AMV-RT, cDNA is formed. The RNase H hydrolyses the RNA from the RNA-DNA hybrid, which results in a single strand of DNA to which the upstream primer can anneal. The AMV-RT synthesizes, through its DNA polymerase acti- vity, a second DNA strand producing a double-stranded DNA intermediate with a transcriptionally active promoter sequence. The T 7 RNA polymerase generates from this intermediate single-stranded RNA copies (100-1000) which serve as a template in the now cyclic phase of NASBAR.

Major advantages of NASBAR over PCR are that NASBAR is performed isothermally which precludes the use of a special thermocycler, and that no separate R T step is required for RNA amplification. NASBAR has already been successfully applied to the detection of RNA viruses (Kievits et al. 1991). Detection levels are 10 molecules of HIV-1 RNA in a model system with in vitro generated HIV-1 RNA as input.

The comparison of 16s rRNA sequences is a powerful method for the systematic classification of micro-organisms (Olsen et al. 1986; Woese 1987). The phylogenetic analysis of the genus of Campylobacter is supported by this tech- nique (Paster and Dewhirst 1988; Thompson et al. 1988). This suggests that the 16s rRNA sequences include a large potential to allow for identification at the species level. This study investigated the use of the NASBAR amplification technique to identify Camp. jejuni, Camp. coli and Camp. lari based on a specific region of the 16s rRNA.

MATERIALS AND METHODS

Bacterial strains and growth conditions

The bacterial strains used in this study are listed in Table 1. Campylobacter spp. were grown on Mueller-Hinton (Oxoid) medium, supplemented with defibrinated horse blood, in an atmosphere of 5% 0,/10% C02/85% N, (OCN atmosphere) at 37°C. Other bacteria were grown on Tryptic Soy agar (TSA; LabM) plates and were incubated aerobically at 37°C with the exception of the Pseudomonas spp. and Shewanella putrefaciens, which were incubated at 30°C. Subsequently, a 24-h-old culture of Campylobacter spp. in Brucella broth (BB; BBL) and an l8-h-old culture of the other Gram-negative bacteria in Brain Heart Infu- sion broth (Gibco) was made. The number of colony- forming units (cfu) in 1 ml of the enrichment cultures was determined after dilution with physiological saline (04iS% NaCl) and plating on Campylobacter Charcoal Differential agar (CCDA; Oxoid) for Camp. jejuni, Camp. coli and Camp. lari and on TSA for the Gram-negative bacteria.

696 M. UYTTENDAELE ET A L .

Table 1 Bacterial species tested by the NASBAR procedure

Species Strain

Campylobacter colr Campylobacter fetus wbsp fetus Campylobacter fetus subsp venerealis Campylobacter hyotntestrnalts Campylobacter jejunt subsp doylei Campylobacter lari Campylobacter sputorum biovar sputorum Campylobacter sputorum biovar fecalis Campylobacter sputorum biovar bububs Campylobacter upsaltensis Campylobacter jtyunt subsp jejuni Campylobacter jejunr subsp J e j U n l

Crfrobacter freundrr Enterobacter cloaca Escherrchta colr Shewanella putrejm tens Klebsrella oxytoca Proteus culRarrs Pseudomonas aeruginosa Pseudomonas /luorescens Salmonella enterrttdis

LMG 6440 LMG 6442 LMG 6443 LMG 7817 LMG 8843 LMG 8846 LMG 7795 LMG 6617 LiMG 6447 LMG 8850 LMG 6444 LMG 6629 LMG 3246 LMG 2783 LMG 8223 LMG 2250 LMG 3055 LMG 2096 LMG 8029 LMG 1794 LMG 10395

LMG, Culture collection of the Laboratory for Microbiology, University of Gent, Belgium.

Campylobacter s p p . were isolated from chicken legs using Preston enrichment for 24 h at 42'C under OCN atmo- sphere and streaking on CCDA. Nineteen suspected colo- nies were characterized as Camp. j e jun i by conventional microbiological identification methods (spiral-shaped mor- phology, positive catalase and oxidase activity, no dextrose utilization, hippurate hydrolysis, nalidixic acid-sensitive and cephalotin-resistant, growth at 37"Cand 42"CunderOCN atmosphere but not at 30°C, no aerobic growth at 37°C). These strains were included in the study to explore the use of NASBAR on food isolates of Camp. jejuni. They were treated in a similar fashion to the reference strains of Campylobacter.

Nucleic acid isolation

The enrichment culture of the bacteria (1.0 ml) was used for lysis and nucleic acid isolation according to the protocol described by Boom et a f . (1990). In brief, the bacteria were added to 9.0 ml of a guanidinium thiocyanate (GuSCN) lysis solution (5-25 mol 1-' GuSCN, 50 mmol 1-l Tris/ HCl, pH 6.4, 20 mmol 1-' EDTA, 1.3 % w/v Triton-X- 100) and mixed intens to facilitate rapid lysis. Activated silica (70 p1 silica (Sigma); 1 g ml-' suspension in 0.1 mol 1-' HCl) was added to bind the released nucleic acid. The nucleic acid-silica complex was washed twice with GuSCN washing solution (5.25 mol 1- ' GuSCN; 50 mmol I- ' Tris/HCl, pH 6.4), twice with 70% (v/v) ethanol and once with acetone. After drying the silica at 56"C, nucleic acids were eluted from the silica with 100 pl RNase-free H,O and stored at - 20°C.

Selection of primers and probes

The primers and probe used in this study are listed in Table 2. They were chosen from a partial 16s rRNA sequence alignment of Campylobacter species (Romaniuk et al. 1987). From the published sequence data of Camp. jejuni these primers were calculated to be 123 nucleotides apart. The primerset (Primer 1 OT1118; Primer 2 OT1547) sandwiches a variable sequence in Campylobacter species. In this stretch of RNA a sequence specific for Camp. jejuni, Camp. coli and Camp. lari occurs. On the basis of this characteristic sequence an oligonucleotide (OT1559) was synthesized for identification.

NASBA~

The NASBAR reactions were performed as described by Kievits et a f . (1991) with some modifications. The final volume of the reaction mixture was 25 p l . First, a 21 pl volume prereaction mixture was assembled so that the final concentration for 25 pl would be: 40 mmol I - ' Tris/HCl, pH 8.5, 12 mmol 1-' MgCl,, 42 mmol 1-1 KC1, 5 mmol I - ' DTT, 1 mmol I - ' of each deoxynucleotide tri- phosphate, 2 mmol I - ' of each nucleotide triphosphate, 15% (v/v) dimethylsulphoxide, 12 U RNA guard

Table 2 Sequences of primers and probe Oligo Sequence* used in amplification of 16s rRNA

PI OT1118 5' A A T TCT AAT ACG ACT CAC T A T AGG GAG AGT GTG ACT GAT CAT CCT CTC A 3'

5' GAC .4AC AGT TGG AAA CGA CTG CTA A T A 3' 5' CTG CTT AAC ACA AGT TGA GTA GG 3'

P2 OT1547 Probe 031559

* Sequences are located in the partial 16s rRNA sequence obtained by using a universal primer complementary to positions 519-536 of Escherichia coli 16s rRNA (Romaniuk et al. 1987).

NASBAR IDENTIFICATION OF CAMPYLOBACTER 697

(Pharmacia), 0.2 pmol l - ' Primer 1 and 0-2 pmol 1-' Primer 2. After the addition of 2 pl of target RNA the tubes were incubated for 5 min at 65°C in order to uncoil the tertiary and secondary structures of the 16s rRNA. The reaction mixtures were then directly transferred to 41°C. After 2 min, 2 pl of an enzyme mix was added to the tubes, resulting in a final volume of 25 pl, containing 0.1 pg bovine serum albumin pl-', 38 U T 7 RNA polymerase (Pharmacia), 8 U AMV-RT (Seikagaku, Rockville, MD, USA) and 0.1 U RNase H (Pharmacia). Isothermal amplifi- cation of the RNA target was performed by incubation of these samples at 41°C for 1.5 h. Reaction mixtures contain- ing no target nucleic acid, but 2 p1 RNase-free H,O instead, served as negative controls. The amplification pro- ducts were directly processed for detection analysis by agarose electrophoresis or enzyme-linked gel assay (ELGA) (see below).

Agarose detection and enzyme-linked gel assay

Agarose detection. For detection of the amplification products, 7.5 p1 of each reaction mixture was loaded onto a 2% agarose (Pronarose, SphaeroQ, Leiden, The Netherlands) gel containing ethidium bromide (Sambrook et al. 1989). The separated products were visualized with U.V. light.

Enzyme-linked gel assay. A rapid non-radioactive 'in- solution' hybridization assay (ELGA) was developed to identify the NASBAR products with species-specific oligo- nucleotide probes (ELGA probes) 5'-labelled with horse- radish peroxidase (HRP). Hybridization was performed without the need for prior denaturation of the NASBAR products, since the product of NASBAR is mainly single- stranded RNA. Stringent hybridization conditions are com- pulsory to distinguish the mismatches in a target by the ELGA probe. After hybridization excess non-hybridized ELGA probes were separated from the homologous hybri- dized product by vertical gel electrophoresis. The free ELGA probes and the hybridized products could be directly visualized in the acrylamide gel by incubating the gel directly in substrate solution for HRP. Because of its lower mobility the homologous hybridized product migrates in the gel above the free ELGA probe.

An ELGA hybridization was performed by mixing 1 p1 of the NASBAR reaction product with 4 p1 hybridization solution (final concentration of this reaction mix was 1 x SSC (0.15 mol I- ' NaCl; 0.015 mol 1-' sodium citrate), 0.01 % bromophenol blue, 0.01 YO xylene cyanol and 10" molecules of HRP-labelled probe). The hybri- dization reaction mixtures were incubated for 10 min at 60°C. Hybridization reaction mixtures (2.5 p1) were then

directly applied on a 7% acrylamide gel, containing 0.04% (w/v) dextran sulphate. After electrophoresis, the hybrid- ized amplification products and the ELGA probes in the gel were visualized by staining with 40 ml of substrate solu- tion (80 pg 3,3',5,5'-tetramethylbenzidine ml - and 04025% H,O, in 0.01 mol 1-' sodium citrate buffer, pH 5.0). The gel was incubated in the substrate solution for approximately 10 min at room temperature before blue- stained bands appeared. The gel was fixed by incubation in 50% (v/v) methanol overnight at room temperature.

Sensitivity of NASBA~

The sensitivity of the NASBAR technique was investigated with a Camp. jejuni LMG 6444 model system. This was done by adding a 10-fold serial dilution of purified total nucleic acid, derived from 1 ml of a Camp. jejuni enrich- ment broth, to the amplification reactions. In addition, a 10-fold serial dilution, starting from an enrichment of Camp. jejuni, was made in BB. Nucleic acid was extracted from this dilution serial and amplified. In order to investi- gate the influence of an accompanying bacterial flora on the sensitivity of detection of Camp. jejuni using NASBAR, a 10-fold dilution series of Camp. jejuni cfu was made and a mix of the Gram-negative bacteria in Table 1 was added to each of the test tubes resulting in the presence of approx. lo6 cfu of Gram-negative bacteria. Nucleic acid was extracted and amplified.

RESULTS

Specificity of N A S B A ~

The NASBAR primers (OT 1547 and OT 11 18) directed the amplification of an approximately 200 nt sequence of the 16s rRNA containing a region which is variable in Campy- lobactev. The amplification product was visualized on agarose gel (Fig. 2a).

Identification of the NASBAR products was done with the ELGA technique, an 'in-solution' hybridization assay. Electrophoresis of the hybridization reaction mixtures is necessary to discriminate between free ELGA probe (fast mobility) and ELGA probe that has specifically hybridized to the NASBAR product (slow mobility). Both products could be visualized directly in an acrylamide gel (Fig. 2b). Probe OT1559 hybridized to both strains of Camp. jejuni. No hybridization occurred at the negative control notwith- standing the presence of amplification product on the ethi- dium bromide-stained agarose gel.

The specificity of NASBAR in combination with ELGA is shown in Fig. 3. Probe OT1559 hybridized with ampli- fied RNA from Camp. jejuni subsp. jejuni LMG 6444,

698 M. UYTTENDAELE ET A L .

Fig. 2 (a) Ethidium bromide-stained agarose gel showing the NASBAR amplification products of Campylobacterjejuni LMG 6444 (lanes A and B) and Camp. jejuni LMG 6629 (lanes C and D) with primer set OT1547/0T1118. M, Marker; and E, amplification product of the negative control sample. (b) Identification of Camp. jejuni LMG 6444 (lanes 2 and 3) and Camp. jejani LMG 6629 (lane 4 and 5) using the ELGA probe OT1559. After 'in solution' hybridization (ELGA) of the NASBAR product, the hybridization reaction mixtures were run in a 7% acrylamide gel, to separate the free ELGA probe (fast mobility) from the homologous hybridized NASBAR product (slow mobility). The negative control sample used in this assay (lane 6) contained no target. Lane 1 contained free ELGA probe

Camp. jtjuni subsp. jejunt L M G 6629, Camp. jejuni subsp. doylei LMG 8843, Camp. ro l i L M G 6440 and Camp. lari LMG 8846. Hybridization did not occur between amplifi- cation products derived from other members of the Cumpjj- lobacter species (Fig. 3a), neither did the probe hybridize to amplification products of other Gram-negative bacteria which can be found in food products (Fig. 3b).

Sensitivity of N A S B A ~

Nucleic acid derived from a BB enrichment containing 5 x 10' cfu ml- ' Camp. jejuni was diluted and amplified. The amount of nucleic acid serving as input is expressed relative to the corresponding number of rRNA molecules, presuming 10' rRNA molecules per cell (Olsen et al. 1986). The nucleic acid isolated from 1 ml of BB enrichment of Cump. jejuni containing 5 x 10' cfu was finally eluted in 100 pl of water and 2 p1 was used for an amplification reaction. This means that nucleic acid derived from 1 ml of the enrichment corresponded to approximately 10' rRNA molecules. The amount of nucleic acid amplified ranged from 1012 to 10' corresponding rRNA molecules. A clearly detectable signal (Fig. 4a) was visualized on an acrylamide

Fig. 3 Specificity of NASBAR in combination with the ELGA probe OT1559. (a) NASBAR was performed on nucleic acid derived from different Campylobacter species. Lanes : 1, Campylobacter coli; 2, Camp. fetus subsp. fe tus; 3 , Camp. fetus subsp. venerealis; 4, Camp. hyoiniesiinalis; 5 , Camp. jejuni subsp. doylei; 6, Camp. lari; 7 , Camp. spuiorum biovar sputorum; 8 ; Camp. sputorum biovar fecalis; 9, Camp. spuiorum biovar bubulus; 10, Camp. upsaliensis; 1 1 , Camp. jejuni subsp. jejuni LMG 6444; 12, Camp.jejuni subsp. jejuni LMG 6629; 13, negative control. (b) NASBAR was performed on nucleic acid derived from different Gram-negative bacteria. Lanes: 1, Shewanella putrefaciens; 2, Pseudornonas Jluorescens ; 3, Ps. aeruginosa ; 4, Klebsiella oxyioca ; 5 , Enterobacter cloaca ; 6, Citrobacter freundii; 7, Escherichia coli; 8, Salmonella enteritidis; 9, Proteus vulgaris; 10, Campylobacier jejuni LMG 6444; 11 , negative control; 12, Camp. coli; 13, free ELGA probe

gel for an amount of nucleic acid corresponding to lo", lo", lo'', lo9, and 10' rRNA molecules. For further dilu- tions of nucleic acid ranging from lo7 to 10' rRNA mole- cules two blue stained bands were visible. The upper indicated the presence of hybridized probe, the lower band represented unhybridized probe. This means that the amount of amplification product was not sufficient to bind all of the probe added, but it still proved the presence of specific amplification product. A weak signal indicating hybridization can be noticed for nucleic acid input corres- ponding to 10' rRNA molecules. For five of the 10 repeat- ed amplification reactions a weak upper band appeared in the case of nucleic acid input corresponding to lo2 rRNA molecules and for four of the 10 in the case of 10' rRNA molecules. These last results are, therefore, not repro- ducible. For nucleic acid corresponding to 10' rRNA mol- ecules a clearly positive hybridization was again seen. This

NASBAR IDENTIFICATION OF CAMPYLOBACTER 699

Fig. 4 Sensitivity of NASBAR. (a) Analysis of NASBAR reactions containing a dilution serial of total nucleic acid purified from Campylobacterjejuni LMG 6444 (5 x 10' cfu). Lanes 1 and 17, free ELGA probes; lane 2, the derived nucleic acid undiluted corresponding to approx. 10'' rRNA molecules; lanes 3-14, 10-'-10-'' of the total isolated nucleic acid as input, corresponding to approx. 10"-lOo rRNA molecules; lanes 15 and 16, negative control samples. (b) Analysis of NASBAR reactions performed on nucleic acid isolated from a dilution serial of Camp. jejuni LMG 6444. The number of bacteria per sample was as follows: lane 2,3 x lo7; lane 3,3 x to6; lane 4,3 x 10'; lane 5, 3 x lo4; lane 6,3 x lo3; lane 7 ,3 x 10'; lane 8 , 3 x 10'; lane 9, 3 x 10'. Lanes 1 and 12 contained free ELGA probe, lane 10 and 11 represent negative control samples. (c) Analysis of NASBAR reactions performed on nucleic acid isolated from a suspension of Gram-negative bacteria (listed in Table 1) containing diminishing numbers of Camp. jejuni LMG 6444. The number of Gram-negative bacteria in each of the samples was 4 x lo6. Lanes 1 and 14 contained free ELGA probe, lanes 2 and 13 represent negative control samples and lane 3 used nucleic acid derived from the suspension of Gram-negative bacteria without addition of Camp. jejuni. The number of Camp. jejuni LMG 6444 was as follows: lane 4,6 x 10'; lane 5,6 x 10'; lane 6 , 6 x 10'; lane 7, 6 x lo3; lane 8 , 6 x lo4; lane 9,6 x lo5; lane 10,6 x lo6; lane 11,6 x lo7; lane 12,6 x 10'

Fig. 5 Identification of Campylobacter jejuni strains isolated from chicken products. Lanes 1 and 16, free ELGA probe; lanes 2-11 correspond to Camp. jejuni isolates 1-10; lane 12, Camp. jejuni LMG 6629; lane 13 used an input of nucleic acid derived from a suspension of Gram-negative bacteria (Table 1); lanes 14 and 15, negative control

was probably due to contamination during the input of the reaction. However, the two negative controls showed only a band referring to unhybridized probe.

In the following experiment an enrichment broth of Camp. jejuni containing 3 x lo7 cfu ml-' was diluted in BB and subsequently the 10-fold dilution serial was used for nucleic acid extraction. A clearly detectable signal was visualized on an acrylamide gel for the amplification product of nucleic acid derived from 3 x lo7, 3 x lo6, 3 x lo5, 3 x lo4, 3 x lo3, 3 x 10' and 3 x 10' cfu of Camp. jejuni. No hybridization occurred in the case of 3 x 10' cfu and the negative controls.

In order to investigate the effect of an accompanying bacterial flora on the sensitivity of detection, a 10-fold dilu- tion serial of an enrichment of Camp. jejuni containing 6 x 10' cfu ml- was made in the presence of 4 x lo6 cfu of Gram-negative bacteria, a detection level of 6 cfu of Camp. jejuni was reached (Fig. 4c). A clearly detectable hybridization signal was seen with amplification product derived from 6 x 10' to 6 x lo3 cfu of Camp. jejuni. Amplification product derived from 6 x 10' to 6 x 10' also demonstrated an upper band, indicating hybridization, but a lower blue stained band corresponding to unhybridized probe was also visible. The negative control, with RNase-free H,O as input, and amplification product of nucleic acid derived from the test tube containing only Gram-negative bacteria and no Camp. jejunz, showed no hybridization.

ldentiflcatlon of different Campy/obacter/e/un/ isolates

Probe OT1559 was based on the 16s rRNA sequence of a single Camp. jejuni strain. Therefore, Camp. jejuni strains were isolated from chicken legs, chicken breast and whole

700 M . UYTTENDAELE E T A L

chicken obtained from different commercial chicken slaughter houses by the conventional method : enrichment in Preston medium followed by CCDA isolation and confir- mation to species level. The 19 Camp. jejuni isolates were tested in this study. NASBAR in combination with ELGA and probe OT1559 allowed identification of the 19 Camp. jejuni strains. Results of ELGA for 10 of the 19 isolates are shown in Fig. 5.

DISCUSSION

The NASBAR primers allowed amplification of a Camp. jejuni, Camp. coli and Camp. lari specific region situated in the 16s rRNA. An amplification product was clearly visible (Fig. 2a), also in the negative controls. A possible explana- tion is that the enzymes contain nucleic acid sequences which allow the primers to anneal and permit synthesis of non-specific amplification product. When the negative control was run on an ELGA acrylamide gel no upper blue stained band could be seen. This indicated a non-specific amplification product that does not hybridize with a specific probe. Thus identification of Camp. jejuni, Camp. roli and Camp. Iari cannot be supported by the pres- ence or absence of amplification product on an ethidium bromide-stained gel. A subsequent hybridization step is necessary. Combination of the NASBAR amplification with ELGA hybridization and detection assay allowed specific identification of the Campylobacter enteropathogens Camp. jejuni, Camp. coli and Camp. lari (Fig. 3). Previous results using the combination of NASBAR and ELGA were obtained by Van der Vliet et al. (1993), who also used a 16s rRNA sequence and the NASBAR amplification technique to successfully identify Mycobacterium species.

With the NASBAR amplification system an input of as few as 1000 rRNA molecules produces sufficient specific amplification product to allow for a reproducible hybrid- ization signal (Fig. 4a). I t was even possible to detect as small an amount of nucleic acid as that corresponding to 100 or 10 rRNA molecules, although this was not repro- ducible. This supported previous results by Kievits et al. (1991) who were able to detect as little as 10 molecules of HIV-1 RNA with in vitro generated RNA as input. A side effect of the high sensitivity of the primer set applied is the increased chance of contamination as the transfer of a few hundred rRNA molecules between the different samples during the input of the NASRAR method may be sufficient to cause false positive results. With regard to the above- mentioned results of %ASBAR, it should be possible, with the combination of NASBAR and ELGA, to detect a single cell of Camp. jejuni (approx. lo5 rRNA molecules gathered in 100 pl of RNase-free H,O or approx. lo3 rRNA mole- cules serving as input for an amplification reaction). When a BB dilution of Camp. jejunt, containing 30 cfu ml-', was

used for nucleic acid isolation, a positive hybridization signal could be seen on ELGA after amplification (Fig. 4b). Previous studies mentioned the use of PCR technology in combination with DNA probes for the identification of campylobacters (Giesendorf et al. 1992; Oyofo et aI. 1992). When the NASBAR technique was used the same level of detection could be reached as with PCR. Giesendorf et al. (1992) mentioned a detection limit of 12.5 cfu. Oyofo et al. (1992) detected as little DNA as the equivalent of four bac- teria. The detection level of Camp. jejuni in the NASBAR model system was 30 cfu.

In the last experiment, which tested the sensitivity of the NASBAR reaction in the presence of other Gram-negative bacteria, six bacteria (Fig. 4c) could be detected while, in the amplification of the nucleic acid derived from the dilu- tion serial of a pure culture, three bacteria (Fig. 4b) showed no hybridization. Possibly the 1 ml dilution, used for nucleic acid extraction, should contain 3 cfu as determined by plating out 1 ml of the same dilution on CCDA, but in fact it is statistically possible that there were no bacteria present in the 1 mi used for nucleic acid extraction. The detection level of Camp. jejuni with NASBAR and ELGA, however, is situated closely around the order of 10 cells. With the serial dilution of a pure reference culture (Fig. 4b), an input of 300 and 30 cfu of Camp. jejuni clearly showed only an upper band in the ELGA corresponding to hybridized probe. In contrast, when Camp. jejuni was spiked in a background of 4 x lo6 Gram-negative bacteria the amount of amplified RNA was not sufficient to bind all the probe but a positive signal was still clearly visible (Fig. 4c).

The correct identification by NASBAR of the 19 Camp. jejuni isolates from various sources shows the potential general applicability of this method. Further studies are necessary to evaluate whether this method can be used for the detection of Camp. jejuni, Camp. coli and Camp. lari in foods.

ACKNOWLEDGEMENTS

MU is indebted to the National Fund of Scientific Research (Belgium) for Scientific Research for a position as research assistant.

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NASBAR IDENTIFICATION OF CAMPYLOBACTER 701

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