Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis as a reliable evidence for suspected Shigella spp. outbreaks

Abstract Background Shigellosis remains a serious public health problem and an important cause of morbidity and mortality worldwide. The aim of this study was to characterize fliC and the genetic relatedness of Shigella spp. isolated during a one-year period from children in a suspected outbreak in Tehran, Iran. Methods and results Fifty Shigella spp. were isolated from 3779 stool samples of children with diarrhea (prevalence rate: 1.32%). Among the isolates, 92% were characterized as Shigella sonnei, while 6% and 2% were identified as S. flexneri and S. boydii, respectively. S. dysenteriae was not recovered from the patients. All isolates were negative for fliC except for Shigella standard strains. The enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) profiles allowed differentiating the 50 isolates into 5 ERIC types, which were grouped into five clusters (ET1-ET5). Computer-assisted clustering of the strains showed a high degree of similarity among the isolates. Conclusion In conclusion, given the clonal correlation of the Shigella strains isolated in this study and the lack of fliC among them, we propose that probably a single or limited fliC-defected Shigella clone spread and caused the outbreak.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Antioxidant capacity of several Iranian, wild and cultivated strains of the button mushroom

The white button mushroom, Agaricus bisporus, is the most commonly grown mushroom in Iran; however, there is a significant shortage of research on its antioxidant activity and other medicinal properties. The aim of this study was to evaluate antioxidant capacity of the methanolic extracts from four cultivated strains and four Internal Transcribed Spacer (ITS)-identified, Iranian wild isolates of A. bisporus. Evaluations were made for total phenols, flavonoids and anthocyanins, and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. Overall, results showed that all the wild isolates exhibited significantly lower DPPH-derived EC₅₀, compared to the cultivated strains (p < 0.05). A relatively high relationship was observed between total phenols and flavonoids or anthocyanins (r² > 0.60). However, these constituents could not statistically differentiate the group of wild samples from the cultivated ones, and there was low correlation with the DPPH-derived EC₅₀s (r² < 0.40). In conclusion, comparisons showed that wild isolate 4 and cultivated strains A15 and H1 had higher antioxidant capacity than the others (p < 0.05). This result identifies these mushrooms as good candidates for further investigation.

Detection of Encephalitozoon spp. from Human Diarrheal Stool and Farm Soil Samples in Korea

Microsporidia are eukaryotic organisms that cause zoonosis and are major opportunistic pathogens in HIV-positive patients. However, there is increasing evidence that these organisms can also cause gastrointestinal and ocular infections in immunocompetent individuals. In Korea, there have been no reports on human infections with microsporidia to date. In the present study, we used real-time PCR and nucleotide sequencing to detect Encephalitozoon intestinalis infection in seven of 139 human diarrheal stool specimens (5%) and Encephalitozoon hellem in three of 34 farm soil samples (8.8%). Genotype analysis of the E. hellem isolates based on the internal transcribed spacer 1 and polar tube protein genes showed that all isolates were genotype 1B. To our knowledge, this is the first report on human E. intestinalis infection in Korea and the first report revealing farm soil samples as a source of E. hellem infection. Because microsporidia are an important public health issue, further large-scale epidemiological studies are warranted.

A Case of Fasciola hepatica Infection Mimicking Cholangiocarcinoma and ITS-1 Sequencing of the Worm

Fascioliasis is a zoonotic infection caused by Fasciola hepatica or Fasciola gigantica. We report an 87-year-old Korean male patient with postprandial abdominal pain and discomfort due to F. hepatica infection who was diagnosed and managed by endoscopic retrograde cholangiopancreatography (ERCP) with extraction of 2 worms. At his first visit to the hospital, a gallbladder stone was suspected. CT and magnetic retrograde cholangiopancreatography (MRCP) showed an intraductal mass in the common bile duct (CBD) without proximal duct dilatation. Based on radiological findings, the presumed diagnosis was intraductal cholangiocarcinoma. However, in ERCP which was performed for biliary decompression and tissue diagnosis, movable materials were detected in the CBD. Using a basket, 2 living leaf-like parasites were removed. The worms were morphologically compatible with F. hepatica. To rule out the possibility of the worms to be another morphologically close species, in particular F. gigantica, 1 specimen was processed for genetic analysis of its ITS-1 region. The results showed that the present worms were genetically identical (100%) with F. hepatica but different from F. gigantica.

A known expressed sequence tag, BM742401, is a potent lincRNA inhibiting cancer metastasis

Long intergenic non-coding RNAs (lincRNAs) have historically been ignored in cancer biology. However, thousands of lincRNAs have been identified in mammals using recently developed genomic tools, including microarray and high-throughput RNA sequencing (RNA-seq). Several of the lincRNAs identified have been well characterized for their functions in carcinogenesis. Here we performed RNA-seq experiments comparing gastric cancer with normal tissues to find differentially expressed transcripts in intergenic regions. By analyzing our own RNA-seq and public microarray data, we identified 31 transcripts, including a known expressed sequence tag, BM742401. BM742401 was downregulated in cancer, and its downregulation was associated with poor survival in gastric cancer patients. Ectopic overexpression of BM742401 inhibited metastasis-related phenotypes and decreased the concentration of extracellular MMP9. These results suggest that BM742401 is a potential lincRNA marker and therapeutic target.

Retention of a recombinant GFP protein expressed by the yellow fever 17D virus in the E/NS1 intergenic region in the endoplasmic reticulum

The flaviviral envelope proteins, E protein and precursor membrane protein, are mainly associated with the endoplasmic reticulum (ER) through two transmembrane (TM) domains that are exposed to the luminal face of this compartment. Their retention is associated with the viral assembly process. ER-retrieval motifs were mapped at the carboxy terminus of these envelope proteins. A recombinant yellow fever (YF) 17D virus expressing the reporter green fluorescent protein (GFP) with the stem-anchor (SA) region of E protein fused to its carboxy terminus was subjected to distinct genetic mutations in the SA sequence to investigate their effect on ER retention. Initially, we introduced progressive deletions of the stem elements (H1, CS and H2). In a second set of mutants, the effect of a length increase for the first TM anchor region was evaluated either by replacing it with the longer TM of human LAMP-1 or by the insertion of the VALLLVA sequence into its carboxy terminus. We did not detect any effect on the GFP localisation in the cell, which remained associated with the ER. Further studies should be undertaken to elucidate the causes of the ER retention of recombinant proteins expressed at the intergenic E/NS1 region of the YF 17D virus polyprotein.

Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol

The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Optimización de la producción de biomasa usando glicerol crudo, de una cepa mutante de Yarrowia lipolytica con actividad incrementada de lipasa. La levadura Yarrowia lipolytica acumula aceites y produce una lipasa extracelular al crecer en diferentes fuentes de carbono, entre ellas el glicerol, principal subproducto de la creciente industria del biodiésel. En el presente trabajo, se optimizó mediante la metodología de superficies de respuesta la producción de biomasa de una nueva cepa mutante de Y. lipolytica, empleando medios con glicerol derivado de la industria del biodiésel como principal fuente de carbono. Esta cepa presentó características morfológicas y perfil de ácidos grasos distintivos, y una mayor actividad de lipasa extracelular. Para obtener una producción significativa de lipasa extracelular, fue necesario el agregado de una fuente orgánica de nitrógeno y de 1 g/l de aceite de oliva. Se utilizaron los diseños estadísticos de Plackett-Burman y central compuesto para la selección y la optimización de las fermentaciones en frascos agitados; los máximos valores de biomasa y de lipasa obtenidos fueron de 16,1 g/l y 12,2 unidades/ml, respectivamente. Luego, el bioproceso en lote optimizado se escaló a biorreactores aireados, y los valores de actividad específica de lipasa alcanzados después de haberse consumido el 95 % del glicerol fueron tres veces más altos que los obtenidos en los cultivos en frascos agitados. En suma, se desarrolló un bioproceso sostenible para la obtención de biomasa y de una actividad de lipasa extracelular, que a la vez maximiza el uso de subproductos de la industria del biodiésel.

Molecular variability in Brycon cf. pesu Müller and Troschel, 1845 (Characiformes: Characidae) from the Araguaia-Tocantins basin

Brycon pesu is a small-sized fish distributed throughout the Amazon and Orinoco Basins and other coastal basins of northeastern South America. Brycon cf. pesu specimens from the Araguaia-Tocantins Basin are currently separated into two morphotypes, Brycon sp1 and Brycon sp2, owing to different coloration of their anal fin. Brycon sp2 has a reddish margin stripe on the anal fin which morphologically distinguishes it from Brycon sp1. In the present research, nuclear and mitochondrial markers were used to test the hypothesis that the Brycon sp1 and Brycon sp2 morphotypes are distinct species. Specimens from the two morphotypes were collected from the Lajeado Hydroelectric Plant and the Palmas River in the Araguaia-Tocantins Basin. Thirty-five loci obtained by the amplification of five inter-simple sequence repeat primers were analyzed but no species-specific bands were detected. Electrophoretic profiles obtained from 5S rDNA non-transcribed spacer amplification failed to show any differentiation in morphotypes. These results were corroborated by nucleotide sequence analysis of the mtDNA control region, in which 24 polymorphic nucleotide sites, representing a polymorphism rate of only 5%, were detected. The low rates of polymorphism detected by inter-simple sequence repeat, non-transcribed spacer and mtDNA D-loop markers strongly reject the hypothesis that the two morphotypes Brycon sp1 and Brycon sp2 represent distinct species within Brycon cf. pesu. Further studies are needed to obtain conclusive data on the notion that the coloration of the anal fin is an intraspecific polymorphism, possibly related to environmental factors.

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90,6 percent of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.

Differential diagnosis of Trichostrongylus and hookworm eggs via PCR using ITS-1 sequence

Trichostrongylus eggs observed in cellophane-thick smears are difficult, in practice, to distinguish from hookworm eggs. In order to overcome these limitations, a molecular approach was conducted. A Trichostrongylus colubriformis adult worm was obtained from a human in Laos, which was identified morphologically. ITS-1 sequence of this worm was determined, and found to be most similar with that of T. colubriformis among the Trichostrongylus spp. reported so far. Then, this sequence was compared with those of human hookworm species, Ancylostoma duodenale and Necator americanus, and species-specific oligonucleotide primers were designed. Polymerase chain reaction (PCR) using these primers evidenced specifically amplified PCR products of Trichostrongylus sp., A. duodenale and N. americanus from the eggs of each (520 bp, 690 bp, and 870 bp, respectively). A species-specific PCR technique can be developed in order to study the epidemiology of Trichostrongylus spp. and hookworms in endemic areas.

Differential diagnosis of Trichostrongylus and hookworm eggs via PCR using ITS-1 sequence

Trichostrongylus eggs observed in cellophane-thick smears are difficult, in practice, to distinguish from hookworm eggs. In order to overcome these limitations, a molecular approach was conducted. A Trichostrongylus colubriformis adult worm was obtained from a human in Laos, which was identified morphologically. ITS-1 sequence of this worm was determined, and found to be most similar with that of T. colubriformis among the Trichostrongylus spp. reported so far. Then, this sequence was compared with those of human hookworm species, Ancylostoma duodenale and Necator americanus, and species-specific oligonucleotide primers were designed. Polymerase chain reaction (PCR) using these primers evidenced specifically amplified PCR products of Trichostrongylus sp., A. duodenale and N. americanus from the eggs of each (520 bp, 690 bp, and 870 bp, respectively). A species-specific PCR technique can be developed in order to study the epidemiology of Trichostrongylus spp. and hookworms in endemic areas.

Molecular phylogenetic analysis of Vibrio cholerae O1 El Tor strains isolated before, during and after the O 139 outbreak based on the inter-genomic heterogeneity of the 16S-23S rRNA intergenic spacer regions.

We have cloned, sequenced and analysed all the five classes of the intergenic (16S-23S rRNA) spacer region (ISR) associated with the eight rrn operons (rrna-rrnh) of Vibrio cholerae serogroup O1 El Tor strains isolated before, during and after the O 139 outbreak. ISR classes 'a' and 'g' were found to be invariant, ISR-B (ISRb and ISRe) exhibited very little variation, whereas ISR-C (ISRc, ISRd, and ISRf) and ISRh showed the maximum variation. Phylogenetic analysis conducted with all three ISR classes (ISR-B, ISR-C and ISRh) showed that the pre-O 139 serogroup and post-O 139 serogroup O1 El Tor strains arose out of two independent clones, which was congruent with the observation made by earlier workers suggesting that analyses of ISR-C and ISR-h, instead of all five ISR classes, could be successfully used to study phylogeny in this organism.