Analysis of biofilm production and expression of adhesion structures of circulating Clostridioides difficile strains from Mexico.

INTRODUCTION: Clostridioides difficile biofilms are believed to protect the pathogen from antibiotics, in addition to potentially contributing to recurrent infections. METHODOLOGY: Biofilm production of 102 C. difficile isolates was determined using the crystal violet staining technique, and detachment assays were performed. The expression levels of cwp84 and slpA genes were evaluated by real-time PCR on selected isolates. RESULTS: More than 70% of isolates (75/102) were strong biofilm producers, and the highest detachment of biofilm was achieved with the proteinase K treatment (>90%). The overall mean expression of cwp84 was higher in RT027 than in RT001 (p=0.003); among strong biofilm-producing strains, the slpA expression was lower in RT027 than in RT001 (p<0.000). CONCLUSIONS: Proteins seem to have an important role in the biofilm's initial adherence and maturation. slpA and cwp84 are differentially expressed by C. difficile ribotype and biofilm production level.

Analysis of biofilm production and expression of adhesion structures of circulating Clostridioides difficile strains from Mexico.

INTRODUCTION: Clostridioides difficile biofilms are believed to protect the pathogen from antibiotics, in addition to potentially contributing to recurrent infections. METHODOLOGY: Biofilm production of 102 C. difficile isolates was determined using the crystal violet staining technique, and detachment assays were performed. The expression levels of cwp84 and slpA genes were evaluated by real-time PCR on selected isolates. RESULTS: More than 70% of isolates (75/102) were strong biofilm producers, and the highest detachment of biofilm was achieved with the proteinase K treatment (>90%). The overall mean expression of cwp84 was higher in RT027 than in RT001 (p=0.003); among strong biofilm-producing strains, the slpA expression was lower in RT027 than in RT001 (p<0.000). CONCLUSIONS: Proteins seem to have an important role in the biofilm's initial adherence and maturation. slpA and cwp84 are differentially expressed by C. difficile ribotype and biofilm production level.

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

First case of autochthonous Clostridium difficile PCR ribotype 027 detected in Spain.

INTRODUCTION: Clostridium difficile ribotype 027 (Cd027) has caused outbreaks in the United States, Canada, and Europe since 2001. In Spain, the importance of Cd027 is still unknown. In 2007, we began active surveillance of Cd027 to determine its incidence in our hospital. METHODS: From January 2007 to April 2012, isolates of C. difficile by multiplex PCR were studied to detect toxin genes. Binary toxin-positive isolates were characterized using PCR-ribotyping. Cd027 were further characterized by toxino-typing, sequencing of tcdC gene, and MLVA (multilocus-variable-number-tandem-repeat-analysis). RESULTS: Only 8 strains were Cd027 from 3666 isolates of C. difficile analyzed during the study period. These strains were isolated from 4 patients: a Spanish patient previously hospitalized in the UK, a pregnant laboratory technician, a British tourist, and a Spanish patient without epidemiological antecedents for acquiring Cd027. MLVA typing of Cd027 isolates revealed 4 different patterns. The first patient had 2 episodes of diarrhea caused by different Cd027. The strains from the first episode of patient 1 and the strain from patient 2 were grouped in the same clonal cluster (these cases were previously published as laboratory transmission), while strains from patients 3 and 4 were genetically unrelated to each other, and to the strains from patients 1 and 2. CONCLUSION: We report the first finding of an autochthonous case of non-severe Cd027 infection. Our results indicate that Cd027 diarrhea is uncommon in our area, and it appears mainly as imported cases. MLVA typing enables us to distinguish different genotypes among our Cd027 isolates.