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The first case of antibiotic-associated colitis by Clostridium difficile PCR ribotype 027 in Korea.

Tae CH, Jung SA, Song HJ, Kim SE, Choi HJ, Lee M, Hwang Y, Kim H, Lee K - J. Korean Med. Sci. (2009)

Bottom Line: Recently, a highly virulent strain of C. difficile polymerase chain reaction ribotype 027 was found in North America, Europe, and Japan.After discharge, she was maintained on probiotics and rifaximin for 3 weeks.She had no relapse for 6 months.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul, Korea.

ABSTRACT
Clostridium difficile (C. difficile) is a common causative agent of pseudomembranous colitis (PMC). C. difficile-associated diarrhea (CDAD) ranges from mild diarrhea to life threatening PMC. Recently, a highly virulent strain of C. difficile polymerase chain reaction ribotype 027 was found in North America, Europe, and Japan. A 52-yr-old woman with anti-tuberculosis medication and neurogenic bladder due to traffic accident experienced five episodes of C. difficile PMC after taking antibiotics for pneumonia along with septic shock and acute renal failure. She was readmitted to the intensive care unit and treated with oral vancomycin with refractory of oral metronidazole, inotropics and probiotics for over 60 days. C. difficile isolated both at the first and the last admission was identified as C. difficile ribotype 027 by ribotyping, toxinotyping, and tcdC gene sequencing, which turned out the same pathogen as the epidemic hypervirulent B1/NAP1 strain. This is the first case of C. difficile PCR ribotype 027 in Korea. After discharge, she was maintained on probiotics and rifaximin for 3 weeks. She had no relapse for 6 months.

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PCR products and restriction patterns of C. difficile ribotype 027 isolates from the patient. (A) Identical PCR ribotype profiles obtained from both the first and the last isolates. Lanes: M, 100 bp DNA ladder; 1, the last isolate; 2, the first isolate. (B) Types of restriction patterns by B1 and A3 PCR showing toxinotype III. Lanes: M, DNA size marker; 1, 4, Acc I restriction patterns of B1 fragment; 2, 5, HincII restriction patterns of B1 fragment; 3, 6, EcoRI restriction patterns of A3 fragment.
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Figure 4: PCR products and restriction patterns of C. difficile ribotype 027 isolates from the patient. (A) Identical PCR ribotype profiles obtained from both the first and the last isolates. Lanes: M, 100 bp DNA ladder; 1, the last isolate; 2, the first isolate. (B) Types of restriction patterns by B1 and A3 PCR showing toxinotype III. Lanes: M, DNA size marker; 1, 4, Acc I restriction patterns of B1 fragment; 2, 5, HincII restriction patterns of B1 fragment; 3, 6, EcoRI restriction patterns of A3 fragment.

Mentions: PCR ribotyping was performed using the method described by Stubbs et al. (7). Briefly, DNA was extracted by boiling bacteria suspension with 5% Chelex-100 (Sigma Co., St Louis, MO, USA). The supernatant (10 µL) of the DNA extract was added to a 40 µL PCR mixture containing 1.5 U of Taq polymerase (AmpliTaq Gold, Applied Biosystems, Foster City, CA, USA), 0.4 mM dNTP, 4.0 mM MgCl2, 10 mM Tris-HCl pH 8.30, 50 mM KCl (1X PCR Buffer II, Applied Biosystems) and 20 ρmol of each primer (5'-CTGGGGTGAAGTCGTAACAAGG-3'and 5'-GCGCCCTTTGTAGCTTGACC-3'). Reaction mixtures were subjected to 40 cycles of denaturation at 95℃ for 1 min, annealing at 55℃ for 1 min, and extension at 72℃ for 2 min and a last extension at 72℃ for 7min. A molecular size standard (100 bp; Invitrogen, Carlsbad, CA, U.S.A.) was run with an electrophoresis of amplification products, and analyzed with Molecular Analyst software (Bio-Rad Laboratory, Hercules, CA, U.S.A.). PCR ribotyping revealed that both the first and the last isolates were the same type as C. difficile ribotype 027 (Fig. 4A). Toxinotyping of C. difficile isolates was performed by the method of Rupnik et al. (8). Restriction patterns of this patient's isolates by B1 and A3 PCRs revealed toxinotype III (Fig. 4B). Sequencing of the tcdC gene of both the first and the last isolates using the primer pairs (5'-TCTCTACAGCTATCCCTGGT-3'and 5'-AAAAATGAGGGTAACGAATTT-3') (9) showed both 18-bp deletions and a single nucleotide deletion at position 117 (Fig. 5), identical to the tcdC-sc1 genotyping classified by Curry et al. (10).


The first case of antibiotic-associated colitis by Clostridium difficile PCR ribotype 027 in Korea.

Tae CH, Jung SA, Song HJ, Kim SE, Choi HJ, Lee M, Hwang Y, Kim H, Lee K - J. Korean Med. Sci. (2009)

PCR products and restriction patterns of C. difficile ribotype 027 isolates from the patient. (A) Identical PCR ribotype profiles obtained from both the first and the last isolates. Lanes: M, 100 bp DNA ladder; 1, the last isolate; 2, the first isolate. (B) Types of restriction patterns by B1 and A3 PCR showing toxinotype III. Lanes: M, DNA size marker; 1, 4, Acc I restriction patterns of B1 fragment; 2, 5, HincII restriction patterns of B1 fragment; 3, 6, EcoRI restriction patterns of A3 fragment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698204&req=5

Figure 4: PCR products and restriction patterns of C. difficile ribotype 027 isolates from the patient. (A) Identical PCR ribotype profiles obtained from both the first and the last isolates. Lanes: M, 100 bp DNA ladder; 1, the last isolate; 2, the first isolate. (B) Types of restriction patterns by B1 and A3 PCR showing toxinotype III. Lanes: M, DNA size marker; 1, 4, Acc I restriction patterns of B1 fragment; 2, 5, HincII restriction patterns of B1 fragment; 3, 6, EcoRI restriction patterns of A3 fragment.
Mentions: PCR ribotyping was performed using the method described by Stubbs et al. (7). Briefly, DNA was extracted by boiling bacteria suspension with 5% Chelex-100 (Sigma Co., St Louis, MO, USA). The supernatant (10 µL) of the DNA extract was added to a 40 µL PCR mixture containing 1.5 U of Taq polymerase (AmpliTaq Gold, Applied Biosystems, Foster City, CA, USA), 0.4 mM dNTP, 4.0 mM MgCl2, 10 mM Tris-HCl pH 8.30, 50 mM KCl (1X PCR Buffer II, Applied Biosystems) and 20 ρmol of each primer (5'-CTGGGGTGAAGTCGTAACAAGG-3'and 5'-GCGCCCTTTGTAGCTTGACC-3'). Reaction mixtures were subjected to 40 cycles of denaturation at 95℃ for 1 min, annealing at 55℃ for 1 min, and extension at 72℃ for 2 min and a last extension at 72℃ for 7min. A molecular size standard (100 bp; Invitrogen, Carlsbad, CA, U.S.A.) was run with an electrophoresis of amplification products, and analyzed with Molecular Analyst software (Bio-Rad Laboratory, Hercules, CA, U.S.A.). PCR ribotyping revealed that both the first and the last isolates were the same type as C. difficile ribotype 027 (Fig. 4A). Toxinotyping of C. difficile isolates was performed by the method of Rupnik et al. (8). Restriction patterns of this patient's isolates by B1 and A3 PCRs revealed toxinotype III (Fig. 4B). Sequencing of the tcdC gene of both the first and the last isolates using the primer pairs (5'-TCTCTACAGCTATCCCTGGT-3'and 5'-AAAAATGAGGGTAACGAATTT-3') (9) showed both 18-bp deletions and a single nucleotide deletion at position 117 (Fig. 5), identical to the tcdC-sc1 genotyping classified by Curry et al. (10).

Bottom Line: Recently, a highly virulent strain of C. difficile polymerase chain reaction ribotype 027 was found in North America, Europe, and Japan.After discharge, she was maintained on probiotics and rifaximin for 3 weeks.She had no relapse for 6 months.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul, Korea.

ABSTRACT
Clostridium difficile (C. difficile) is a common causative agent of pseudomembranous colitis (PMC). C. difficile-associated diarrhea (CDAD) ranges from mild diarrhea to life threatening PMC. Recently, a highly virulent strain of C. difficile polymerase chain reaction ribotype 027 was found in North America, Europe, and Japan. A 52-yr-old woman with anti-tuberculosis medication and neurogenic bladder due to traffic accident experienced five episodes of C. difficile PMC after taking antibiotics for pneumonia along with septic shock and acute renal failure. She was readmitted to the intensive care unit and treated with oral vancomycin with refractory of oral metronidazole, inotropics and probiotics for over 60 days. C. difficile isolated both at the first and the last admission was identified as C. difficile ribotype 027 by ribotyping, toxinotyping, and tcdC gene sequencing, which turned out the same pathogen as the epidemic hypervirulent B1/NAP1 strain. This is the first case of C. difficile PCR ribotype 027 in Korea. After discharge, she was maintained on probiotics and rifaximin for 3 weeks. She had no relapse for 6 months.

Show MeSH
Related in: MedlinePlus