Detection of Campylobacter jejuni by Multiplex PCR and Patterns of Pulsed-Field Gel Electrophoresis
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1 Campylobacter jejuni Detection of Campylobacter jejuni by Multiplex PCR and Patterns of Pulsed-Field Gel Electrophoresis Jae-Kyoo Lee*, **, Kwang-Yup Kim**, Myoung-Sook Koo***, Dong-Eun Yong****, and Eui-Chong Kim*, **** Clinical Research Institute, Seoul National University Hospital, Seoul*; Department of Food Science and Technology, Graduate School of Chungbuk National University, Cheongju**; Department of Clinical Pathology, Kangnam General Hospital Public Corporation***; Department of Clinical Pathology, Yonsei University College of Medicine****; Department of Clinical Pathology, Seoul National University College of Medicine*****, Seoul Korea Background: Campylobacter is the most common bacterial cause of food-borne infection in developed countries, and handling or eating of contaminated poultry products was reported as the major cause of human campylobacteriosis in sporadic cases. This study was performed to investigate the prevalence of Campylobacter in patients with diarrhea and raw chickens of grocery, and identify the species by multiplex PCR and determine the genotypes of isolates by SmaI pulsedfield gel electrophoresis(pfge) profiles. Methods: Eight hundred and fifty six stool specimens obtained from 773 hospitalized patients with diarrhea and 16 raw chickens purchased from grocery were tested. Karmali s charcoal based selective medium and Campylobacter enrichment broth were used for isolation of Campylobacter from patients and chicken, respectively. And membrane filter method with sheep blood agar was also used in both two cases. Isolates were indentified with PCR, PCR-RFLP, and biochemical test. And genotypes were determined with SmaI PFGE profile analysis. Results: A total of 13 Campylobacter strains(1.7%) were isolated from 856 stool specimens of 773 patients with diarrhea, nine isolates were C. jejuni and four were C. coli. All of 16 raw chickens were contaminated with Campylobacter spp., and both of C. jejuni and C. coli were detected from eight chickens. From the SmaI-digested PFGE profile analysis of nine C. jejuni strains and four C. coli strains isolated from patients, eight types and four types of PFGE profile were obtained, respectively. And 15 types and seven types of PFGE profile were obtained from 23 of C. jejuni and 11 of C. coli which strains were isolated from chicken samples, respectively. The several isolates CM Tel : Fax : e Campylobacter FoodNet
2 showing the different PFGE patterns were detected in the same chicken. Three PFGE patterns of C. jejuni isolated from patients were observed in the chickens. One type of C. coli PFGE profiles of the patient s isolates were the same as that of chicken. Conclusions: The prevalence of Campylobacter infection is not high compared to the other countries, but most of raw chickens are contaminated with Campylobacter spp. Several genotypes of C. jejuni and C. coli are contaminated in the single chicken. The PFGE patterns of some human isolates are the same as those of chicken so that human infection may be originated from the chicken. But the reason of low infection rate in human in spite of the very high contamination rate of chicken should be clarified in the near future (Korean J Clin Microbiol 2002;5(1):35-41) Key words: Campylobacter, Diarrhea, Chicken, Multiplex PCR, PCR-RFLP, Pulsed-field Gel Electrophoresis Campylobacter 21.7(10 Salmonella 12.4, Shigella 8.5, E. coli O157:H7 2.8 [1]. Campylobacter, C. jejuni C. coli ,, [1-3]. 1-8, 3.,,,,,,. 3-5., Guillain Barre syndrome Reiter syndrome [3-5]. Campylobacter., 50%., 40 [3,6]. Campylobacter ribotyping pulsedfield gel electrophoresis(pfge), flagellin typing, subtyping PFGE. PFGE SmaI, KpnI SalI, SacII, BssHII [7-9]. Campylobacter spp multiplex PCR PCR-restriction fragment length polymorphism(rflp) PFGE , , Cary-Blair transport medium(bbl). Cary-Blair Transport medium : Cary-Blair Transport medium 12.6 g 121, 15 1% calcium chloride 9 ml 15 ml cap-tube 7 ml FBP solution : Ferrous sulfate 2.5 g, sodium bisulfite(sigma) 2.5 g sodium pyruvate(sigma) 2.5 g 100 ml 10 ml. Karmali s antibiotic supplements stock solution : Sodium pyruvate(sigma) 1000 mg, vancomycin(sigma) 200 mg, cefoperazone(sigma) 320 mg cycloheximide(sigma) 1000 mg 100ml 10 ml -70. CSM(Charcoal-based selective medium, Karmali s formula) : Columbia agar base(bbl) g activated charcoal powder(sigma) 2 g 485 ml, 0.5% hemin solution 3.2 ml 121, , FBP solution 5 ml Karmali s antibiotic supplements stock solution 10 ml Petri dish. BAP(Blood agar plate) : Korea Media LTD (Brain-Heart Infusion agar base, 5% defibrinated sheep blood).
3 Campylobacter CEB(Campylobacter enrichment broth with Karmali s antibiotic supplements) : Brucella broth base(bbl) 14 g 485 ml, 0.5% hemin solution 3.2 ml 121, , FBP solution 5 ml antibiotic supplements stock solution 10 ml 250 ml media-bottle 100 ml. (Membrane filter method) : 4 BAP 37. Membrane filter(millipore, 0.65 pore size, 47 mm diameter, mixed cellulose ester) BAP, 300 L membrane filter 37 1, filter candle jar. Campylobacter : Cary-Blair transport medium BHI broth 2 ml tube vortex 1 CSM, 300 L BAP. CSM BAP Candle-jar Campylobacter : 25 g 100 ml CEB, candle jar 37 shaking incubator 50 rpm ml 100 ml CEB 37, 50 rpm L 1,800 L 0.45% saline 10, 10 1,000 ( 10,000 ) BAP filter. 42 3, BAP,, 1. Multiplex PCR : C. jejuni C. coli multiplex PCR. primer Linton [10], C. jejuni hippuricase gene HIP400F (5 -GAA GAG GGT TTG GGT GGT G-3 ) HIP1134R (5 -AGC TAG CTT CGC ATA ATA ACT TG-3 ), C. coli aspartokinase gene downstream open reading frame CC18F (5 -GGT ATG ATT TCT ACA AAG CGA G-3 ) CC519R (5 -ATA AAA GAC TAT CGT CGC GTG-3 ), C. jejuni C. coli rrna gene CCCJ609F (5 -AAT CTA ATG GCT TAA CCA TTA-3 ) CCCJ1442R (5 -GTA ACT AGT TTA GTA TTC CGG-3 ) 3, 735 bp, 500 bp 854 bp. Gram Campylobacter BAP 100 L, CBT lysis buffer(15% Chelex-100, 1% Brij 58, 1% Tween 20) 15 13,000 rpm 3 DNA template. PCR Tris-Cl (ph8.3) 10 mm, KCl 50 mm, MgC l2 1.5 mm, dntp (Roche) 0.2 mm, Taq polymerase 2.5 U(Roche), primer 0.2 pmol 45 L, DNA template 5 L 50 L , 52 30, cycle, denaturation 94 3, extension % agarose (50 g EtBr/mL). PCR-RFLP : Multiplex-PCR C. jejuni C. coli PCR-RFLP. PCR primer Marshall [11] CAH16S 1a (5 -AAT ACT TGC AAG TCG AAC GA-3 ) CAH16S 1b (5 -TTA ACC CAA CAT CTC ACG AC-3 ) Campylobacter spp. 16S rrna gene target, 1004 bp. DNA template PCR multiplex-pcr, 94 30, 52 30, cycle, denaturation 94 3, extension % agarose QIAEX II Gel Extraction kit (QIAGEN). DdeI (New England Biolabs), BsrI (New England Biolabs). 3% agarose UV, Marshall. PFGE : PulseNet (CDC, USA) rapid PFGE protocol[12]., BAP 42, phosphate buffered saline (PBS, 0.01 M phosphate buffer, ph 7.4, 0.85% NaCl) McFarland No ml 200 L PBS proteinase K 50 L (10 mg/ml stock solution) 1.5 ml microcentrifuge tube TE buffer (10 mm Tris, 1 mm EDTA, ph 8.0) (200 L) 1% SKG agarose (SeaKem Gold agarose, FMC) plug mold. plug 5 ml cell lysis buffer (50 mm Tris, 50 mm EDTA, ph 8.0, 1% N-lauroylsarcosine, 0.1 mg proteinase K/mL) 50 ml tube lysis. Lysis plug 20 1, TE buffer 3. plug SmaI buffer 10 dialysis 40 U SmaI (Takara) SmaI buffer 100 L 25 3 digestion.
4 Table 1. Incidence of Campylobacter spp. from patients by sex of patients and species of isolates Species % Incidence (no. of isolates) (no. of isolates) Male Female Total (n=440) (n=333) (n=773) C. jejuni(9) 0.9 (4) 1.5 (5) 1.2 (9) C. coli(4) 0.7 (3) 0.3 (1) 0.5 (4) Total(13) 1.6 (7) 1.8 (6) 1.7 (13) (A) Fig. 2. Multiplex-PCR for identification of C. jejuni and C. coli. M : 100 bp ladder ; lanes 1,2, and 3 : C. jejuni ATCC 33291, H775BC, and C20-S, respectively ; lanes 4, 5, and 6 : C. coli ATCC 33559, H480C, and C20-M, respectively. Both C. jejuni and C. coli could be amplified with primers CCCJ609F and CCCJ1442R showing 854 bp PCR product. C. jejuni, showing 735 bp band amplified with hippuricase gene-specific primers HIP400F and HIP1134R, could be differentiated with C. coli showing 500 bp band amplified with primers CC18F and CC519R. (B). agarose gel scanning, Molecular Analyst 1.0(Bio-rad) program. Similarity band based similarity coefficient dice coefficient, clustering Ward algorithm. Fig. 1. Incidence of Campylobacter infection from patients with diarrhea. (A) By month ; (B) By age group. plug size marker (lambda ladder PFG marker low range PFG marker, New England Biolabs) 0.5X TBE 1% SKG agarose gel well, 1% low melting agarose (Sea Plaque GTG, FMC) agarose CHEF mapper TM (Bio-Rad), auto algorithm 14, initial switch time 6.75s, final switch time 38.35s, gradient 6V/cm, included angle 120, running time 18h. agarose EtBr (3 /ml) 15, Campylobacter spp., 1.7%. Multiplex PCR-RFLP hippurate 9 C. jejuni, 4 C. coli. Table 1, Fig Campylobacter. C. jejuni 6, C. coli 2, C. jejuni C. coli 8. Campylobacter C. jejuni C. coli multiplex PCR (Fig. 2)., 1 (H812C) 1 (C16-S) PCR band (854 bp), band (735 bp 500 bp), PCR-RFLP C. jejuni C. coli hippurate C. jejuni.
5 Campylobacter (A) (B) Fig. 3. SmaI-digested PFGE restriction profile of isolates. (A) C. jejuni ; (B) C. coli. Dendrograms were constructed with band based similarity coefficient (dice coefficient) analysis and Ward clustering. PFGE Fig. 3. C. jejuni 9 C. coli 4 8 4, C. jejuni 23 C. coli SmaI. C6-S C6- M, C8-S1 C8-S1, C12-S C12-L1, C12-L2, C13-L C13-M C. jejuni. C7-S C8-M, C17-M C18-S, C19-S, L C20-L. C6-M C13-M, C7-M C10, C6-L C19-L. H963B, H865C C14-M, H940B C20-S, H944B C13-M, H635C C6-L, C19-L C20-L.
6 C. jejuni 1.2% [13] 0.8%, [2] 0.5%, C. jejuni C. coli 69% 31% Campylobacter. 1.6%, 1.8% (Table 1) %. Campylobacter. Campylobacter., [14] 33.7%, [15] 55% C. jejuni. Campylobacter Salmonella,. Campylobacter (CEB) CSM (Karmali s charcoal-based selective medium)., 10. (CSM) Campylobacter ,000 10,000 Campylobacter, C. coli C. jejuni. SmaI PFGE profile C6-S C6-M, C8-S1 C8-S1, C12-S C12-L1, C12-L2, C13-L C13-M C. jejuni. C. jejuni. C7-S C8-M, C17-M C18-S, C19-S, L C20-L PFGE pattern.. PFGE. C6-M C13-M, C7-M C10, C6-L C19-L.,. H963B, H865C C14-M. H940B C20-S, H944B C13-M, H635C C6-L, C19-L, C20-L., Campylobacter. : Campylobacter,., multiplex PCR, PFGE. : Charcoal-based selective medium Campylobacter enrichment broth,, multiplex PCR PCR-RFLP,. SmaI PFGE. : Campylobacter 1.7%, C. jejuni 9, C. coli Campylobacter, 8 C. jejuni C. coli. SmaI C. jejuni 9 C. coli 4 8 4, C. jejuni 23 C. coli C. jejuni 3. C. coli. : Campylobacter
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