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Solis-Sanchez A.,National Autonomous University of Mexico | Hernandez-Chinas U.,National Autonomous University of Mexico | Navarro-Ocana A.,National Autonomous University of Mexico | De La Mora J.,National Autonomous University of Mexico | And 2 more authors.
Virology Journal

Background: Epidemics and pandemics of cholera, a diarrheal disease, are attributed to Vibrio cholerae serogroups O1 and O139. In recent years, specific lytic phages of V. cholerae have been proposed to be important factors in the cyclic occurrence of cholera in endemic areas. However, the role and potential participation of lytic phages during long interepidemic periods of cholera in non-endemic regions have not yet been described. The purpose of this study was to isolate and characterize specific lytic phages of V. cholerae O1 strains. Methods: Sixteen phages were isolated from wastewater samples collected at the Endhó Dam in Hidalgo State, Mexico, concentrated with PEG/NaCl, and purified by density gradient. The lytic activity of the purified phages was tested using different V. cholerae O1 and O139 strains. Phage morphology was visualized by transmission electron microscopy (TEM), and phage genome sequencing was performed using the Genome Analyzer IIx System. Genome assembly and bioinformatics analysis were performed using a set of high-throughput programs. Phage structural proteins were analyzed by mass spectrometry. Results: Sixteen phages with lytic and lysogenic activity were isolated; only phage ØVC8 showed specific lytic activity against V. cholerae O1 strains. TEM images of ØVC8 revealed a phage with a short tail and an isometric head. The ØVC8 genome comprises linear double-stranded DNA of 39,422 bp with 50.8 % G + C. Of the 48 annotated ORFs, 16 exhibit homology with sequences of known function and several conserved domains. Bioinformatics analysis showed multiple conserved domains, including an Ig domain, suggesting that ØVC8 might adhere to different mucus substrates such as the human intestinal epithelium. The results suggest that ØVC8 genome utilize the "single-stranded cohesive ends" packaging strategy of the lambda-like group. The two structural proteins sequenced and analyzed are proteins of known function. Conclusions: ØVC8 is a lytic phage with specific activity against V. cholerae O1 strains and is grouped as a member of the VP2-like phage subfamily. The encoding of an Ig domain by ØVC8 makes this phage a good candidate for use in phage therapy and an alternative tool for monitoring V. cholerae populations. © 2016 Solís-Sánchez et al. Source

Cazares-Dominguez V.,Laboratorio Of Investigacion En Bacteriologia Intestinal | Ochoa S.A.,Laboratorio Of Investigacion En Bacteriologia Intestinal | Cruz-Cordova A.,Laboratorio Of Investigacion En Bacteriologia Intestinal | Rodea G.E.,Laboratorio Of Investigacion En Bacteriologia Intestinal | And 5 more authors.
Frontiers in Microbiology

Staphylococcus aureus is an opportunistic pathogen that colonizes human hosts and causes a wide variety of diseases. Two interacting regulatory systems called agr (accessory gene regulator) and sar (staphylococcal accessory regulator) are involved in the regulation of virulence factors. The aim of this study was to evaluate the effect of vancomycin on hld and spa gene expression during the exponential and post-exponential growth phases in multidrug-resistant (MDR) S. aureus. Methods: Antibiotic susceptibility was evaluated by the standard microdilution method. The phylogenetic profile was obtained by pulsed-field gel electrophoresis (PFGE). Polymorphisms of agr and SCCmec (staphylococcal cassette chromosome mec) were analyzed by multiplex polymerase chain reaction (PCR). The expression levels of hld and spa were analyzed by reverse transcription-PCR. An enzyme-linked immunosorbent assay (ELISA) was performed to detect protein A, and biofilm formation was analyzed via crystal violet staining. Results: In total, 60.60% (20/33) of S. aureus clinical isolates were MDR. Half (10/20) of the MDR S. aureus isolates were distributed in subcluster 10, with >90% similarity among them. In the isolates of this subcluster, a high prevalence (100%) for the agrII and the cassette SCCmec II polymorphisms was found. Our data showed significant increases in hld expression during the post-exponential phase in the presence and absence of vancomycin. Significant increases in spa expression, protein A production and biofilm formation were observed during the post-exponential phase when the MDR S. aureus isolates were challenged with vancomycin. Conclusion: The polymorphism agrII, which is associated with nosocomial isolates, was the most prevalent polymorphism in MDR S. aureus. Additionally, under our study conditions, vancomycin modified hld and spa expression in these clinical isolates. Therefore, vancomycin may regulate alternative systems that jointly participate in the regulation of these virulence factors. © 2015 Cázares-Domínguez, Ochoa, Cruz-Córdova, Rodea, Escalona, Olivares, Olivares-Trejo, Velázquez-Guadarrama and Xicohtencatl-Cortes. Source

Sepulveda-Gonzalez M.E.,National Polytechnic Institute of Mexico | Sepulveda-Gonzalez M.E.,Laboratorio Of Investigacion En Bacteriologia Intestinal | Parra-Ortega B.,National Polytechnic Institute of Mexico | Betancourt-Cervantes Y.,National Polytechnic Institute of Mexico | And 3 more authors.
Revista Iberoamericana de Micologia

Background: The Saccharomyces cerevisiae vacuole is actively involved in the mechanism of autophagy and is important in homeostasis, degradation, turnover, detoxification and protection under stressful conditions. In contrast, vacuolar proteases have not been fully studied in phylogenetically related Candida glabrata. Aims: The present paper is the first report on proteolytic activity in the C. glabrata vacuole. Methods: Biochemical studies in C. glabrata have highlighted the presence of different kinds of intracellular proteolytic activity: acid aspartyl proteinase (PrA) acts on substrates such as albumin and denatured acid hemoglobin, neutral serine protease (PrB) on collagen-type hide powder azure, and serine carboxypeptidase (CpY) on N-benzoyl-tyr-pNA. Results: Our results showed a subcellular fraction with highly specific enzymatic activity for these three proteases, which allowed to confirm its vacuolar location. Expression analyses were performed in the genes CgPEP4 (CgAPR1), CgPRB1 and CgCPY1 (CgPRC), coding for vacuolar aspartic protease A, neutral protease B and carboxypeptidase Y, respectively. The results show a differential regulation of protease expression depending on the nitrogen source. Conclusions: The proteases encoded by genes CgPEP4, CgPRB1 and CgCPY1 from C. glabrata could participate in the process of autophagy and survival of this opportunistic pathogen. © 2014 Asociación Española de Micología. Source

Cazares-Dominguez V.,Laboratorio Of Investigacion En Bacteriologia Intestinal | Cruz-Cordova A.,Laboratorio Of Investigacion En Bacteriologia Intestinal | Ochoa S.A.,Laboratorio Of Investigacion En Bacteriologia Intestinal | Escalona G.,Laboratorio Of Investigacion En Bacteriologia Intestinal | And 5 more authors.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important opportunistic pathogen that causes both healthcare- and community-acquired infections. An increase in the incidence of these infections may lead to a substantial change in the rate of vancomycin usage. Incidence of reduced susceptibility to vancomycin has been increasing worldwide for the last few years, conferring different levels of resistance to vancomycin as well as producing changes in the cell wall structure. The aim of the present study was to determine the effect of vancomycin on cell wall thickening in clinical isolates of vancomycin-tolerant (VT) MRSA obtained from pediatric patients. From a collection of 100 MRSA clinical isolates from pediatric patients, 12% (12/100) were characterized as VT-MRSA, and from them, 41.66% (5/ 12) exhibited the heterogeneous vancomycin-intermediate S . aureus (hVISA) phenotype. Multiplex-PCR assays revealed 66.66% (8/12), 25% (3/12), and 8.33% (1/12) of the VTMRSA isolates were associated with agr group II, I, and III polymorphisms, respectively; the II-mec gene was amplified from 83.3%(10/12) of the isolates, and the mec IVa gene was amplified from 16.66% (2/12) of the isolates. Pulsed field electrophoresis (PFGE) fingerprint analysis showed 62% similarity among the VT-MRSA isolates. Thin transverse sections analyzed by transmission electron microscopy (TEM) revealed an average increase of 24 nm (105.55%) in the cell wall thickness of VT-MRSA compared with untreated VT-MRSA isolates. In summary, these data revealed that the thickened cell walls of VT-MRSA clinical isolates with agr type II and SCCmec group II polymorphisms are associated with an adaptive resistance to vancomycin. © 2015 Cázares-Domínguez et al. Source

Cruz-Cordova A.,Laboratorio Of Investigacion En Bacteriologia Intestinal | Espinosa-Mazariego K.,Laboratorio Of Investigacion En Bacteriologia Intestinal | Ochoa S.A.,Laboratorio Of Investigacion En Bacteriologia Intestinal | Saldana Z.,Laboratorio Of Investigacion En Bacteriologia Intestinal | And 13 more authors.
Frontiers in Microbiology

Background: Enterotoxigenic Escherichia coli (ETEC) colonize the human intestinal mucosa using pili and non-pili colonization factors (CFs). CS21 (also designated Longus) is one of the most prevalent CFs encoded by a 14 kb lng DNA cluster located in a virulence plasmid of ETEC; yet limited information is available on the prevalence of CS21 positive ETEC isolates in different countries. The aim of this study was to evaluate the prevalence of CS21 among ETEC clinical isolates from Mexican and Bangladeshi children under 5 years old with diarrhea and to determine the phenotypic and genotypic features of these isolates. Methods: ETEC clinical isolates positive to lngA gene were characterized by genotype, multidrug-resistance, self-aggregation, biofilm formation, and adherence to HT-29 cell line. Results: A collection of 303 E. coli clinical isolates were analyzed, the 81.51% (247/303) were identified as ETEC, 30.76% (76/247) were st+/lt+, and 25.10% (62/247) were positive for the lngA gene. Among the lngA+ ETECs identified, 50% of isolates (31/62) were positive for LngA protein. The most frequent serotype was O128ac:H12 found in 19.35% (12/62) of lngA+ ETEC studied. Multidrug-resistance (MDR) lngA+ ETEC isolates was identified in 65% (39/60), self-aggregation in 48.38% (30/62), and biofilm formation in 83.87% (52/62). ETEC lngA+ isolates were able to adhere to HT-29 cells at different levels. Two lngA isogenic mutants were constructed in the ETEC E9034A and ETEC73332 clinical isolate, showing a 77% and 98% reduction in adherence, respectively with respect to the wild type. Conclusion: ETEC isolates that have the lngA gene showed features associated with self-aggregation, and adherence to HT-29 cells, important characteristics in the human gut colonization process and pathogenesis. © 2014 2014 Cruz-Córdova, Espinosa-Mazariego, Ochoa, Saldaña, Rodea, Cázares-Domínguez, Rodríguez-Ramírez, Eslava-Campos, Navarro-Ocaña, Arrellano-Galindo, Hernández-Castro, Gómez-Duarte, Qadri and Xicohtencatl-Cortes. Source

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