JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1999, p. 510–517 0095-1137/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 37, No. 3
Identification of the Enteropathogens Campylobacter jejuni and Campylobacter coli Based on the cadF Virulence Gene and Its Product MICHAEL E. KONKEL,* SEAN A. GRAY, BONG J. KIM, STEVEN G. GARVIS,
AND
JULIE YOON
Department of Microbiology, Washington State University, Pullman, Washington 99164-4233 Received 16 July 1998/Returned for modification 30 August 1998/Accepted 12 November 1998
Campylobacter jejuni and Campylobacter coli are common causes of gastroenteritis in humans. Infection with C. jejuni or C. coli is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for C. jejuni and C. coli isolates based on the cadF virulence gene and its product. The cadF gene from C. jejuni and C. coli encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates with primers designed to amplify an internal fragment of the cadF gene. PCR was then used to amplify Campylobacter DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-C. jejuni 37-kDa serum. In addition, methanol-fixed samples of whole-cell C. jejuni and C. coli were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the cadF gene and its product are conserved among C. jejuni and C. coli isolates and that a PCR assay based on the cadF gene may be useful for the detection of Campylobacter organisms in food products. lobacter adhesin to fibronectin, which aids in the binding of C. jejuni and C. coli to intestinal epithelial cells (12). CadF is an outer membrane protein with an apparent molecular mass of 37 kDa. Previous work indicated that a rabbit antiserum raised against the CadF protein reacted with a 37-kDa protein in all C. jejuni isolates (n 5 15) tested, as judged by immunoblot analysis. In addition, antibodies reactive against the CadF protein were present in convalescent serum from C. jejuni-infected individuals (n 5 5). Collectively, these data suggested that the CadF protein was conserved among C. jejuni isolates and that a variety of assays could be developed based on the detection of the cadF virulence gene and its product. The primary aims of this study were to determine whether the cadF gene and protein are conserved among a group of diverse C. jejuni and C. coli isolates and to develop assays for the specific detection of Campylobacter organisms. The diversity of the C. jejuni isolates used in this study was assessed at the phenotypic level by biotyping and serotyping and at the genotypic level by pulsed-field gel electrophoresis (PFGE).
Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are recognized as a major cause of gastrointestinal disease, with between 2 and 8 million cases of campylobacteriosis, resulting in an estimated 200 to 800 deaths, per year in the United States. Infection with C. jejuni or C. coli is characterized by the sudden onset of fever, abdominal cramps, and diarrhea with blood and leukocytes (3, 4). Despite the worldwide prevalence of Campylobacter infections, relatively few PCR assays have been described which are based on the amplification of target genes encoding putative virulence determinants (8, 9). There are many possible sources of infection with C. jejuni and C. coli, as they are part of the normal intestinal flora in a wide range of birds and mammals. Large-scale outbreaks of human campylobacteriosis are rare and are usually linked to the consumption of polluted water or raw milk. Sporadic cases of campylobacteriosis are more common and are associated with the consumption of undercooked chicken. In the United States, case-control studies have attributed 48 to 70% of the sporadic infections to the consumption of Campylobacter-contaminated chickens (6, 10). The percentage of Campylobactercontaminated chicken carcasses varies, often between 50 and 90%, depending on the time of year and the number of carcasses tested. One study found that as many as 98% of chicken carcasses may be contaminated with C. jejuni by the time of sale (23). This finding is not surprising given the potential for chickens to be heavily cross-contaminated during mechanized processing (1, 23). The identification of Campylobacter genes encoding potential virulence determinants may prove to be invaluable for the detection and identification of Campylobacter species in food products and to diagnose Campylobacter-infected individuals. We recently identified an adhesin termed CadF, for Campy-
MATERIALS AND METHODS Bacterial isolates and growth conditions. The bacterial isolates used in this study are listed in Table 1. C. jejuni, C. coli, Campylobacter hyointestinalis, and Helicobacter pylori isolates were cultured on Mueller-Hinton agar plates containing 5% citrated bovine blood (MH-blood) in a 11.5% CO2 incubator at 37°C. All isolates were passaged every 24 to 48 h. Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens, Shigella dysenteriae, S. flexneri, Streptococcus agalactiae, and Streptococcus pyogenes were cultured on Luria-Bertani agar plates (10 g of Bacto Tryptone, 5 g of yeast extract, 5 g of sodium chloride, and 15 g of Bacto Agar per liter) in a 37°C incubator. Biochemical tests. C. jejuni (hippurate positive) and C. coli (hippurate negative) were tested for hippurate hydrolysis by the rapid method of Hwang and Ederer (11). Streptococcus agalactiae and S. pyogenes were used as positive and negative controls, respectively. Isolates were tested for H2S production as described by Skirrow and Benjamin (22) with the modifications made by Lior (15). S. typhimurium and S. flexneri served as positive and negative controls, respectively. The isolates were also tested for DNase activity with DNase test agar medium (Difco Laboratories, Detroit, Mich.). S. marcescens and E. aerogenes were used as positive and negative controls, respectively. In addition to undergoing all three biochemical tests performed in our laboratory, most of the C.
* Corresponding author. Mailing address: Department of Microbiology, Washington State University, Pullman, WA 99164-4233. Phone: (509) 335-5039. Fax: (509) 335-1907. E-mail:
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TABLE 1. Detection of the cadF virulence gene and its producta Strain
PCR product
Detection by dot blot
Immunoblot detection
Source or reference
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
ATCC Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Arizona Washington Washington Washington Washington Washington Washington Washington Washington Washington Washington Minnesota Minnesota Minnesota Minnesota Minnesota Minnesota M. Blaser L. Tompkins L. Tompkins
1 2 1 1 1 1
1 2 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1 1
Arizona L. Tompkins L. Tompkins L. Tompkins L. Tompkins L. Tompkins
2 2 2 2 2 2
ND ND ND ND ND ND
2 2 2 2 2 2
2 2 2 2 2 2
ATCC ATCC ATCC ATCC H. Olander D. Francis
Serotype
Biotype
C. jejuni isolates 33560 F38011 M129 H70100 M95 M98 M125 M128 M369 M521 M48789 W52400 X34578 St. Joseph St. M 3143 St. M F1474 St. M T6644 St. M W726 UMC T1393 T8531 TGH3611 KLC 100 KLC 101 KLC 102 KLC 106 KLC 108 KLC 109 KLC 110 KLC 111 KLC 112 KLC 114 E95-412 (blood) E96-1009 (blood) E97-2653 (blood) E97-2796 E97-2805 E97-2845 78-27 81116 81176
99 90 36 32 7 UT UT 1 Rough, UT 7 5 77 UT Rough, UT UT 36 36 16 18 36 9 2 99 36 76 ND 36 7 4 36 6 2 18 36 UT 38 2 1 6 5
I I I II I II II I I I II II II I I I I I III II II II I II I ND II I II I II I I I II II I I III I
1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
C. coli isolates M275 T1138 T1631 2144 T2234 INN-18383
29 12 ND 97 105 105
I II ND I I I
F2B-R1B
Other isolates C. hyointestinalis 35217 H. pylori 43504 P. aeruginosa PA01 S. dysenteriae 13313 S. typhimurium 85-102840 E. coli H30 a
F2B-R1C
UT, untypeable; ND, not done; 1, positive; 2, negative.
jejuni and C. coli isolates were also tested for H2S production and DNase hydrolysis (16) at the National Laboratory for Enteric Pathogens in Canada. PFGE. PFGE was performed as outlined by Chang and Taylor (5) with minor modifications. C. jejuni cells were harvested from MH-blood agar plates in TE buffer (50 mM Tris, 2 mM EDTA [pH 8.0]), and cell densities were adjusted to an optical density at 600 nm (OD600) of 2.0. Four hundred microliters of each bacterial suspension was added to 700 ml of 1.3% low-melting-point agarose (Bio-Rad Laboratories, Hercules, Calif.) that had been boiled and cooled to
50°C. One hundred microliters of the mixture was pipetted into agarose gel molds presized to fit the wells made by the PFGE combs. The agarose blocks were removed from the molds and incubated in 1 ml of ESP buffer (500 mM EDTA, 1% N-lauroyl sarcosine, 0.1 mg of proteinase K ml21) at 50°C for 48 h. Following digestion, the agarose blocks were washed twice in TE plus 1 mM phenylmethylsulfonyl fluoride for 20 min each time at 37°C and then two more times in TE without phenylmethylsulfonyl fluoride for 20 min each time at 37°C. Each agarose block, containing 4 to 8 mg of DNA, was then preincubated in 1 ml
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of 13 restriction endonuclease buffer for 1 h at 37°C. Following preincubation, the buffer was removed and replaced with 150 ml of 13 restriction endonuclease buffer containing 2 ml of either KpnI or SalI restriction endonuclease. The reaction mixtures were incubated at 37°C for 12 h. Following incubation, the agarose plugs were loaded into a pulsed-field gel. The restricted DNAs were separated in 1% agarose (pulsed-field certified; Bio-Rad Laboratories) which had been prepared with 0.53 TBE (0.089 M Tris base, 0.089 M boric acid, 0.002 M EDTA [pH 8.0]), in 0.53 TBE running buffer. Typical run parameters consisted of a reorientation angle of 120 degrees with a constant voltage of 180 V and a constant temperature of 14°C. Actual run and pulse times varied depending on the restriction endonuclease used, the sizes of the restricted fragments, and the region of the gel determined to be of greatest interest. For the enzyme SalI, a run time of 23 h and a ramped pulse time of 15 to 105 s was used, whereas for the enzyme KpnI, a run time of 20 h and a ramped pulse time of 5 to 75 s was used. The gels were stained for 1 h in 3 mg of ethidium bromide ml21 in deionized water and destained for 1 h in water. PCR and analysis of amplified products. Bacteria were harvested from agar plates and suspended in 200 ml of water. The amplification reaction was performed in a volume of 100 ml containing 10 ml of the bacterial suspension, 10 ml of 103 PCR buffer minus Mg, 8 ml of a mixture of the four deoxyribonucleotides (final concentration, 2.0 mM [each] deoxynucleoside triphosphate), 3 ml of 50 mM stock of MgCl2, 5 ml of the forward and reverse primers (100 pmol each), 48.5 ml of water, and 0.5 ml (2.5 U) of Taq DNA polymerase (Gibco BRL). The forward (cadF-F2B) and reverse (cadF-R1B) primers were selected after sequencing the cadF genes from C. jejuni F38011 and M129 and one C. coli isolate, M275. The cadF-F2B primer (59-TTG AAG GTA ATT TAG ATA TG-39) corresponds to nucleotides 101 to 120, and the cadF-R1B primer (59-CTA ATA CCT AAA GTT GAA AC-39) corresponds to nucleotides 497 to 478, with a mismatch at nucleotide 489 of the cadF gene from C. coli M275. Samples were subjected to 30 cycles of PCR. Each cycle consisted of a denaturing step (1 min; 94°C), primer annealing (1 min; 45°C), and chain extension (3 min; 72°C) PCRamplified products (15 ml) were resolved in 1.5% agarose gels and visualized by staining with ethidium bromide. Amplification of Campylobacter DNA from store-bought chickens. Five hundred microliters of fluid, collected from each of the plastic bags in which the chickens were packaged, was mixed with an equal volume of phenol-chloroformisoamyl alcohol (25:24:1). Each suspension was vortexed and centrifuged for 5 min at 3,000 3 g. The aqueous phase was collected, mixed with an equal volume of chloroform-isoamyl alcohol (24:1), vortexed, and centrifuged for 5 min at 3,000 3 g. Two hundred microliters of the aqueous phase was collected, and nucleic acids were precipitated by standard protocols. The resultant pellet was suspended in 50 ml of water. Twenty microliters of each sample was then subjected to PCR analysis as described above. Southern and dot blot hybridization analyses. Southern hybridization analysis was performed with purified chromosomal DNA and agarose-resolved PCRamplified products as previously described (13, 18). The chromosomal DNAs were digested with the restriction endonucleases BglII and Sau3AI, separated in 0.8% agarose gels, and transferred to GeneScreen membranes (New England Nuclear, Boston, Mass.). The products amplified from the extracts of the chicken carcasses by PCR were also transferred to GeneScreen membranes after separation in 1.5% agarose gels. The blots were incubated with the cadF-F2B–cadFR1B PCR product from C. jejuni F38011, which had been nick translated with [a-32P]dCTP (NEN). For dot blot hybridization, bacteria were harvested from agar plates, pelleted by centrifugation at 6,000 3 g, and suspended in phosphate-buffered saline (PBS) at an OD540 of 0.18. One milliliter of each bacterial suspension was then centrifuged at 6,000 3 g for 10 min. The bacterial pellet was suspended in 200 ml of lysis buffer (0.4 M Tris HCl [pH 8], 100 mM EDTA, 1.0% sodium dodecyl sulfate [SDS], and 200 mg of proteinase K) and incubated for 2 h at 60°C. DNA was extracted with 200 ml of phenol-chloroform (1:1) saturated with 10 mM Tris HCl–1 mM EDTA–100 mM NaCl and then extracted with an equal volume of chloroform. The supernatant was retained, and 0.1 volume of 3 M NaOH was added to each sample. The samples were incubated for 1 h at 65°C, allowed to cool to 20°C, and neutralized to pH 7 by adding 1 volume of 2 M ammonium acetate. Sixty microliters of the solution was vacuum transferred to a GeneScreen membrane with a Minifold II slot blot vacuum apparatus (Schleicher & Schuell, Inc., Keene, N.H.). Following crosslinking of the DNA to the membrane, the membrane was incubated with the nick-translated cadF-F2B–cadF-R1B PCR probe under conditions outlined elsewhere (13, 18). SDS-PAGE and immunoblot analysis. Bacterial whole-cell extracts (an equivalent of 0.1 OD600 units) were solubilized in single-strength electrophoresis sample buffer and incubated at 95°C for 5 min. Proteins were separated in SDS–12.5% polyacrylamide gel electrophoresis (PAGE) minigels with the discontinuous buffer system described by Laemmli (14) and electrophoretically transferred to polyvinylidene fluoride membranes (Immobilon P; Millipore Corp., Bedford, Mass.). The membranes were washed three times in PBS and incubated for 18 h at 4°C with a 1:250 dilution of the rabbit anti-C. jejuni 37-kDa serum in PBS (pH 7.4)–0.01% Tween 20 (PBS-Tween) containing 20% fetal bovine serum. Bound antibodies were detected with peroxidase-conjugated goat anti-rabbit immunoglobulin G and 4-chloro-1-naphthol (Sigma) as the chromogenic substrate.
J. CLIN. MICROBIOL. Indirect immunofluorescence assays. C. jejuni cells were harvested from MHblood agar plates in PBS, pelleted by centrifugation, and suspended to approximately 108 bacteria ml21 in PBS. Also prepared was a suspension of bovine erythrocytes, which contained approximately 107 cells per ml in PBS. Equal volumes of the bacterial and erythrocyte suspensions were mixed, and 20 ml of the mixture was allowed to air dry on glass slides. The slides were immersed in methanol for 5 min, rinsed five times in PBS, and incubated in a humidified petri dish at 37°C for 1 h with a 1:50 dilution of the rabbit anti-C. jejuni 37-kDa serum in PBS-Tween. The slides were then immersed five times for 1 min each rinse in PBS and incubated in a humidified petri dish at 37°C for 1 h with a 1:100 dilution of an affinity-purified fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin G (H plus L chains) antibody in PBS. Following incubation, the slides were again immersed five times in PBS. A drop of PBS-glycerol (1:1) was then placed on the surface of each slide, and a coverslip was added. Samples were examined with a Nikon inverted microscope equipped with a krypton-argon laser (Bio-Rad). Images were captured with the Bio-Rad 1024 laser scanning confocal microscopy imaging system and processed with Photoshop 4.0 software (Adobe Systems, Inc., Mountain View, Calif.). Other analytical methods. Based on the sequence of the cadF gene from C. coli M275 (previously C. jejuni M275), the cadF genes from C. jejuni F38011 and M129 were PCR amplified with the cadF-F38 forward primer (59-ATG AAA AAG TTA TTA CTA TGT TTA GG-39) and the cadF-R20 reverse primer (59-AGG ATA AAT TTA GCA TCC-39). DNA sequencing was performed with a double-stranded DNA cycle-sequencing kit (Life Technologies Inc., Gaithersburg, Md.) according to the supplier’s instructions. Sequencing primers were synthesized by Ransom Hill Bioscience, Inc. (Ramona, Calif.) and Life Technologies Inc. Samples were heated to 95°C for 5 min prior to electrophoresis in 8% polyacrylamide–8M urea sequencing gels in TBE (0.089 M Tris base, 0.089 M boric acid, 0.002 M EDTA [pH 8.0]). After electrophoresis, the gels were transferred to 3MM paper (Whatman), dried, and analyzed by autoradiography. Nucleotide sequence accession numbers. The sequences of the cadF genes from C. jejuni F38011 and M129 have been deposited in the GenBank database and given accession no. AF104303 and AF104302, respectively.
RESULTS Characterization of Campylobacter isolates. To ensure that the bacteria used in this study represented a diverse group of C. jejuni and C. coli isolates, most were subjected to biotyping, serotyping, and PFGE (Table 1). The biotyping scheme used was devised by Lior (15) and allows for the differentiation of C. jejuni isolates into four biotypes and C. coli isolates into two biotypes based on their hydrogen sulfide production and their ability to hydrolyze DNA. Of the 39 C. jejuni isolates tested, 22 (56.4%) belonged to biotype I (H2S2 DNase2), 15 (38.5%) belonged to biotype II (H2S2 DNase1), and 2 (5.1%) belonged to biotype III (H2S1 DNase2). C. jejuni isolates belonging to biotype IV (H2S1 DNase1) were not identified. Of the five C. coli isolates biotyped, four were identified as biotype I (H2S2 DNase2) and one was identified as biotype II (H2S2 DNase1). The Campylobacter isolates were also subjected to serotyping by the slide agglutination test, which employs viable bacteria (17). This serotyping scheme is based on heat-labile antigens, with a total of 130 antisera currently available. Sixteen different serotypes were observed among the 39 C. jejuni isolates tested. The most commonly identified C. jejuni serotype was type 36, occurring six times. Seven isolates were found to be untypeable with the antisera currently available. PFGE was performed to characterize the genomic diversity in the C. jejuni isolates used in this study. Initially, C. jejuni isolates were analyzed by PFGE following digestion of chromosomal DNAs with the restriction enzyme SalI. A representative gel is presented in Fig. 1. PFGE of SalI-restricted C. jejuni chromosomal DNA commonly yielded four to six fragments ranging in size from 38.5 to 1,125 kb. Based on the relative PFGE patterns following SalI digestion, the isolates were subdivided into eight groups. Isolates within a group exhibited the same relative PFGE pattern, as determined by the size and number of DNA fragments obtained. The isolates yielding identical PFGE patterns or differing by the sizes of one or two bands upon SalI digestion were then analyzed by PFGE following digestion with KpnI. Consistent with previous
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FIG. 1. DNA polymorphism among C. jejuni isolates demonstrated by PFGE analyses of SalI-digested chromosomal DNAs. (A) The eight SalI patterns observed. (B) Pictorial of the eight representative SalI-PFGE profiles. Lanes: 1, F38011; 2, ATCC 33560; 3, W52400; 4, 81116; 5, X34578; 6, St. M M3143; 7, E97-2796; 8, KLC106. Size standards, in kilobases, are shown between the two panels.
studies (7), digestion of the C. jejuni DNA with the restriction enzyme KpnI commonly yielded fragments ranging from 48.5 to 365 kb. None of the C. jejuni isolates showing the same or similar patterns by SalI digestion yielded identical PFGE patterns upon KpnI digestion, suggesting that every isolate was unique. Using the eight isolates presented in Fig. 1, the size of the C. jejuni genome was estimated to range between 1.855 and 2.028 Mb by determining the size of the genomic DNA fragments after digestion with the SalI restriction enzyme. Selection of primers and PCR amplification. Using the cadF-F38 and cadF-R20 primers flanking the 59 and 39 ends of the cadF gene from C. coli M275, the cadF genes from C. jejuni isolates F38011 and M129 were amplified by PCR and sequenced (Fig. 2). Comparison of the nucleotide sequences of the cadF genes from these three Campylobacter isolates revealed that the C. coli cadF gene shows 87.4 and 84.7% identity with the cadF genes from C. jejuni F38011 and M129, respectively (Table 2). The cadF genes from C. jejuni F38011 and M129 exhibited 98.6% identity with one another. Several primers were selected, based on the alignment of the cadF genes from the one C. coli and two C. jejuni isolates, and used to attempt to amplify the cadF genes from all of the C. jejuni isolates. Preliminary analyses with a limited number of C. jejuni isolates revealed that only two primers, designated cadF-F2B and cadF-R1B, amplified the expected 400-bp product. Further analyses revealed that a 400-bp fragment of DNA could be PCR amplified with the cadF-F2B and cadF-R1B primers from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates (Table 1). In total, the cadF gene was amplified from 43 of the 46 (93.5%) Campylobacter isolates by PCR with the cadF-F2B and cadF-R1B primers. A band was not detected in C. hyointestinalis, H. pylori, P. aeruginosa, S. dysenteriae, S. typhimurium, and E. coli isolates subjected to PCR testing with the cadF-F2B and cadF-R1B primers. A PCR product was not observed from two C. jejuni isolates (M48789 and KLC100) and one C. coli isolate (T1138) with the cadF-F2B and cadF-R1B primers. To determine if these isolates contained a copy of the cadF gene, Southern hybridization analysis was performed with purified chromosomal DNAs
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from C. jejuni F38011, M48789, and KLC100 and C. coli M275 and T1138. The blots were probed with the cadF-F2B–cadFR1B PCR product from C. jejuni F38011. Hybridizing bands were observed with each of the isolates tested (not shown). All of the bacterial isolates listed in Table 1 were also subjected to dot blot analysis with the nick-translated cadF-F2B–cadF-R1B PCR product from C. jejuni F38011 as a probe. A band was obtained with each of the 40 C. jejuni and 6 C. coli isolates but not with any of the other bacterial isolates tested (Fig. 3A and Table 1). Further inspection of the sequences of the cadF genes from C. coli M275 and the two C. jejuni isolates F38011 and M129 revealed several sites containing stretches of bases which appeared to be unique in the C. coli cadF gene. Several primers were selected from these dissimilar sites and used in combination with the cadF-F2B primer to attempt to specifically amplify the cadF gene from only C. coli isolates. A portion of the cadF gene was successfully amplified by PCR from five of the six C. coli isolates, but none of the C. jejuni isolates, using the cadF-F2B and cadF-R1C primer pair (Fig. 2 and Table 1). While additional testing of C. coli isolates is required to validate the usefulness of these primers, these data suggest that a PCR assay specific for C. coli isolates may be developed based on the differences in the nucleotide sequences in the cadF genes from C. coli and C. jejuni isolates. In any case, all PCR assays described below were performed with the cadF-F2B and cadF-R1B primers, as one of the aims of this work was to develop a PCR assay to detect both C. jejuni and C. coli isolates in food products. Sensitivity of the PCR assay. The sensitivity of the PCR assay with the cadF-F2B and cadF-R1B primers was assessed by preparing 10-fold serial dilutions (106 to 100) of C. jejuni F38011 in Eagle’s minimal essential medium. The viable number of C. jejuni cells in each sample was quantitated by plating the bacterial suspensions on MH-blood agar plates. A 400-bp product was visualized from as few as 100 bacteria (not shown). Amplification of a portion of the cadF gene from chicken carcasses. To determine the efficacy of the PCR assay with the cadF-F2B and cadF-R1B primers in detecting Campylobacter organisms in food products, seven chicken carcasses were initially purchased from five local grocery stores. The fluid was collected from the plastic sack in which each chicken was wrapped and, following the extraction protocol outlined in Materials and Methods, subjected to PCR analyses. All seven carcasses, representing four different brand names, yielded a 400-bp product, as judged by PCR amplification with the cadFF2B and cadF-R1B primers coupled with agarose gel electrophoresis (not shown). The number of samples tested by PCR was expanded by purchasing a total of 20 more chicken carcasses from two different grocery stores. A 400-bp product was amplified with the cadF-F2B and cadF-R1B primers from 19 of the 20 chicken carcasses. The nick-translated cadF-F2B–cadF-R1B PCR product from C. jejuni F38011 hybridized to the 400-bp product amplified from each of the 19 chicken fluids, as judged by Southern hybridization analysis (Fig. 3B). Based on a single attempt in which an aliquot of the fluid (100 ml) collected from each plastic sack was spread onto a Campy-Cephex agar plate (24) immediately after the carcasses were purchased, viable Campylobacter organisms were isolated from 12 of the 20 (60%) carcasses tested. Immunodetection of CadF. One of the goals of this study was to determine whether the CadF protein could be detected in Campylobacter isolates with a rabbit anti-37-kDa serum. A representative gel and immunoblot of five Campylobacter and three non-Campylobacter isolates is shown in Fig. 4. A 37-kDa
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FIG. 2. Alignment of the complete cadF gene from C. coli with the PCR-amplified cadF genes from C. jejuni isolates M129 and F38011. The 59 and 39 ends of the cadF genes from C. jejuni F38011 and M129 were not determined. The boxes indicate identical nucleotides in the cadF genes of at least two of the three Campylobacter isolates. Based on the regions of identity found within the cadF genes from the three Campylobacter isolates, two primers were chosen for developing a PCR-based assay. Indicated are the cadF-F2B forward and cadF-R1B reverse primers corresponding to nucleotides 101 to 120 and 478 to 497 of the C. coli cadF gene. Also indicated are the cadF-R1C, cadF-F38, and cadF-R20 primers.
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TABLE 2. Comparison of cadF nucleotide and deduced amino acid sequences from C. coli M275, C. jejuni F38011, and C. jejuni M129 Nucleotide sequence data Organism
C. coli M275 C. jejuni F38011 C. jejuni M129 a
Predicted polypeptide data
No. of nucleotides
% G1C
% Identity
No. of amino acids
981 861a 861a
34.6 31.8 32.2
100 87.4 84.7
326 287a 287a
Molecular mass (kDa)
% Similarity (% identity)
36.7
100 (100) 89.2 (83.3) 88.9 (83.3)
Computer analysis performed with a partial nucleotide sequence.
band was detected in the whole-cell extracts of every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with the rabbit anti-37-kDa serum (Table 1). In contrast, a reactive band was not detected in the whole-cell extracts of C. hyointestinalis, H. pylori, P. aeruginosa, S. dysenteriae, S. typhimurium, or E. coli. These findings indicate that the CadF protein is conserved in size and antigenicity among C. jejuni and C. coli isolates. To determine whether Campylobacter organisms could be detected by an indirect-immunofluorescence microscopy assay, several C. jejuni isolates and two non-Campylobacter isolates were reacted with the rabbit anti-37-kDa serum followed by incubation with a fluorescein isothiocyanate-labeled goat antirabbit serum. The staining procedure demonstrated that the rabbit anti-37-kDa serum bound efficiently to C. jejuni but not to the bacteria used as controls (not shown).
FIG. 3. Detection of the cadF gene in C. jejuni and C. coli organisms and food products. (A) A variety of Campylobacter and non-Campylobacter isolates subjected to dot blot analysis with the nick-translated cadF-F2B–cadF-R1B PCR product from C. jejuni F38011. C. jejuni isolates are located in wells 1 through 40, C. coli isolates are in wells 41 through 46, and other gram-negative bacteria are in wells 47 through 52. Wells: 1, 33560; 2, F38011; 3, M129; M129; 4, H70100; 5, M95; 6, M98; 7, M125; 8, M128; 9, M369; 10, M521; 11, M48789; 12, W52400; 13, X34578; 14, St. Joseph; 15, St. M 3143; 16, St. M F1474; 17, St. M T6644; 18, St. M W726; 19, UMC T1393; 20, UMC T8531; 21, UMC TGH3611; 22, KLC100; 23, KLC101; 24, KLC102; 25, KLC106; 26, KLC108; 27, KLC109; 28, KLC110; 29, KLC111; 30, KLC112; 31, KLC114; 32, MINN E96-1009; 33, MINN E972653; 34, MINN E97-2796; 35, MINN E97-2805; 36, MINN E97-2845; 37, MINN E97-95412; 38, 78-27; 39, 81116; 40, 81176; 41, M275; 42, T1138; 43, T1631; 44, T2144; 45, T2232; 46, INN-18383; 47, C. hyointestinalis 35217; 48, H. pylori 43504; 49, P. aeruginosa PA01; 50, S. dysenteriae 13313; 51, S. typhimurium 85-102840; 52, E. coli H30. (B) Autoradiograph of the PCR-amplified products from 20 chicken carcasses. The DNA fragments were run in a 1.5% agarose gel, transferred to a GeneScreen membrane, and hybridized with the nick-translated cadF-F2B-cadF-R1B PCR product from C. jejuni F38011. Lanes: 1 to 20, chicken carcass extracts; 21, no-template control; 22, E. coli.
DISCUSSION The phenotypic and genotypic diversity of the collection of Campylobacter isolates used in this study was evaluated by biotyping, serotyping, and PFGE. Of the 39 C. jejuni isolates tested, 56.4% were found to be biotype I, 38.5% were biotype II, and 5.1% were biotype III. No biotype IV C. jejuni isolates were identified. These results are consistent with the biotyping results obtained by Lior (15), who found that a majority of C. jejuni clinical isolates belong to biotype I or II. Lior noted that only 4.0 and 2.7% of the 1,195 C. jejuni isolates from humans were found to be biotype III and biotype IV, respectively. Most of the C. jejuni and C. coli strains were also serotyped by using the slide agglutination assay, which is based on heat-labile antigens. Sixteen different serotypes were noted, with serotype 36 being the most common among the 39 C. jejuni isolates tested. Serotype 36 has previously been noted as a common serotype in the United States (19). Further analysis revealed that 8 of the 10 most common serotypes (serotypes 1, 2, 4, 6, 7, 9, 11, 16, 17, and 36) found in the United States were represented among the collection of C. jejuni isolates used in this study. The genotypic variation of the C. jejuni isolates was also examined by comparing the DNA banding pattern following digestion with SalI or KpnI and resolving the fragments by PFGE. The Campylobacter isolates used in this study were unique in that no two isolates exhibited identical PFGE patterns with both the SalI and KpnI restriction endonucleases. It is noteworthy that even though three serotype 36 C. jejuni strains were isolated from the same geographical region over a relatively short time, none of these three isolates appeared to be clonally or epidemiologically related to one another, as judged by their PFGE banding profiles. Despite advances in isolation methods, Campylobacter spp.
FIG. 4. Representative gel, stained with Coomassie brilliant blue R-250 (CBB-R250), and immunoblot showing the detection of the CadF protein in the whole-cell extracts of various C. jejuni and C. coli isolates. Bacterial whole-cell extracts (25 mg per lane) were separated in 12.5% SDS-PAGE gels, transferred to polyvinylidene fluoride membranes, and reacted with a 1:250 dilution of the rabbit anti-37-kDa serum. A 37-kDa band, corresponding to the CadF protein, was detected in every C. jejuni and C. coli isolate tested. Lanes: 1, C. jejuni M129 (biotype I); 2, C. jejuni H70100 (biotype II); 3, C. jejuni UMC T1393 (biotype III); 4, C. coli M275 (biotype I); 5, C. coli T1138 (biotype II); 6, S. dysenteriae 13313; 7, S. typhimurium 85-102840; 8, E. coli H30. The relative positions of the standards are given in kilodaltons on the left.
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remain labor intensive to culture and identify. Here we report the specific identification of a diverse group of C. jejuni and C. coli isolates based on PCR amplification of the cadF virulence gene. The cadF genes from C. jejuni and C. coli isolates encode a 37-kDa outer membrane protein that promotes the organism’s binding to fibronectin (12). In total, 93.5% of the C. jejuni and C. coli isolates tested with the cadF-F2B and cadF-R1B primers yielded the expected 400-bp PCR product. The specificity of the assay was determined to be 100%, as no PCR products were observed from whole-cell lysates of C. hyointestinalis or non-Campylobacter isolates. Using a series of 10-fold serial dilutions of viable C. jejuni organisms, the sensitivity of the PCR was determined to be 100 CFU. These findings suggested that this PCR assay might be useful for the detection of Campylobacter isolates in food products. Previous reports have indicated that ingestion of as few as 500 Campylobacter organisms may be sufficient to cause disease (2, 20). Other reports have also indicated that as many as 90% of the fresh, whole chickens purchased at local grocery stores are contaminated with Campylobacter organisms. This finding is not surprising given the potential for extensive crosscontamination during the slaughtering process (1, 23). Of the 20 carcasses tested, viable Campylobacter organisms were isolated from 60% (12 of 20). PCR detection of Campylobacter organisms proved to be much more sensitive than plating the fluids collected from the plastic sacks on Campy-Cephex agar plates, as 96.3% (26 of 27) of the store-bought chickens tested positive, as judged by the amplification of a 400-bp fragment with the cadF-F2B and cadF-R1B primers. These data indicate that a majority of chicken carcasses tested were contaminated with Campylobacter organisms and represented possible sources of food-borne contamination. The CadF protein was found to be conserved in size and antigenicity among all C. jejuni and C. coli isolates tested (n 5 46), as judged by immunoblot analysis with the rabbit anti-37kDa serum. No cross-reactivity was observed with the rabbit anti-37-kDa serum upon screening the whole-cell extracts of C. hyointestinalis or the non-Campylobacter strains. The rabbit anti-37-kDa serum also reacted with C. jejuni on glass slides in an indirect-immunofluorescence microscopy assay. Collectively, these data suggest that the CadF protein may be useful in developing several assays, such as a direct-fluorescence antibody test and an enzyme-linked immunosorbent assay with CadF-coated plates, to determine whether an individual has been infected with C. jejuni or C. coli. In this regard, we have previously found that antibodies present in convalescent antiserum from C. jejuni-infected individuals recognize the 37-kDa protein (12). An enzyme-linked immunosorbent assay might be helpful in identifying individuals diagnosed with GuillainBarre´ syndrome (21, 25, 26) who have previously been infected with C. jejuni. In summary, one of the aims of this work was to assess whether the cadF virulence gene was conserved among a diverse group of C. jejuni and C. coli isolates in order to develop a specific assay for the direct detection of Campylobacter organisms. The cadF gene was amplified from 93.5% of the C. jejuni and C. coli isolates subjected to PCR. In addition, a 37-kDa immunoreactive protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-37-kDa antiserum. The rabbit anti-37-kDa antiserum was also used to detect methanol-fixed C. jejuni and C. coli organisms by an indirect-immunofluorescence microscopy assay. A PCR and immunofluorescence assay, based on the detection of the cadF virulence gene and its product, may prove useful for the detection of pathogenic C. jejuni and C. coli in food products or in stool samples from infected indi-
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viduals. An advantage of the assays is that neither requires the bacteria to be cultured. The detection of Campylobacter DNA from 26 of the 27 carcasses tested suggests that a PCR assay based on the Campylobacter cadF virulence gene might be useful in monitoring the number of Campylobacter-contaminated carcasses and in helping to establish control methods. We believe the data presented here are promising and warrant further evaluation in field or clinical situations. ACKNOWLEDGMENTS We thank Wendy Johnson and David Woodward (National Laboratory for Enteric Pathogens, Laboratory Centre for Disease Control, Ottawa, Ontario, Canada) for serotyping and biotyping the C. jejuni and C. coli isolates used in this study. We also thank Johanna Skoropinski for assistance with PCR amplification, SDS-PAGE, and immunoblot analyses. Finally, we thank Chris Grant and Tom Schwan for critically reviewing the manuscript. This work was supported by grants from the National Institutes of Health (1R01 DK50567-01A1) and the USDA National Research Initiative Competitive Grants Program (USDA/NRICGP no. 9601496) awarded to M.E.K. J. Skoropinski was supported by the Howard Hughes Fellowship Program. REFERENCES 1. Berndtson, E., M. Tivemo, and A. Engvall. 1992. Distribution and numbers of Campylobacter in newly slaughtered broiler chickens and hens. Int. J. Food Microbiol. 15:45–50. 2. Black, R. E., M. M. Levine, M. L. Clements, T. P. Hughes, and M. J. Blaser. 1988. Experimental Campylobacter jejuni infection in humans. J. Infect. Dis. 157:472–479. 3. Blaser, M. J., I. D. Berkowitz, F. M. LaForce, J. Cravens, L. B. Reller, and W. L. Wang. 1979. Campylobacter enteritis: clinical and epidemiological features. Ann. Intern. Med. 91:179–185. 4. Blaser, M. J., J. G. Wells, R. A. Feldman, R. A. Pollard, and J. R. Allen. 1983. Campylobacter enteritis in the United States. A multicenter study. Ann. Intern. Med. 98:360–365. 5. Chang, N., and D. E. Taylor. 1990. Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580. J. Bacteriol. 172:5211–5217. 6. Deming, M. S., R. V. Tauxe, P. A. Blake, S. E. Dixon, B. S. Fowler, T. S. Jones, E. A. Lockamy, C. M. Patton, and R. O. Sikes. 1987. Campylobacter enteritis at a university: transmission from eating chicken and from cats. Am. J. Epidemiol. 126:526–534. 7. Gibson, J., E. Lorenz, and R. J. Owen. 1997. Lineages within Campylobacter jejuni defined by numerical analysis of pulsed-field gel electrophoretic DNA profiles. J. Med. Microbiol. 46:157–163. 8. Gonzalez, I., K. A. Grant, P. T. Richardson, S. F. Park, and M. D. Collins. 1997. Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant. J. Clin. Microbiol. 35:759–763. 9. Harmon, K. M., G. M. Ransom, and I. V. Wesley. 1997. Differentiation of Campylobacter jejuni and Campylobacter coli by polymerase chain reaction. Mol. Cell. Probes 11:195–200. 10. Harris, N. V., N. S. Weiss, and C. M. Nolan. 1986. The role of poultry and meats in the etiology of Campylobacter jejuni/coli enteritis. Am. J. Public Health 76:407–411. 11. Hwang, M.-N., and G. M. Ederer. 1975. Rapid hippurate hydrolysis method for presumptive identification of group B streptococci. J. Clin. Microbiol. 1:114–115. 12. Konkel, M. E., S. G. Garvis, S. L. Tipton, D. E. Anderson, Jr., and W. Cieplak, Jr. 1997. Identification and molecular cloning of a gene encoding a fibronectin-binding protein (CadF) from Campylobacter jejuni. Mol. Microbiol. 24:953–963. 13. Konkel, M. E., R. T. Marconi, D. J. Mead, and W. Cieplak, Jr. 1994. Cloning and expression of the hup gene encoding a histone-like protein of Campylobacter jejuni. Gene 146:83–86. 14. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. 15. Lior, H. 1984. New, extended biotyping scheme for Campylobacter jejuni, Campylobacter coli, and “Campylobacter laridis.” J. Clin. Microbiol. 20:636– 640. 16. Lior, H., and A. Patel. 1987. Improved toluidine blue-DNA agar for detection of DNA hydrolysis by campylobacters. J. Clin. Microbiol. 25:2030–2031. 17. Lior, H., D. L. Woodward, J. A. Edgar, L. J. Laroche, and P. Gill. 1982. Serotyping of Campylobacter jejuni by slide agglutination based on heatlabile antigenic factors. J. Clin. Microbiol. 15:761–768. 18. Marconi, R. T., D. S. Samuels, and C. F. Garon. 1993. Transcriptional
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