Tipificación molecular de aislados de vibrio cholerae O1 causantes de dos brotes de cólera en Venezuela / Molecular typing of vibrio cholerae O1 isolates causing two outbreaks of cholera in Venezuela

En Venezuela, en junio de 1996, se reportó que los casos de cólera eran causados por V. cholerae O1 serotipo Ogawa. A finales de 1998 se detectó un segundo brote de cólera causado por V. cholerae O1 serotipo Inaba resistente a la ampicilina y el trimetoprim-sulfametoxazol. Para estudiar las relaciones entre las cepas se examinaron veinticinco aislados de Vibrio cholerae O1 obtenidos desde 1996 a 2000 en Venezuela, para determinar la presencia de genes de virulencia y perfiles genómicos. Mediante la reacción en cadena de la polimerasa se determinó la presencia de genes de virulencia. Para determinar el perfil genómico de los aislamientos se utilizó ribotipificación y electroforesis en gel de campo pulsado (PFGE). Todos los aislados resultaron positivos para los genes ctxA, ctxB, zot y ace. El análisis RFLP de los genes RNAr mostró un único patrón de ribotipo V. El análisis de PFGE mostró una similitud de 91,5% independientemente del año o lugar de aislamiento, lo que indica la relación genómica entre los aislados. En conjunto, los datos sugieren que la cepa de V. cholerae O1 resistente a los antibióticos que apareció en 1998 surgió de la cepa epidémica anterior o de otro estrechamente relacionado con el clon anterior, con cambio de serotipo y ganancia de determinantes de resistencia a antibióticos. Es muy importante monitorear continuamente la aparición de la variantes porque mejorará la comprensión de la evolución de nuevos clones de V. cholerae

In Venezuela, cholera reported in June 1996 was caused by V. cholerae O1 serotype Ogawa. Second outbreak of cholera caused by V. cholerae O1 serotype Inaba, resistant to ampicillin and trimethoprim- Sulfamethoxazole, was notify at the end of 1998. Twenty-five isolates of Vibrio cholerae O1 obtained from 1996 to 2000 in Venezuela were examined to study the relationships between strains, presence of virulence genes and genomic profiles. Presence of virulence genes was detected by Polymerase Chain Reaction. Ribotyping and pulsed-field gel electrophoresis (PFGE) were used to determine the genomic profile of isolates. All isolates shown PCR product for ctxA, ctxB, zot and ace genes. RFLP analysis of rRNA gene showed one unique pattern from ribotype V. PFGE analysis revealed a similarity of 91.5%, regardless year or place of isolation, suggesting genomic relatedness among them. Overall, these data suggest that antibiotic resistant V. cholerae O1 El Tor strain that appeared in 1998 emerged from the previous epidemic strain or from another closely related to the previous clone. It is important the continuous monitor the emergence of variants because it will improve our understanding of the evolution of new clones V. cholerae

Genomic profile of antibiotic resistant, classical ctxB positive Vibrio cholerae O1 biotype El Tor isolated in 2003 and 2005 from Puri, India: A retrospective study.

Objectives: To examine eight strains of Vibrio cholerae O1 isolated in 2003 and 2005 from Puri, India, for antibiotic susceptibility, presence of virulence and regulatory genes, cholera toxin (CT) production, CTX arrangement and genomic profiles. Materials and Methods: Bacterial strains were tested for antibiotic susceptibility using disc diffusion assay. Polymerase chain reaction determined the presence of antibiotic resistance, virulence and regulatory genes. To determine the type of cholera toxin subunit B (ctxB), nucleotide sequencing was performed. Southern hybridisation determined the number and arrangement of CTXΦ. Ribotyping and pulsed‑field gel electrophoresis (PFGE) were used to determine the genomic profile of isolates. Results: All the eight strains, except one strain, showed resistant to nalidixic acid, sulphamethoxazole, streptomycin and trimethoprim and possessed the sullI, strB, dfrA1 and intSXT genes. All the strains carried the toxin‑co‑regulated pilus pathogenicity island, the CTX genetic element, the repeat in toxin and produced CT. Restriction fragment length polymorphism (RFLP) analysis showed that V. cholerae O1 possess a single copy of the CTX element flanked by tandemly arranged RS element. Nucleotide sequencing of the ctxB gene showed the presence of classical ctxB. RFLP analysis of conserved rRNA gene showed two ribotype patterns. PFGE analysis also showed at least three PFGE patterns, irrespective of year of isolations, indicating the genomic relatedness among them. Conclusion: Overall, these data suggest that classical ctxB‑positive V. cholerae O1 El Tor strains that appeared in 2003 continue to cause infection in 2005 in Puri, India, and belong to identical ribotype(s) and/or pulsotype(s). There is need to continuous monitor the emergence of variant of El Tor because it will improve our understanding of the evolution of new clones of variant of V. cholerae.

PCR-ribotyping for genotyping Clostridium dif ficile clinical isolates / 国际检验医学杂志

Objective To investigate the genotype and production of toxin A and B of C .difficile clinical isolates collected from Sydney ,Australia .Methods Sixty‐eight C .difficile clinical isolates were collected from Westmead Hospital ,the University of Sydney ,which were genotyped by using PCR‐ribotyping ,and toxin A ,B coding gene tcdA ,tcdB were detected by using PCR meth‐od .Results Thirty‐one PCR‐ribotypes (RTs) were confirmed in the 68 C .difficile clinical isolates ,RT014 (19 .1% ) and RT002 (11 .8% ) were the common genotypes .Sixty‐four of 68 (94 .1% ) isolates contained tcdA and tcdB for toxin A and B .Conclusion The common prevalent PCR‐ribotypes of C .difficile were RT014 and RT002 in Sydney ,most of the C .difficile clinical isolates contained toxin A and B .

Clinical and Microbiologic Characteristics of Clostridium difficile Infection Caused by Binary Toxin Producing Strain in Korea

BACKGROUND: Binary toxin-producing Clostridium difficile infections (CDI) are known to be more severe and to cause higher case fatality rates than those by binary toxin-negative isolates. There has been few data of binary toxin-producing CDI in Korea. Objective of the study is to characterize clinical and microbiological trait of CDI cause by binary-toxin producing isolates in Korea. MATERIALS AND METHODS: From September 2008 through January 2010, clinical characteristics, medication history and treatment outcome of all the CDI patients were collected prospectively. Toxin characterization, PCR ribotyping and antibiotic susceptibility were performed with the stool isolates of C. difficile. RESULTS: During the period, CDI caused by 11binary toxin-producing isolates and 105 toxin A & toxin B-positive binary toxin-negative isolates were identified. Comparing the disease severity and clinical findings between two groups, leukocytosis and mucoid stool were more frequently observed in patients with binary toxin-positive isolates (OR: 5.2, 95% CI: 1.1 to 25.4, P = 0.043; OR: 7.6, 95% CI: 1.6 to 35.6, P = 0.010, respectively), but clinical outcome of 2 groups did not show any difference. For the risk factors for acquisition of binary toxin-positive isolates, previous use of glycopeptides was the significant risk factor (OR: 6.2, 95% CI: 1.4 to 28.6, P = 0.019), but use of probiotics worked as an inhibitory factor (OR: 0.1, 95% CI: 0.0 to 0.8; P = 0.026). PCR ribotypes of binary toxinproducing C. difficile showed variable patterns: ribotype 130, 4 isolates; 027, 3 isolates; 267 and 122, 1 each isolate and unidentified C1, 2 isolates. All 11 binary toxin-positive isolates were highly susceptible to clindamycin, moxifloxacin, metronidazole, vancomycin and piperacillin-tazobactam, however, 1 of 11 of the isolates was resistant to rifaximin. CONCLUSIONS: Binary toxin-producing C. difficile infection was not common in Korea and those isolates showed diverse PCR ribotypes with high susceptibility to antimicrobial agents. Glycopeptide use was a risk factor for CDI by those isolates.

Emergence of Clostridium difficile Ribotype 027 in Korea / 대한진단검사의학회지

BACKGROUND: Clostridium difficile infection (CDI) has markedly risen and is associated with hypervirulent ribotype 027 outbreaks in North America and Europe since 2003. The aims of this study were to determine the prevalence of ribotype 027 among C. difficile isolates in Korea, to characterize the ribotype 027 isolates, and to determine the clinical severity of CDI in patients infected with these isolates. METHODS: A total of 1,251 isolates of C. difficile recovered from stool specimens of suspected CDI patients at two tertiary-care hospitals and one commercial laboratory between 2002 and 2009. Genes for toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA and cdtB) were detected by PCR. Mutation in the tcdC gene was detected by sequencing after PCR amplification. For molecular genotyping, we performed PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and multilocus variable-number tandem-repeat analysis (MLVA). Minimum inhibitory concentrations of moxifloxacin were determined using Etest strips (AB bioMerieux, Sweden). RESULTS: We identified 7 isolates as ribotype 027. These isolates had the same tcdC mutation as the epidemic strain, and 6 of them were resistant to moxifloxacin. The isolates were categorized into 3 different PFGE types and 7 different MLVA types. All the 7 cases had occurred sporadically. CONCLUSIONS: C. difficile ribotype 027 is uncommon, but it has emerged in Korea. The spread of this ribotype should be closely monitored in order to avoid an outbreak of CDI in Korea.

The First Case of Antibiotic-associated Colitis by Clostridium difficile PCR Ribotype 027 in Korea

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.