Gerardo Vázquez-Marrufo

Determination of α- and β-amanitin in clinical urine samples by Capillary Zone Electrophoresis

Amanitins are toxins found in species of the mushroom genera Amanita, Lepiota and Galerina. Intoxication after ingestion of these mushrooms can be fatal with an estimated 20% of mortality rate. An early diagnosis is necessary in order to avoid invasive and expensive therapy and to improve patients prognosis. In this paper, a Capillary Zone Electrophoresis method was developed and validated to determine alpha- and beta-amanitin in urine in less than 7 min using 5 mM, pH 10 borate buffer as background electrolyte. The separation conditions were: capillary: 75 microm I.D., 41 cm effective length, 48 cm total length, 25 degrees C, 20 KV and PDA detection at 214 nm. Sample treatment for analysis only required urine dilution in background electrolyte. The method was validated following established criteria and was found to be selective, linear in the range 5-100 ng/ml. Intra- and inter-day precision and accuracy were within required limits. Limit of detection (LOD) and limit of quantification (LOQ) were 1.5 and 5 ng/ml, respectively. Eight urine samples from suspected cases of intoxication with amanitins were analyzed after 2 years of storage at -20 degrees C, and beta-amanitin was determined in two samples with concentrations of 53 and 65 ng/ml, respectively. The method here described includes the use of non-aggressive reagents to the capillary or the system and is the first Capillary Electrophoresis method used to determine amanitins in clinical samples.

Structural and Phylogenetic Analysis of Laccases from Trichoderma: A Bioinformatic Approach

The genus Trichoderma includes species of great biotechnological value, both for their mycoparasitic activities and for their ability to produce extracellular hydrolytic enzymes. Although activity of extracellular laccase has previously been reported in Trichoderma spp., the possible number of isoenzymes is still unknown, as are the structural and functional characteristics of both the genes and the putative proteins. In this study, the system of laccases sensu stricto in the Trichoderma species, the genomes of which are publicly available, were analyzed using bioinformatic tools. The intron/exon structure of the genes and the identification of specific motifs in the sequence of amino acids of the proteins generated in silico allow for clear differentiation between extracellular and intracellular enzymes. Phylogenetic analysis suggests that the common ancestor of the genus possessed a functional gene for each one of these enzymes, which is a characteristic preserved in T. atroviride and T. virens. This analysis also reveals that T. harzianum and T. reesei only retained the intracellular activity, whereas T. asperellum added an extracellular isoenzyme acquired through horizontal gene transfer during the mycoparasitic process. The evolutionary analysis shows that in general, extracellular laccases are subjected to purifying selection, and intracellular laccases show neutral evolution. The data provided by the present study will enable the generation of experimental approximations to better understand the physiological role of laccases in the genus Trichoderma and to increase their biotechnological potential.

Genetic diversity and population structure of Escherichia coli from neighboring small-scale dairy farms.

The genetic diversity and population structure of Escherichia coli isolates from small-scale dairy farms were used to assess the ability of E. coli to spread within the farm environment and between neighboring farms. A total of 164 E. coli isolates were obtained from bovine feces, bedding, cow teats and milk from 6 small-scale dairy farms. Ward’s clustering grouped the isolates into 54 different random amplified polymorphic DNA (RAPD) types at 95% similarity, regardless of either the sample type or the farm of isolation. This suggests that RAPD types are shared between bovine feces, bedding, cow teats, and milk. In addition, transmission of RAPD types between the studied farms was suggested by the Ward grouping pattern of the isolates, Nei’s and AMOVA population analyses, and genetic landscape shape analysis. For the first time, the latter analytical tool was used to assess the ability of E. coli to disseminate between small-scale dairy farms within the same producing region. Although a number of dispersal mechanisms could exist between farms, the genetic landscape shape analysis associated the flow of E. coli RAPD types with the movement of forage and milking staff between farms. This study will aid in planning disease prevention strategies and optimizing husbandry practices.

Investigation of a food-borne Salmonella Oranienburg outbreak in a Mexican prison

INTRODUCTION Gastroenteritis outbreaks in prisons represent a public health risk worldwide. Identifying and characterizing the etiological agents of gastroenteritis outbreaks in prisons is important for implementing effective prevention and infection control measures. We present the first studied case of a gastroenteritis outbreak in a Mexican prison. METHODOLOGY Rectal swab samples were obtained from affected inmates. Standard microbiological techniques were used for isolating Salmonella enterica. Isolates were typed by PCR assays of DNA repetitive elements (ERIC, BOX, REP) and RAPD. Antibiotic resistance profiles were performed by the Kirby-Bauer method. RESULTS S. enterica serotype Oranienburg was responsible for the outbreak affecting 150 inmates. All patients presented diarrhea, and 70% of them also presented vomiting, with no fatal cases. The origin of the outbreak was undetermined due to the difficulty of gathering epidemiological information, but was likely the result of consumption of shrimp broth or a cantaloupe melon beverage. REP, BOX, and ERIC analyses of 26 serotype Oranienburg strains resulted in Simpson discrimination index (D) values of 0, 0.5507, and 0.5661, respectively. The D values from DG93-RAPD analyses and from the combined ERIC-BOX-DG93 markers were 0.7753 and 0.6092, respectively. All strains showed multiresistance to antibiotics. CONCLUSIONS This is the only studied case of a gastroenteritis outbreak in a Mexican prison, and of the first such outbreak caused by serotype Oranienburg. The combined ERIC, BOX, and RAPD markers adequately assessed the genotype diversity of analyzed strains. Penitentiary personnel or inmates involved in outbreaks might spread multiresistant strains outside of the facility.

MULTI-TYPING OF ENTEROBACTERIA HARBORING LT AND ST ENTEROTOXIN GENES ISOLATED FROM MEXICAN CHILDREN

Enterotoxigenic Escherichia coli is the most common cause of diarrhea in children younger than 5 years in the developing world. We used 16S rRNA gene sequencing, the Biolog® system, and an Amplified Ribosomal DNA Restriction Analysis (ARDRA) to identify 69 enterobacteria isolated from the feces of healthy children up to 12 years old and 54 enterobacteria isolated from stool samples obtained from children up to 5 years old with diarrhea from Morelia, Michoacán, Mexico. In the diarrheic group, 18 isolates belonged to the enterotoxigenic pathotype, 1 isolate had both LT (heat labile toxin) gene and ST (heat stable toxin) gene, and 17 had the ST gene. The identity of most of the strains harboring the ST gene was E. coli, and 3 of the strains were identified as Morganella morganii. The ST toxin gene of one of the strains identified as M. morganii showed 100% identity with an ST toxin gene of E. coli. The ARDRA was a very useful tool to differentiate between E. coli and M. morganii. The phenotypic and genetic analyses of the isolates using the Biolog® system and Random Amplified Polymorphic DNA, respectively, showed physiological variation among the studied strains and genetic differences between subgroups.

Typing and selection of wild strains of Trichoderma spp. producers of extracellular laccase

Using the ITS region and the gene tef1, 23 strains of the genus Trichoderma were identified as belonging to the species T. harzianum (n = 14), T. olivascens (n = 1), T. trixiae (n = 1), T. viridialbum (n = 1), T. tomentosum (n = 2), T. koningii (n = 1), T. atroviride (n = 1), T. viride (n = 1), and T. gamsii (n = 1). Strains expressing extracellular laccase activity were selected by decolorization/oxidation assays in solid media, using azo, anthraquinone, indigoid, and triphenylmethane dyes, and the phenolic substances tannic acid and guaiacol. No strain decolorized Direct Blue 71 or Chicago Blue 6B, but all of them weakly oxidized guaiacol, decolorized Methyl Orange, and efficiently oxidized tannic acid. Based in decolorization/oxidation assays, strains CMU‐1 (T. harzianum), CMU‐8 (T. atroviride), CMU‐218 (T. viride), and CMU‐221 (T. tomentosum) were selected for evaluating their extracellular laccase activity in liquid media. Strain CMU‐8 showed no basal laccase activity, while strains CMU‐1, CMU‐218, and CMU‐221 had a basal laccase activity of 1,313.88 mU/mL, 763.88 mU/mL, and 799.53 mU/mL, respectively. Addition of sorghum straw inhibited laccase activity in strain CMU‐1 by 34%, relative to the basal culture, while strains CMU‐8, CMU‐21, and CMU‐221 increased their laccase activity by 1,321.5%, 64%, and 47%, respectively. These results show that assayed phenolic substrates are good tools for selecting laccase producer strains in Trichoderma. These same assays indicate the potential use of studied strains for bioremediation processes. Straw laccase induction suggests that analyzed strains have potential for straw delignification in biopulping and other biotechnological applications.

Identification and characterization of the biotechnological potential of a wild strain of Paraconiothyrium sp

The isolation and characterization of fungal strains from poorly described taxa allows undercover attributes of their basic biology useful for biotechnology. Here, a wild fungal strain (CMU‐196) from recently described Paraconiothyrium genus was analyzed. CMU‐196 was identified as Paraconiothyrium brasiliense by phylogenetic analysis of the rDNA internal transcribed spacer region (ITS). CMU‐196 metabolized 57 out of 95 substrates of the Biolog FF microplates. Efficient assimilation of dextrins and glycogen indicates that CMU‐196 is a good producer of amylolytic enzymes. It showed a remarkably assimilation of α‐d‐lactose, substrate described as inducer of cellulolytic activity but poorly assimilated by several fungi. Metabolically active mycelium of the strain decolorized broth supplemented with direct blue 71, Chicago sky blue and remazol brilliant blue R dyes. The former two dyes were also well removed from broth by mycelium inactivated by autoclaving. Both mycelia had low efficiency for removing fuchsin acid from broth and for decolorizing wastewater from the paper industry. CMU‐196 strain showed extracellular laccase activity when potato dextrose broth was supplemented with Cu+2, reaching a maximum activity of 46.8 (±0.33) U L−1. Studied strain antagonized phytopathogenic Colletotrichum spp. fungi and Phytophthora spp. oomycetes in vitro, but is less effective towards Fusarium spp. fungi. CMU‐196 antagonism includes overgrowing the mycelia of phytopathogens and growth inhibition, probably by hydrosoluble extracellular metabolites. The biotechnological potential of strain CMU‐196 here described warrants further studies to have a more detailed knowledge of the mechanisms associated with its metabolic versatility, capacity for environmental detoxification, extracellular laccase production, and antagonism against phytopathogens.

Ensayo de PCR multiplex para la detección en leche del género Salmonella, la subespecie I y el serotipo Typhimurium

espanolDebido a la importancia social y economica de las enfermedades gastrointestinales causadas por alimentos contaminados con Salmonella enterica, es necesario contar con sistemas de deteccion rapidos, sensibles y especificos para la deteccion oportuna de dicho patogeno. En este trabajo se presenta un protocolo de PCR de punto final para la identificacion de S. enterica en leche, que utiliza los pares de iniciadores STM3098-f2/STM3098-r2, STM4057-f/STM4057-r y STM4497-f/STM4497-r previamente validados como especificos del genero Salmonella, de la subespecie I y del serotipo Typhimurium, respectivamente. El protocolo presentado permite la deteccion de 1 UFC/25 ml de leche contaminada artificialmente, con un periodo de pre-enriquecimiento de 12 h, en ensayos de PCR simple y multiplex con los tres pares de iniciadores. El protocolo disenado puede ser aplicado en sistemas de vigilancia sanitaria para la deteccion de S. enterica en leche. EnglishGastrointestinal diseases caused by foodstuffs contaminated with Salmonella enterica implicate important social and economic issues, making it necessary to have fast, sensitive, and specific systems for opportune detection of the pathogen. An endpoint PCR protocol for identification of S. enterica in milk is presented using primer pairs previously validated as specific to the genus Salmonella (STM3098-f2/STM3098-r2), subspecies enterica (subspecies I) (STM4057-f/STM4057-r), and serotype Typhimurium (STM4497-f/ STM4497-r). This protocol allowed for detection of 1 CFU/25 mL of spiked milk after a 12 h pre-enrichment period by means of simple and multiplex PCR assay with the three used primer pairs. The designed protocol can be applied in sanitary survey programs for detection of S. enterica in milk.