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.

Resistance patterns, ESBL genes, and genetic relatedness of Escherichia coli from dogs and owners

Abstract Antimicrobial resistance in Escherichia coli isolated from pet dogs can be considered a potential threat of infection for the human population. Our objective was to characterize the resistance pattern, extended spectrum beta-lactamase production and genetic relatedness of multiresistant E. coli strains isolated from dogs (n = 134), their owners (n = 134), and humans who claim to have no contact with dogs (n = 44, control), searching for sharing of strains. The strains were assessed for their genetic relatedness by phylogenetic grouping and pulsed-field gel electrophoresis. Multiresistant E. coli strains were isolated from 42 (31.3%) fecal samples from pairs of dogs and owners, totaling 84 isolates, and from 19 (43.1%) control group subjects. The strains showed high levels of resistance to ampicillin, streptomycin, tetracycline, trimethoprim and sulfamethoxazole regardless of host species or group of origin. The blaTEM, blaCTX-M, and blaSHV genes were detected in similar proportions in all groups. All isolates positive for bla genes were ESBL producers. The phylogenetic group A was the most prevalent, irrespective of the host species. None of the strains belonging to the B2 group contained bla genes. Similar resistance patterns were found for strains from dogs, owners and controls; furthermore, identical PFGE profiles were detected in four (9.5%) isolate pairs from dogs and owners, denoting the sharing of strains. Pet dogs were shown to be a potential household source of multiresistant E. coli strains.

Genetic relatedness of Candida albicans bloodstream infection clinical isolates in Malaysia

Aims: The aim of this study was to investigate the genetic relatedness of the most prevalent Candida bloodstream infection (BSI) species in in a Malaysian population via Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) fingerprinting. Methodology and results: The genomic DNA of 43 Candida BSI blood culture samples obtained from Universiti Malaya Medical Centre (UMMC) was isolated, after which species identification was carried out using PCR with ITS-1 and ITS-4 pan-fungal primers in conjunction with CHROMagar™ Candida. The predominant Candida species in the BSI samples is Candida albicans (14 out of 43 isolates). RAPD-PCR on these 14 C. albicans clinical isolates was performed using PST as the arbitrary primer. Data analysis using MEGA found an overall non-relatedness of these 14 clinical isolates [average similarity coefficient (SAB) value 0.733±0.172]. Following in-depth analysis, five of the 14 isolates were observed to be identical (SAB values of 1.00 each), four isolates had SAB values of 0.80-0.99, indicating that they are highly similar, but are non-identical, while five isolates are unrelated (SAB lower than 0.80). This suggests that microevolution might have occurred and that these clinical isolates may possibly belong to different strains. Conclusion, significance and impact of study: A fair degree of genetic heterogeneity was found among the 14 C. albicans isolates from UMMC. To our knowledge, this is the first report on the genetic profiles of C. albicans bloodstream infection isolates from Malaysia, warranting further studies in the possible evolutionary trends within this Candida species in Malaysia. Keywords: Randomly Amplified Polymorphic DNA PCR (RAPD-PCR), Candida albicans, Candida bloodstream infections, Genetic relatedness, DNA fingerprinting

Molecular Analysis of Exophiala Species Using Molecular Markers

Genetic relatedness of medically important Exophiala species such as E. dermatitidis, E. mansonii, and three E. jeanselmei varieties: jeanselmei, lecanii-corni, and heteromorpha was examined using PCR-RFLP (restriction fragment length polymorphism) of ribosomal DNA, M-13, (GTG)5, and nucleotide sequences of ribosomal ITS (internal transcribed space) II regions. Three E. jeanselmei varieties showing distinct band patterns for each DNA markers as well as different nucleotide sequences of ribosomal ITS II regions could be considered as a separate species. E. dermatitidis and E. mansonii demonstrated the identical band patterns of RFLP of ribosomal DNA, M-13, and (GTG)5 markers. However, nucleotides sequences of ribosomal ITS II region were different between these two species.

Genetic Relatedness of Korean Isolates of Pythium Using Restriction Fragment Length Polymorphism of PCR Amplified Ribosomal DNA and M-13 Marker

Twenty-five isolates of Pythium species were identi6ed and classified on the basis of RFLP(restriction fragment length polymorphism) of ITS(internal transcribed spacer) region in ribosomal DNA. and M-13 PCR markers. Eight restriction endonucleases were used to investigate the genetic relatedness among isolates P. graminicola and P. arrhenomanes as well as P. aphanidermatum and P. delience produced identical band patterns with all restriction endonucleases used and M-13 markers. P. myriotylum and P. catenulatum also formed tight cluster on the basis of RFLP of ribosomal DNA but produced distinct band pattern with M-13 PCR markers. No intraspecitic variations were observed with RFLP of ITS region in ribosomal DNA. Molecular analysis based on M-13 marker demonstrated that each species produced distinct band patterns. Intraspecific variation of P. ultimum and P. torulosum was observed with M-13 markers.