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Kim, Kim, Choi, So, Shim, Kim, Park, Seong, and Chong: Cytolethal Distending Toxin Production, Genotypes and Atimicrobial Susceptibility of Campylobacter jejuni Isolates from Diarrhea Patients and Chickens
Original Article

J Bacteriol Virol 2008;38(4):207-219.

Published online: 18 January 2008

DOI: https://doi.org/10.4167/jbv.2008.38.4.207

Cytolethal Distending Toxin Production, Genotypes and Atimicrobial Susceptibility of Campylobacter jejuni Isolates from Diarrhea Patients and Chickens

Shin-Moo Kim1, ∗, Eun-Cheol Kim2, Mi-Rae Choi1, Hyung-Ah So3, Eun-Sook Shim1, Eun-Sook Kim4, 5, 6, Seong-Chan Park2, Chi-Nam Seong2, Yunsop Chong7

1Department of Clinical Laboratory Science, Wonkwang Health Science College, Iksan, Korea

2Department of Biology, Sunchon National University, Sunchon, Korea

3Department of Microbiology, Jenllabukdo Instituite of Health & Environmental Reach, Jeonju, Korea

4Vestibulocochlear Research Center, Wonkwang University School of Medicine, Iksan, Korea

5Department of Microbiology, Wonkwang University School of Medicine, Iksan, Korea

6Division of Biological Sciences, Graduate School, Chonbuk National University, Jeonju, Korea

7Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea

∗Corresponding author: Shin-Moo Kim. Department of Clinical Laboratory Science. Wonkwang Health Science College, Iksan 570-750, Korea. Phone: +82-63-840-1211, Fax: +82-63-840-1211 e-mail: smkim1211@hanmail.net

∗∗ This study was supported by research fund from Wonkwang Health Science College, 2008.

      Revised 7 November 200811 December 2008       Accepted 11 December 2008

Copyright © 2008 Korean Society for Legal Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Campylobacter jejuni isolates from diarrhea patients and chickens in 2008 in Iksan, Korea were tested for biochemical characteristics, and for possession of genes hipO, mutated gyrA, and cdtB. Among the chickens tested 52% carried C. jejuni. All 28 patient isolates and 48 chickens isolates had typical biochemical characteristics, except for nalidixic acid resistance. All isolates from patients and chickens were resistant to ciprofloxacin, and had mutated gyrA gene indicating good correlation of the two tests. Analysis of pulsed-field gel electrophoresis (PFGE) pattern of SmaI-restricted DNA of 53 isolates showed 14 clusters. Twenty-eight patient isolates and two chicken isolates (57%) showed an identical pattern (cluster 9). Chicken isolates C37 and C48 (cluster 2), C31 and C33 (cluster 3), C29, C34, C35, and C36 (cluster 4), and C43, C44 (cluster 6) had identical patterns. All patient isolates, compared to 87% and 80% of chicken isolates, were susceptible to amikacin and chloramphenicol, respectively. Antibiotics with the lowest MIC90 were imipenem, gentamicin, and erythromycin, whereas, those with the highest were ampicillin and tetracycline. In conclusion, C. jejuni carriage rate of chickens in Iksan, Korea, was high, all 28 isolates from patients and two from chickens were an identical clone, whereas isolates from patients and remaining chickens were different clones with only 62% similarity, all isolates had hipO and cdtB genes, and all isolates were resistant to ciprofloxacin.

Keywords: Campylobacter jejuni, Genotype, cdtB, Antimicrobial Susceptibility

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Figure 1.
Agarose gel electrophoresis of PCR products generated by primer pairs for hipO gene for identification of C. jejuni. (A) Lane 1, to 12, C. jejuni; M, size marker. (B) Lane 13 to 24, C. jejuni; M, size marker.
jbv-38-207f1.tif
Figure 2.
Agarose gel electrophoresis of PCR products generated by primer pairs for mutated gryA gene for detection of ciprofloxacin-resistant C. jejuni. Lane 1 to 12, C. jejuni; M, size marker.
jbv-38-207f2.tif
Figure 3.
Agarose gel electrophoresis of PCR products generated by primer pairs for cdtB gene for detection of cytolethal distending toxin. Lane 13 to 25, C. jejuni; M, size marker.
jbv-38-207f3.tif
Figure 4.
PFGE of SmaI-restricted DNA from C. jejuni isolates from diarrhea patients and chickens the dendrogram was based on the unweighted-pair group method using arithmetic averages from the Dice coefficient.
jbv-38-207f4.tif
Table 1.
Sequence of oligonucleotides used in this study
Target gene Direction Sequence (5′ to 3′) PCR amplicon size (bp)
hipO Forward 5′-ACTTCTTTATTGCTTGCTGC-3′ 323
  Reverse 5′-GCCACAACAAGTAAAGAAGC-3′  
gryA Forward 5′-TTT TTA GCA AAG ATT CTG AT-3′ 265
  Reverse 5′-CAA AGC ATC ATA AAC TGC AA-3′  
cdtB Forward 5′-ATCCGCAGCCACAGAAAGCAAATG-3′ 623
  Reverse 5′-GCGGTGGAGTATAGGTTTGTTGTC-3′  
Table 2.
Biochemical and genetic characteristics of C. jejuni isolates from patients and chickens
Source of isolation Hippurate hydrolysis Susceptibility Detection of indicated genes by PCR
Cephalothin Nalidixic acid hipO gryA cdtB
Patient (n=28)a            
H0706-01 + R R + + +
H0706-02 + R R + + +
H0706-03 + R R + + +
H0706-04 + R R + + +
H0706-05 + R R + + +
H0706-06 + R R + + +
H0706-07 + R R + + +
H0706-08 + R R + + +
H0706-09 + R R + + +
H0706-10 + R R + + +
H0706-11 + R R + + +
H0706-12 + R R + + +
H0706-13 + R R + + +
H0706-14 + R R + + +
H0706-15 + R R + + +
H0706-16 + R R + + +
H0706-17 + R R + + +
H0706-18 + R R + + +
H0706-19 + R R + + +
H0706-20 + R R + + +
H0706-21 + R R + + +
H0706-22 + R R + + +
H0706-23 + R R + + +
H0706-24 + R R + + +
H0706-25 + R R + + +
H0706-26 + R R + + +
H0706-27 + R R + + +
H0706-28 + R R + + +
Chicken (n=26)            
C0808-29 + R R + + +
C0808-30 + R R + + +
C0808-31 + R R + + +
C0808-32 + R R + + +
C0808-33 + R R + + +
C0808-34 + R R + + +
C0808-35 + R R + + +
C0808-36 + R R + + +
C0808-37 + R R + + +
C0808-38 + R R + + +
C0808-39 + R R + + +
C0808-40 + R R + + +
C0808-41 + R R + + +
C0808-42 + R R + + +
C0808-43 + R R + + +
C0808-44 + R R + + +
C0808-45 + R R + + +
C0808-46 + R R + + +
C0808-47 + R R + + +
C0808-48 + R R + + +
C0808-49 + R R + + +
C0808-50 + R R + + +
C0808-51 + R R + + +
C0808-52 + R R + + +
C0808-53 + R R + + +
C0808-54 + R R + + +

a The strains from patients were isolated in June, 2007 and those from chickens were in August 2008.

Table 3.
Disk diffusion antimicrobial susceptibility of 54 C. jejuni isolates from patients with diarrhea and chickens
Antimicrobial Patients (N=28)a Chickens (N=26)
agents Sb I R S I R
Amikacin 100 0 0 87 0 13
Ampicillin 0 0 100 31 0 69
Cefotaxime 100 0 0 100 0 0
Cephalothin 0 0 100 0 0 100
Chloramphenicol 100 0 0 80 4 16
Ciprofloxacin 0 0 100 0 0 100
Erythromycin 100 0 0 100 0 0
Gentamicin 100 0 0 100 0 0
Imipenem 100 0 0 100 0 0
Kanamycin 96 4 0 92 0 8
Nalidixic acid 0 0 100 0 0 100
Tetracycline 0 7 93 0 0 100
Co-trimoxazole 0 0 100 4 0 96

a No. of isolates tested.

b S, susceptible; I, intermediate; R, resistant.

Table 4.
Agar dilution antimicrobial susceptibility of C. jejuni from patients with diarrhea and chickens
Source of isolates (No. tested) Antimicrobial agent MIC (μg/mL) % resistant
Range 50% 90%
Patients (28) Ampicillin 128 ~ ≥128 ≥128 ≥128 100
  Cephalothin ≥128 ≥128 ≥128 100
  Ciprofloxacin 8~32 16 16 100
  Clindamycin 2 2 2 0
  Erythromycin <0.5~4 1 4 0
  Gentamicin <0.5~1 ≥0.5 2 0
  Imipenem 0.125 0.125 0.125 0
  Nalidixic acid 128 ~ ≥128 ≥128 ≥128 100
  Tetracycline 32~64 64 64 100
Chicken (26) Ampicillin 16 ~ ≥128 128 ≥128 96
  Cephalothin ≥128 ≥128 ≥128 100
  Ciprofloxacin 16 ~ ≥128 16 128 100
  Clindamycin 2~128 2 32 46
  Erythromycin <0.5~1 ≤0.5 1 0
  Gentamicin 1~2 2 2 0
  Imipenem 0.06~2 0.125 1 0
  Nalidixic acid ≥128 ≥128 ≥128 100
  Tetracycline 6 4 ~ ≥128 ≥128 ≥128 100
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