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Sample records for isolation molecular identification

  1. Molecular identification of clinical Nocardia isolates from India.

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    Rudramurthy, Shivaprakash M; Honnavar, Prasanna; Kaur, Harsimran; Samanta, Palash; Ray, Pallab; Ghosh, Anup; Chakrabarti, Arunaloke

    2015-10-01

    The epidemiology of nocardiosis is evolving with increasing number of Nocardia spp. causing human infection. In recent years, molecular techniques have been used to identify Nocardia spp. There are limited data available on the spectrum of Nocardia spp. isolated from clinical samples in India. Here, a molecular study was carried on 30 clinical isolates maintained in our National Culture Collection to evaluate the techniques used for identifying the agents. The isolates were identified by sequencing two promising genes: the 16S rRNA gene and hsp65. Both hsp65 and the 16S rRNA gene could reliably identify 90 % of Nocardia isolates, i.e. N. farcinica, N. cyriacigeorgica, N. brasiliensis, N. otitidiscaviarum, N. amamiensis and N. pneumoniae. The mean percentage dissimilarity of sequence identification was higher using the hsp65 gene (4 %, range 0-7.9 %) compared with the 16S rRNA gene (2.3 %, range 0-8.9 %). Two isolates that showed ambiguous results in both the short segment of the 16S rRNA gene and hsp65 sequences could be resolved by sequencing a larger fragment (∼1000 bp) of the 16S rRNA gene. Both of these isolates were identified as N. beijingensis with similarities of 99.8 and 100 % compared with the standard strain. Genotyping of N. cyriacigeorgica strains was performed using hsp65 gene sequences and compared with previously described genotypes. Our N. cyriacigeorgica isolates belonged to genotype 1 (n = 4) and genotype 2 (n = 2). The present study highlights a wide spectrum of Nocardia spp. in India and emphasizes the need for molecular techniques for identification to the species level.

  2. Pythium insidiosum: morphological and molecular identification of Brazilian isolates

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    Maria Isabel de Azevedo

    2012-07-01

    Full Text Available Pythium insidiosum is an oomycete belonging to the kingdom Stramenipila and it is the etiologic agent of pythiosis. Pythiosis is a life-threatening infectious disease characterized by the development of chronic lesions on cutaneous and subcutaneous, intestinal, and bone tissues in humans and many species of animals. The identification of P. insidiosum is important in order to implement a rapid and definitive diagnosis and an effective treatment. This study reports the identification of 54 isolates of P. insidiosum of horses, dogs and sheep that presented suspicious clinical lesions of pythiosis from different regions in Brazil, by using morphological and molecular assays. Throughout the PCR it was possible to confirm the identity of all Brazilian isolates as being P. insidiosum.

  3. The isolated Leptospira Spp. Identification by molecular biological techniques

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    Duangjai Suwancharoen

    2017-01-01

    Full Text Available Leptospirosis is a zoonotic disease caused by the bacteria of Leptospira spp. Identification of this bacterium relies on serotyping and genotyping. Data base for animal causative serovars in Thailand is limited. As the unknown serovars are found in the laboratory, they need to be sent overseas for referent identification. To reduce the cost, this research intended to develop a leptospiral identification method which is user–friendly and able to classify efficiently. Ten Leptospira isolations were cultured from urine samples. They were identified by three molecular biological techniques, including Pulsed-Field Gel Electrophoresis (PFGE, Variable Number Tandem Repeat (VNTR and Multilocus Sequence Typing (MLST. These methods were developed and compared to find the most suitable one for leptospiral identification. VNTR was found to be inappropriate since it could not identify the agents and it did not show the PCR product. PFGE and MLST gave the same results of the unknown 1 and 2 which were L.weilii sv Samin st Samin. Unknown 4 showed different results by each technique. Unknown 5 to 10 were likely to be L.meyeri sv Ranarum st ICF and Leptonema illini sv Illini st 3055 by PFGE but MLST could not identify the serovar. However, molecular biological technique for Leptospira identification should be done by several methods in order to confirm the result of each other.

  4. Isolation and molecular identification of yeast strains from “Rabilé” a ...

    African Journals Online (AJOL)

    Isolation and molecular identification of yeast strains from “Rabilé” a starter of local fermented drink. Ibrahim Keita, Marius K Somda, Aly Savadogo, Iliassou Mogmenga, Ousmane Koita, Alfred S Traore ...

  5. Isolation and molecular identification of Lactobacillus brevis from traditional vinegar

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    Zeynab Ebrahimi

    2016-09-01

    Full Text Available Introduction: Vinegar is a popular condiment in the world that different materials and methods have been used to produce it. In Iran natural vinegar is also prepared mostly in a traditional way by using different fruits such as grapes and apples. Natural vinegar has beneficent properties and because of this, it is recommended to be used by traditional and Islamic medicine. Vinegar contains acetic acid bacteria, lactic acid bacteria and yeast. Acetic acid bacteria and yeasts are involved in the production of vinegar and lactic acid bacteria improve the flavor of vinegar. The aim of this study was isolation and identification of lactic acid bacteria especially Lactobacillus brevis from traditional vinegar. Materials and methods: After collecting a few traditional vinegars, the vinegar samples cultured for isolation of lactic acid bacteria on MRS broth and agar media contained nystatin as an anti-yeast antibiotic. Then some microbiological tests including catalase, gram staining and fermentation of carbohydrates were performed. Then, they were cultured at different temperatures, pH and different concentrations of salts. Finally, three isolates bacteria with biochemical properties of Lactobacillus brevis were evaluated by16 srDNA gene amplification. Results: Twelve lactobacilli were isolated from three vinegar samples. All isolated bacteria were catalase-negative and gram-positive. They could be able to grow at pH around 4.5 and 5.6, and at 2, 4 and 5.6% of salt concentrations. Most of the bacteria grew at 15oC, whereas one isolated grew at 45oC. Sequencing and Blast results showed that the three strains are Lactobacillus brevis. Discussion and conclusion: Lactobacillus brevis and Lactobacillus plantrum were found in traditional vinegars. Although isolation of Lactobacillus plantrum from vinegar was reported previously, as far as we could determine, it is for the first time that we could isolate Lactobacillus brevis from vinegar.

  6. Phenotypic and molecular identification of Fonsecaea pedrosoi strains isolated from chromoblastomycosis patients in Mexico and Venezuela.

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    Carolina Rojas, O; León-Cachón, Rafael B R; Pérez-Maya, Antonio Alí; Aguirre-Garza, Marcelino; Moreno-Treviño, María G; González, Gloria M

    2015-05-01

    Chromoblastomycosis is a chronic granulomatous disease caused frequently by fungi of the Fonsecaea genus. The objective of this study was the phenotypic and molecular identification of F. pedrosoi strains isolated from chromoblastomycosis patients in Mexico and Venezuela. Ten strains were included in this study. For phenotypic identification, we used macroscopic and microscopic morphologies, carbohydrate assimilation test, urea hydrolysis, cixcloheximide tolerance, proteolitic activity and the thermotolerance test. The antifungal activity of five drugs was evaluated against the isolates. Molecular identification was performed by sequencing the internal transcribed spacer (ITS) ribosomal DNA regions of the isolated strains. The physiological analysis and morphological features were variable and the precise identification was not possible. All isolates were susceptible to itraconazole, terbinafine, voriconazole and posaconazole. Amphotericin B was the least effective drug. The alignment of the 559-nucleotide ITS sequences from our strains compared with sequences of GenBank revealed high homology with F. pedrosoi (EU285266.1). In this study, all patients were from rural areas, six from Mexico and four from Venezuela. Ten isolates were identified by phenotypic and molecular analysis, using ITS sequence and demonstrated that nine isolates from Mexico and Venezuela were 100% homologous and one isolate showed a small genetic distance. © 2015 Blackwell Verlag GmbH.

  7. First molecular identification and characterization of classical swine fever virus isolates from Nepal.

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    Postel, Alexander; Jha, Vijay C; Schmeiser, Stefanie; Becher, Paul

    2013-01-01

    Classical swine fever (CSF) is a major constraint to pig production worldwide, and in many developing countries, the epidemiological status is unknown. Here, for the first time, molecular identification and characterization of CSFV isolates from two recent outbreaks in Nepal are presented. Analysis of full-length E2-encoding sequences revealed that these isolates belonged to CSFV subgenotype 2.2 and had highest genetic similarity to isolates from India. Hence, for CSFV, Nepal and India should be regarded as one epidemiological unit. Both Nepalese isolates exhibited significant sequence differences, excluding a direct epidemiological connection and suggesting that CSFV is endemic in that country.

  8. Molecular identification of Echinococcus granulosus isolates from ruminants in Greece.

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    Roinioti, Erifylli; Papathanassopoulou, Aegli; Theodoropoulou, Ioanna; Simsek, Sami; Theodoropoulos, Georgios

    2016-08-15

    Cystic echinococcosis is a parasitic disease caused by Echinococcus granulosus, a cestode with worldwide distribution. Data on the circulating Echinococcus granulosus genotypes in Greek livestock is scant. The aim of the present study was to conduct a genetic analysis of 82 Echinococcus granulosus isolates from ruminants in Greece, including areas which until today have not been the subject of studies. The analysis relied on a PCR assay targeting cytochrome c oxidase, subunit 1 gene (CO1), followed by bidirectional sequence analysis of the amplification product. Eighty (n=80) of the 82 (97.6%) isolates were allocated to Echinococcus granulosus sensu stricto (G1-G3) and were classified in 13 distinct haplotypes (9 common and 4 novel) with 12 polymorphic sites. The presence of the dominant haplotype EG1 as was documented in the European populations, was indicated in the country. Almost all regions shared the same common haplotype. In comparison to this predominant haplotype, the number of the nucleotide changes in all the other haplotypes ranged from 1 to 5. All nucleotide changes proved to be transitions (A↔G or C↔T). Two fertile hydatid cysts of sheep origin in different areas (Arkadia, Ilia) of the Peloponnese were identified as Echinococcus canadensis (G7 genotype). Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Molecular identification and characterization of Rhizoctonia solani AG-3 isolates causing black scurf of potato

    International Nuclear Information System (INIS)

    Zaidy, M.E.; Othman, M.R.; Mahmoud, M.

    2018-01-01

    Twenty-six isolates of Rhizoctonia solani AG-3 were collected from four potato growing area of Saudi Arabia. Yield damages due to this infection is reported to range from 7-64% (average of 35%), depending on many factors. Molecular identification of R. solani AG-3 isolates by ITS-regions and characterization was done by inter simple sequence repeat (ISSR) markers. Twenty-six isolates of R. solani used in the current study were isolated from potato fields in four major potato-producing regions of Saudi Arabia. All isolates were inoculated on potato and observations on the percentage of disease incidence were recorded. Genomic DNA extraction of R. solani AG-3 isolates was used by A specific and sensitive PCR and ISSR primers. A single splinter of nearly 500 bp was only amplified once genomic DNA from R. solani AG-3 isolates. Amplicon size of three ISSR primers ranged from 0.3 to 2.8 Kb in isolates. Using the three primers, the tested isolates were separated on the basis of genetic similarity coefficients (GSC). The range of the GSC was beginning at 0.62 and ending at 1.00. In unweighted pair-group method arithmetic averages (UPGMA) analysis, the R. solani isolates grouped into five clusters. The present method provided rapid and reliable detection of R. solani AG-3 isolates. Molecular characterization have great genetic variation in the R. solani AG-3 population, without any correlation between aggressiveness, geographical regions and genetic variation based on ISSR markers. (author)

  10. Isolation, enumeration, molecular identification and probiotic potential evaluation of lactic acid bacteria isolated from sheep milk

    OpenAIRE

    Acurcio, L.B.; Souza, M.R.; Nunes, A.C.; Oliveira, D.L.S.; Sandes, S.H.C.; Alvim, L.B.

    2014-01-01

    Lactic acid bacteria species were molecularly identified in milk from Lacaune, Santa Inês and crossbred sheep breeds and their in vitro probiotic potential was evaluated. The species identified were Enterococcus faecium (56.25%), E. durans (31.25%) and E. casseliflavus (12.5%). No other lactic acid bacteria species, such as lactobacilli, was identified. Most of the isolated enterococci were resistant to gastric pH (2.0) and to 0.3% oxgall. All tested enterococci were resistant to ceftazidime,...

  11. Species identification and molecular typing of human Brucella isolates from Kuwait.

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    Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

    2017-01-01

    Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

  12. Isolation, enumeration, molecular identification and probiotic potential evaluation of lactic acid bacteria isolated from sheep milk

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    L.B. Acurcio

    2014-06-01

    Full Text Available Lactic acid bacteria species were molecularly identified in milk from Lacaune, Santa Inês and crossbred sheep breeds and their in vitro probiotic potential was evaluated. The species identified were Enterococcus faecium (56.25%, E. durans (31.25% and E. casseliflavus (12.5%. No other lactic acid bacteria species, such as lactobacilli, was identified. Most of the isolated enterococci were resistant to gastric pH (2.0 and to 0.3% oxgall. All tested enterococci were resistant to ceftazidime, oxacillin and streptomycin and sensible to clindamycin, erythromycin and penicillin. The resistance to ciprofloxacin, gentamicin, tetracycline and vancomycin varied among tested species. All tested enterococci strongly inhibited (P<0.05 Escherichia coli and Listeria monocytogenes, moderately inhibited E. faecalis and Staphylococcus aureus and did not inhibit Pseudomonas aeruginosa, Salmonella enterica var. Typhimurium and also one E. durans sample isolated from sheep milk. Four samples of E. faecium, one of E. durans and one of E. casseliflavus presented the best probiotic potential.

  13. Molecular identification of Mycobacterium tuberculosis complex isolates from Kermanshah Province, Iran

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    Roghieh Moghaddam

    2016-01-01

    Full Text Available Tuberculosis is one of the most important zoonotic diseases in the world. Rapid diagnosis of the disease and identification of species is extremely important for proper treatment of the disease as some species of the complex are resistant to the first-line of tuberculosis drugs. The aim of present study was molecular identification of Mycobacterium tuberculosis (MTB complex isolates from Kermanshah Province, Iran, which were submitted to the Tuberculosis Reference Laboratory at Razi Vaccine and Serum Research Institute (Tehran, Iran. To identify the genus Mycobacterium, all isolates were subjected to 16S rRNA polymerase chain reaction (PCR, and PCR-IS6110 was subsequently used to confirm that the isolates belonged to MTB complex. Finally, region of difference (RD typing was used to identify the species in the complex. The results of 16S rRNA and IS6110 PCR analysis showed the presence of 543-bp and 245-bp bands, respectively. Furthermore, 146bp, 172bp, 235bp, and 369bp at RD1, RD4, RD9, and RD12, respectively, were observed during RD typing. Thus, based on the results, all isolates were identified as MTB. It is worth mentioning that most tuberculosis cases are identified on the basis of acid-fast bacilli detection, and antibiotic therapy is immediately initiated subsequently. Moreover, it should be noted that some of these acid-fast positive cases might not be of genus Mycobacterium, and thus, the antibiotics prescribed might threaten the health of the patients. Additionally, if the identified bacilli are not within MTB complex, the drug therapy would differ. However, Mycobacterium bovis, which is a member of MTB complex and is resistant to pyrazinamide, requires exact strain identification. Based on the findings, individual isolates should be identified by RD typing methods, which could clearly discriminate the species from each other.

  14. Comparison of biochemical and molecular methods for the identification of bacterial isolates associated with failed loggerhead sea turtle eggs.

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    Awong-Taylor, J; Craven, K S; Griffiths, L; Bass, C; Muscarella, M

    2008-05-01

    Comparison of biochemical vs molecular methods for identification of microbial populations associated with failed loggerhead turtle eggs. Two biochemical (API and Microgen) and one molecular methods (16s rRNA analysis) were compared in the areas of cost, identification, corroboration of data with other methods, ease of use, resources and software. The molecular method was costly and identified only 66% of the isolates tested compared with 74% for API. A 74% discrepancy in identifications occurred between API and 16s rRNA analysis. The two biochemical methods were comparable in cost, but Microgen was easier to use and yielded the lowest discrepancy among identifications (29%) when compared with both API 20 enteric (API 20E) and API 20 nonenteric (API 20NE) combined. A comparison of API 20E and API 20NE indicated an 83% discrepancy between the two methods. The Microgen identification system appears to be better suited than API or 16s rRNA analysis for identification of environmental isolates associated with failed loggerhead eggs. Most identification methods are not intended for use with environmental isolates. A comparison of identification systems would provide better options for identifying environmental bacteria for ecological studies.

  15. Molecular identification of Giardia duodenalis isolates from domestic dogs and cats in Wroclaw, Poland.

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    Piekarska, Jolanta; Bajzert, Joanna; Gorczykowski, Michał; Kantyka, Magdalena; Podkowik, Magdalena

    2016-09-01

    Giardia duodenalis (G. intestinalis) is a common protozoan causing gastrointestinal disorders in many species of mammals. The genus of Giardia has high molecular diversity. Dogs and cats, in addition to their typical infection with assemblages C, D and F, may be a reservoir of zoonotic assemblages (A and B). The aim of this study was a genetic characteristic of Giardia isolates of dogs and cats from the area of Wroclaw (Poland). A total of 128 and 33 faecal samples from dogs and cats, respectively, were analyzed by routine coprological methods. The animals were diagnosed on the presence of G. duodenalis antigens in faeces soluble with the use of SNAP Giardia (IDEXX Laboratories) immunosorbent assay. 27 DNA isolates of Giardia were subjected to molecular identification (PCR-RFLP). The prevalence of G. duodenalis was 21.1% (27/128) in dogs and 15.1% (5/33) in cats. In dogs, C assemblage was present in 18 (81%) positive stool samples, D assemblage in 2 (9%) samples, B assemblage present in one (4.5%), and mixed assemblages (C and D) occurred in one (4.5%) sample. F assemblage was found in 4 (80%) cats' positive stool samples and A assemblage occurred in one case (20%). Confirmation of the presence of A and B zoonotic assemblages suggests that infected pets can be a threat to human health. This study describes for the first time the presence of mixed infections within host-specific C and D assemblages in dogs in Poland.

  16. Isolation and Molecular Identification of Some Blue-Green Algae (Cyanobacteria from Freshwater Sites in Tokat Province of Turkey

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    Tunay Karan

    2017-11-01

    Full Text Available Collected blue-green algae (cyanobacteria from freshwater sites throughout Tokat province and its outlying areas were isolated in laboratory environment and their morphological systematics were determined and also their species identifications were studied by molecular methods. Seven different species of blue-green algae collected from seven different sites were isolated by purifying in cultures in laboratory environment. DNA extractions were made from isolated cells and extracted DNAs were amplified by using PCR. Cyanobacteria specific primers were used to amplify 16S rRNA and phycocyanine gene regions using PCR. Phylogenetic identification of species were conducted by evaluation of obtained sequence analysis data by using computer software. According to species identification by sequence analysis, it was seen that molecular data supports morphological systematics.

  17. Molecular identification and cluster analysis of homofermentative thermophilic lactobacilli isolated from dairy products.

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    Andrighetto, C; De Dea, P; Lombardi, A; Neviani, E; Rossetti, L; Giraffa, G

    1998-10-01

    Twenty-five strains of thermophilic lactobacilli isolated from yoghurt and from semi-hard and hard cheeses (in parallel with nine type or reference strains) were identified and grouped according to their genetic relatedness. Strains were identified by sugar fermentation patterns using the "API 50 CHL" galleries, by species-specific DNA probes in dot-blot hybridization experiments, by amplification and restriction analysis of the 16S rRNA gene (ARDRA) and by polymerase chain reaction (PCR) using species-specific oligonucleotide primers. Strains were classified as Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus, L. helveticus, and L. acidophilus. Strains which were atypical by sugar fermentation patterns were also identified. Most of the strains could not be grouped using carbohydrate fermentation profiles. PCR fingerprinting was used to identify DNA profiles for the 25 lactobacilli. Experimentally obtained PCR profiles enabled discrimination of all strains, which were grouped according to the similarities in their combined patterns. In general, the clustering of the strains corresponded well with species delineation obtained by molecular identification. The dendrogram of genetic relatedness enabled the unambiguous identification of most of the strains which were shown to be atypical by the sugar fermentation profile, except for a discrepancy in one L. delbrueckii subsp. lactis strain and one atypical Lactobacillus sp. strain.

  18. Isolation and identification of Salmonella spp. in environmental water by molecular technology in Taiwan

    Science.gov (United States)

    Kuo, Chun Wei; Hao Huang, Kuan; Hsu, Bing Mu; Tsai, Hsien Lung; Tseng, Shao Feng; Shen, Tsung Yu; Kao, Po Min; Shen, Shu Min; Chen, Jung Sheng

    2013-04-01

    Salmonella spp. is one of the most important causal agents of waterborne diseases. The taxonomy of Salmonella is very complicated and its genus comprises more than 2,500 serotypes. The detection of Salmonella in environmental water samples by routines culture methods using selective media and characterization of suspicious colonies based on biochemical tests and serological assay are generally time consuming. To overcome this drawback, it is desirable to use effective method which provides a higher discrimination and more rapid identification about Salmonella in environmental water. The aim of this study is to investigate the occurrence of Salmonella using molecular technology and to identify the serovars of Salmonella isolates from 70 environmental water samples in Taiwan. The analytical procedures include membrane filtration, non-selective pre-enrichment, selective enrichment of Salmonella. After that, we isolated Salmonella strains by selective culture plates. Both selective enrichment and culture plates were detected by Polymerase Chain Reaction (PCR). Finally, the serovars of Salmonella were confirmed by using biochemical tests and serological assay. In this study, 15 water samples (21.4%) were identified as Salmonella by PCR. The positive water samples will further identify their serotypes by culture method. The presence of Salmonella in environmental water indicates the possibility of waterborne transmission in drinking watershed. Consequently, the authorities need to provide sufficient source protection and to maintain the system for disease prevention. Keywords: Salmonella spp., serological assay, PCR

  19. Isolation and molecular identification of Sunshine virus, a novel paramyxovirus found in Australian snakes.

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    Hyndman, Timothy H; Marschang, Rachel E; Wellehan, James F X; Nicholls, Philip K

    2012-10-01

    This paper describes the isolation and molecular identification of a novel paramyxovirus found during an investigation of an outbreak of neurorespiratory disease in a collection of Australian pythons. Using Illumina® high-throughput sequencing, a 17,187 nucleotide sequence was assembled from RNA extracts from infected viper heart cells (VH2) displaying widespread cytopathic effects in the form of multinucleate giant cells. The sequence appears to contain all the coding regions of the genome, including the following predicted paramyxoviral open reading frames (ORFs): 3'--Nucleocapsid (N)--putative Phosphoprotein (P)--Matrix (M)--Fusion (F)--putative attachment protein--Polymerase (L)--5'. There is also a 540 nucleotide ORF between the N and putative P genes that may be an additional coding region. Phylogenetic analyses of the complete N, M, F and L genes support the clustering of this virus within the family Paramyxoviridae but outside both of the current subfamilies: Paramyxovirinae and Pneumovirinae. We propose to name this new virus, Sunshine virus, after the geographic origin of the first isolate--the Sunshine Coast of Queensland, Australia. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Isolation and molecular identification of endophytic diazotrophs from seeds and stems of three cereal crops.

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    Huawei Liu

    Full Text Available Ten strains of endophytic diazotroph were isolated and identified from the plants collected from three different agricultural crop species, wheat, rice and maize, using the nitrogen-free selective isolation conditions. The nitrogen-fixing ability of endophytic diazotroph was verified by the nifH-PCR assay that showed positive nitrogen fixation ability. These identified strains were classified by 879F-RAPD and 16S rRNA sequence analysis. RAPD analyses revealed that the 10 strains were clustered into seven 879F-RAPD groups, suggesting a clonal origin. 16S rRNA sequencing analyses allowed the assignment of the 10 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Paenibacillus, Enterobacter, Klebsiella and Pantoea. These representative genus are not endophytic diazotrophs in the conventional sense. They may have obtained nitrogen fixation ability through lateral gene transfer, however, the evolutionary forces of lateral gene transfer are not well known. Molecular identification results from 16S rRNA analyses were also confirmed by morphological and biochemical data. The test strains SH6A and MZB showed positive effect on the growth of plants.

  1. Evaluation of molecular markers for Phytophthora ramorum detection and identification using a standardized library of isolates

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    F.N. Martin; M. Coffey; R. Hamelin; P. Tooley; M. Garbelotto; K. Hughes; T. Kubisiak

    2008-01-01

    A number of molecular diagnostic procedures for detection of Phytophthora ramorum have been reported in the literature. In an effort to evaluate the specificity of 10 of these techniques a standardized DNA library for 317 isolates was assembled that included 60 described species as well as 22 taxonomically unclassified isolates. These were sent blind...

  2. Molecular identification and artificial cultivation of a wild isolate of oyster mushroom in Albania

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    Jordan Merkuri

    2016-03-01

    Full Text Available Basidiomata of a wild mushroom macroscopically recognised as Pleurotus ostreatus were observed on an oak trunk in a mixed wood of northern Albania. Pure cultures of the fungus were then obtained on potato-dextrose-agar medium. Molecular analyses of genomic DNA of the fungus confirmed its identification. The rDNA ITS region nucleotide sequence of the studied Pleurotacea matched at 99% those of two P. ostreatus strains already present in NCBI GenBank database. The rDNA ITS nucelotide sequences of two pure cultures of the Albanian P. ostreatus were deposited in EMBL database under the accession numbers LN849458 and LN849459. One of the fungus isolates was subsequently cultivated under protected and semi-natural conditions. Productivity and biological efficiency of the Albanian P. ostreatus ranged from about 10% to 16% and from 33 to 53.33%, respectively. This seems to be the first report on the artificial cultivation of P. ostreatus in Albania and could have, in the next future, a high economic impact on development and diffusion of this important edible mushroom over the country.

  3. Molecular identification, antifungal susceptibility profile, and biofilm formation of clinical and environmental Rhodotorula species isolates.

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    Nunes, Jorge Meneses; Bizerra, Fernando César; Ferreira, Renata Carmona E; Colombo, Arnaldo Lopes

    2013-01-01

    Rhodotorula species are emergent fungal pathogens capable of causing invasive infections, primarily fungemia. They are particularly problematic in immunosuppressed patients when using a central venous catheter. In this study, we evaluated the species distribution of 51 clinical and 8 environmental Rhodotorula species isolates using the ID32C system and internal transcribed spacer (ITS) sequencing. Antifungal susceptibility testing and biofilm formation capability using a crystal violet staining assay were performed. Using ITS sequencing as the gold standard, the clinical isolates were identified as follows: 44 R. mucilaginosa isolates, 2 R. glutinis isolates, 2 R. minuta isolates, 2 R. dairenensis isolates, and 1 Rhodosporidium fluviale isolate. The environmental isolates included 7 R. mucilaginosa isolates and 1 R. slooffiae isolate. Using the ID32C system, along with a nitrate assimilation test, only 90.3% of the isolates tested were correctly identified. In the biofilm formation assay, R. mucilaginosa and R. minuta exhibited greater biofilm formation ability compared to the other Rhodotorula species; the clinical isolates of R. mucilaginosa showed greater biofilm formation compared to the environmental isolates (P = 0.04). Amphotericin B showed good in vitro activity (MIC ≤ 1 μg/ml) against planktonic cells, whereas voriconazole and posaconazole showed poor activity (MIC(50)/MIC(90), 2/4 μg/ml). Caspofungin and fluconazole MICs were consistently high for all isolates tested (≥64 μg/ml and ≥ 4 μg/ml, respectively). In this study, we emphasized the importance of molecular methods to correctly identify Rhodotorula species isolates and non-R. mucilaginosa species in particular. The antifungal susceptibility profile reinforces amphotericin B as the antifungal drug of choice for the treatment of Rhodotorula infections. To our knowledge, this is the first study evaluating putative differences in the ability of biofilm formation among different Rhodotorula

  4. Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches

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    Rodrigues, Paula; Venâncio, Armando; Lima, Nelson

    2011-01-01

    Section Flavi is one of the most significant Sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by MALDI-TOF ICMS, and to evaluate the discriminatory power of the various approaches in species identification. Methods and ...

  5. Molecular Identification and Echinocandin Susceptibility of Candida parapsilosis Complex Bloodstream Isolates in Italy, 2007-2014.

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    Grazia Lovero

    Full Text Available The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2 and Clinical Laboratory Standards Institute (CLSI M27-A3 guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4% were identified as C. parapsilosis, and 27 (16.6% as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis to 11.1% (C. orthopsilosis, were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin

  6. Isolation and identification of molecular species of phosphatidylcholine and lysophosphatidylcholine from jojoba seed meal (Simmondsia chinensis).

    Science.gov (United States)

    Léon, Fabian; Van Boven, Maurits; de Witte, Peter; Busson, Roger; Cokelaere, Marnix

    2004-03-10

    A mixture of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) has been isolated by column chromatography from a jojoba meal (Simmondsia chinensis) extract. The molecular species of both classes could be separated and isolated by C18 reversed phase HPLC. The two major compounds were identified by 1D and 2D (1)H and (13)C NMR, by MS, and by GC-MS as 1-oleoyl-3-lysophosphatidylcholine and 1,2-dioleoyl-3-phosphatidylcholine. Eight other molecular species of LPC and four other molecular species of PC could be assigned by comparison of the mass spectra of the isolated compounds with the spectra of the two major compounds. Complete characterization of the individual molecular species was achieved by GC and GC-MS analysis of the fatty acyl composition from the isolated compounds. The PC/LPC proportion in the phospholipid mixture from three different samples is 1.6 +/- 0.1. LPC is considered to be an important bioactive compound; the results of this study suggest further research for the evaluation of potential health benefits of jojoba meal phospholipids.

  7. Microbiological and molecular identification of bacterial species isolated from nasal and oropharyngeal mucosa of fuel workers in Riyadh, Saudi Arabia.

    Science.gov (United States)

    AlWakeel, Suaad S

    2017-09-01

    This study aimed to determine the bacterial species colonizing the nasal and oropharyngeal mucosa of fuel workers in Central Riyadh, Saudi Arabia on a microbiological and molecular level. Throat and nasal swab samples were obtained from 29 fuel station attendants in the period of time extending from March to May 2014 in Riyadh, Saudi Arabia. Microbiological identification techniques were utilized to identify the bacterial species isolated. Antibiotic sensitivity was assessed for each of the bacterial isolates. Molecular identification techniques based on PCR analysis of specific genomic sequences was conducted and was the basis on which phylogeny representation was done for 10 randomly selected samples of the isolates. Blood was drawn and a complete blood count was conducted to note the hematological indices for each of the study participants. Nineteen bacterial species were isolated from both the nasal cavity and the oropharynx including Streptococcus thoraltensis , alpha-hemolytic streptococci, Staphylococcus hominis , coagulase-negative staphylococci, Leuconostoc mesenteroides , Erysipelothrix rhusiopathiae and several others. We found 100% sensitivity of the isolates to ciprofloxacin, cefuroxime and gentamicin. Whereas cefotaxime and azithromycin posted sensitivities of 85.7% and 91.4%, respectively. Low sensitivities (fuel products may be a contributing factor to bacterial colonization of the respiratory tract in fuel workers.

  8. Molecular identification and pathogenicity of Citrobacter and Serratia species isolated from cultured Oreochromis niloticus

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    Manal I. El-Barbary

    2017-09-01

    Full Text Available This study aimed to isolate and characterize some pathogenic bacterial strains belonging to the family Enterobacteriaceae. They had been isolated from gills, liver, kidney and skin of naturally infected Oreochromis niloticus and had been identified by biochemical test and 16S rRNA gene using four universal primers. Additionally, the isolates were tested for antimicrobial susceptibility, histopathological alterations of liver, kidney and gills and the pathogenicity of the identified isolates for O. niloticus. The results of phylogenetic analysis placed the isolates in the family Enterobacteriaceae (genera Serratia and Citrobacter based on 99% homology. The primer pair (17F and 1390R is the most appropriate pair of universal primers employed for the identification of 16S rRNA gene as it covers as much as possible of the variable regions (Vs. V1 and V2 regions of 16S rRNA gene presented weak evidence of the diversity of the genera Serratia. The mortality rate was 40–60% after challenging O. niloticus by identified isolates, which revealed its sensitivity to ciprofloxacin and norfloxacin. Histological changes showed dilation in sinusoids with severe vacuolar degeneration in the liver, tubular degeneration and hemorrhage between renal tubules with pyknotic nuclei in the kidney, epithelial hyperplasia, aneurism and evident epithelium interstitial edema in gills of O. niloticus. This study concluded that these isolates should be considered as an opportunistic pathogen of O. niloticus. The study also states that the sequencing of 16S rRNA is an important tool for the identification of unknown bacterial species of fish pathogen. Keywords: Citrobacter sp., Serratia sp., Phylogenetic analysis, Histology, Antibiotic sensitivity, Oreochromis niloticus

  9. Identification, characterization and molecular epidemiology of Escherichia coli isolated from lamb and goat kids with diarrhoea

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    Süheyla Türkyılmaz

    2013-01-01

    Full Text Available Neonatal diarrhoea is a serious health problem on commercial farms. Enterovirulent Escherichia coli is a significant aetiological agent of neonatal diarrhoea. In this work, identification and classification of E. coli isolates obtained from lambs and goat kids with diarrhoea were studied along with antibiotic resistance and clonal relationships of enterovirulent strains. A total of 107 E. coli strains isolated from animals on 43 farms were investigated. Specific virulence genes were determined by multiplex and uniplex polymerase chain reaction. Testing of antibiotic susceptibility was carried out by the Vitek II compact system. The relationship of E. coli isolates was determined by enterobacterial repetitive intergenic consensus polymerase chain reaction. A total of 39 (36.4% enterovirulent E. coli strains were identified and of this 19 (48.7% were shiga toxigenic, 12 (30.8% enterotoxigenic and 8 (20.5% enteropathogenic. Three isolates (7.7% were found to be positive for extended spectrum beta lactamase; 10 (25.6% isolates showed multi-drug resistance to antimicrobials. A total of 28 types were detected by enterobacterial repetitive intergenic consensus polymerase chain reaction. Twenty strains had distinct types while 5 types were common for 2 strains and 3 types were common for 3 strains. This is the first current determination of types, clonality and antibiotic resistance of enterovirulent E. coli isolated from small ruminants with diarrhoea. The results of this study showed that the rates of shiga toxigenic, enterotoxigenic and enteropathogenic isolates of E. coli are high in the western part of Turkey. Although these isolates were not clonal, presence of multidrug resistant isolates may cause public health problems.

  10. Molecular identification of Aspergillus and Eurotium species isolated from rice and their toxin-producing ability.

    Science.gov (United States)

    Yazdani, D; Zainal Abidin, M A; Tan, Y H; Kamaruzaman, S

    2011-01-01

    Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp. infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. niger) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it doesn't work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.

  11. Isolation, Characterization, and Molecular Identification of Phosphate Solubilizing Bacteria from Several Tropical Soils

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    Fahrizal Hazra

    2013-03-01

    Full Text Available The objectives of the research were: (i to isolate and characterize of phosphate solubilizing bacteria (PSB and (ii to identify PSB based on molecular amplification of 16S rRNA gene. Soil samples were collected from rhizosphere in Bogor, West Nusa Tenggara, and East Nusa Tenggara. Several stages in this research were: (i isolation PSB in Pikovskaya agar, (ii morphological and biochemical characterization of PSB, (iii measurement of phosphatase enzymes, and (iv measurement of secreting indole acetic acid phytohormone. As many as 29 isolates of PSB have been collected and three isolates of them, namely: P 3.5 (East Nusa Tenggara, P 6.2 (West Nusa Tenggara, and P 10.1 (Citeureup, West Java were chosen for further study. There were many characteristics of isolate P 10.1: (i it had capable to solubilize P with the value of highest solubilization index (1.80, (ii it had the highest phosphatase enzyme (120.40 mg kg-1, and (iii it had the highest pH decrease at each observation for six days. Isolates P 3.5 and P 10.1 were the Gram-negative bacteria with coccus shapes and isolate P 6.2 was a Gram-negative bacteria with bacillus shape. Deoxiribonucleat Acid (DNA amplification of these bacteria employing 16S rRNA primers generated the 1,300bp-PCR product. The results of the analysis of 16S rRNA gene sequences showed that isolates P 3.5 and P 10.1 has 98% similarity with Gluconacetobacter sp. strains Rg1-MS-CO and isolate P 6.2 has 97% similarity with Enterobacter sp. pp9c strains.

  12. Isolation and molecular identification chitinase-producing Streptomyces strains and examination of their in-vitro antagonistic effects

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    Alireza Dehnad

    2015-12-01

    Full Text Available Introduction: The chemical fungicides are used widely in the world. To reduce the application of synthetic fungicides in treating plant diseases, biological methods are considered as an alternative way to control plant diseases. Many actinomycetes, particularly Streptomyces species are biological agents against a broad spectrum of fungal plant pathogens. The purpose of this study was using the kitinolitik actinomycetes isolated from soil of Eastern Azerbaijan province In order to produce biological pesticides. Materials and methods: Soil samples were taken from different areas of Eastern Azerbaijan province. According to Streptomyces morphological features, single colonies were isolated. To identify the bacteria by molecular characteristic, the genomic DNA was extracted and then the sequences of 16S rDNA were replicated. By using specific primers the bacterial isolates containing chitinase gene were screened. The isolates consisted Chitinase enzyme and were antagonistically cultured with Alternaria genus which is a fungal plant pathogen. Results: Out of 60 soil collected samples, 31 Streptomyces bacterial isolates were separated. Four isolates showed positive results to selectivity action of the chitinase enzyme. Treatment of 3 bacterial isolates with 2 pathogenic fungi showed that AE09 is the most effective anti-fungal isolates. Discussion and conclusion: Soils in Eastern Azerbaijan province are rich of Streptomyces bacteria which generate antifungal compounds. Obtaining the Streptomyces bacteria which have chitinase gene, can lead to identification of very effective strains as anti-fungal.

  13. Molecular identification and distribution profile of Candida species isolated from Iranian patients.

    Science.gov (United States)

    Mohammadi, Rasoul; Mirhendi, Hossein; Rezaei-Matehkolaei, Ali; Ghahri, Mohammad; Shidfar, Mohammad Reza; Jalalizand, Nilufar; Makimura, Koichi

    2013-08-01

    A total of 855 yeast strains isolated from different clinical specimens, mainly nail (42%) and vulva-vagina (25%) were identified by a set of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Genomic DNA was extracted from fresh colonies using Whatman FTA Card technology. PCR assays were performed on the complete ribosomal DNA internal transcribed spacer (rDNA-ITS) region for all isolates and species identification was carried out through their specific electrophoretic profiles after digestion with the enzyme MspI. Those isolates suspected as Candida parapsilosis group were then subjected to amplification of the secondary alcohol dehydrogenase (SADH) gene and restriction digestion with NlaIII enzyme. In total, 71.1% of the strains were obtained from females and 28.9% from males. The age group of 31-40 years consisted of the highest frequency of patients with candidiasis. Candida albicans was the predominant species (58.6%) followed by C. parapsilosis (11.0%), C. glabrata (8.3%), C. tropicalis (7.0%), C. kefyr (5.8%), C. krusei (4.4%), C. orthopsilosis (2.1%), and C. guilliermondii (0.6%). A few strains of C. lusitaniae, C. rugosa, C. intermedia, C. inconspicua, C. neoformans and S. cerevisiae were isolated. We could not identify 8 (0.9%) isolates. Candida albicans remains the most frequently species isolated from Iranian patients; however, the number of non-C. albicans Candida species looks to be increasing. The simple and reliable PCR-RFLP system used in the study has the potential to identify most clinically isolated yeasts.

  14. Molecular identification of fungi isolated from bean tissues with anthracnose symptoms

    International Nuclear Information System (INIS)

    Vanegas Berrouet, Katherin M; Gutierrez Sanchez, Pablo A; Marin Montoya, Mauricio A

    2014-01-01

    In this work, endophytic fungi from leaves and pods of bean presenting anthracnose symptoms were isolated from plants collected at different municipalities in the province of Antioquia (Colombia). Isolates were identified by sequencing the RDNA its regions together with the examination of reproductive structures during sporulation in culture media. Colletotrichum lindemuthianum, the causal agent of anthracnose was isolated in all samples showing symptoms of this disease. These results were confirmed by duplex PCR using the specific primers CD1/CD2 and CY1/CY2. Additionally, 17 endophytic fungi were obtained. Fourteen isolates did not sporulate in culture media (Mycelia sterilia) but were identified by phylogenetic analysis of the regions as the Ascomycetes: Leptosphaerulina (3), Diaporthe (3), Gibberella (1), Plectosphaerella (1) and Biscogniauxia (1) and the Mitosporic genera phoma (2), Alternaria (2) and Stemphylium (1) Three isolates were identified combining morphological and molecular analysis as Fusarium (2) and Curvularia lunata (1). This work increases our knowledge of the mycobiota of legume plants and will serve as support of future studies aimed at determining the effect of these fungi on the development of anthracnose as well as other problems affecting the bean crop.

  15. Identification and molecular characterization of Chryseobacterium vrystaatense ST1 isolated from oligomineral water of southeast Serbia

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    Kojić M.

    2012-01-01

    Full Text Available The isolation and molecular characterization of bacterial strains isolated from water sources in the Vlasina Mountain in southeast Serbia, confirmed the presence of a new species Chryseobacterium vrystaatense ST1. This Gram- negative species showed an extremely low level of biochemical reactivity in biochemical tests. The gene for 16S rRNA was amplified by PCR using universal primers and sequenced. Comparison of 16S rRNA gene sequence and phenotypic features indicated that the isolate ST belonged to Chryseobacterium vrystaatense. A BLAST search of sequenced 1088 nucleotides of the 16S rRNA gene with all sequences deposited in the NCBI collection showed the highest similarity (98% with the strain Chryseobacterium vrystaatense sp. nov., designated as strain R-23533. The very high homology of these two strains allowed classification of our strain at the species level, but some differences indicate, and indirectly confirm, that the isolate ST is an authentic representative. On the basis of these results, we could conclude that Chryseobacterium vrystaatense ST was for first time isolated in Serbia, which is particularly important when one bears in mind that there are only three sequences of this species deposited in the NCBI collection.

  16. Microbiological and molecular identification of bacterial species isolated from nasal and oropharyngeal mucosa of fuel workers in Riyadh,

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    Suaad S. AlWakeel

    2017-09-01

    Full Text Available This study aimed to determine the bacterial species colonizing the nasal and oropharyngeal mucosa of fuel workers in Central Riyadh, Saudi Arabia on a microbiological and molecular level. Throat and nasal swab samples were obtained from 29 fuel station attendants in the period of time extending from March to May 2014 in Riyadh, Saudi Arabia. Microbiological identification techniques were utilized to identify the bacterial species isolated. Antibiotic sensitivity was assessed for each of the bacterial isolates. Molecular identification techniques based on PCR analysis of specific genomic sequences was conducted and was the basis on which phylogeny representation was done for 10 randomly selected samples of the isolates. Blood was drawn and a complete blood count was conducted to note the hematological indices for each of the study participants. Nineteen bacterial species were isolated from both the nasal cavity and the oropharynx including Streptococcus thoraltensis, alpha-hemolytic streptococci, Staphylococcus hominis, coagulase-negative staphylococci, Leuconostoc mesenteroides, Erysipelothrix rhusiopathiae and several others. We found 100% sensitivity of the isolates to ciprofloxacin, cefuroxime and gentamicin. Whereas cefotaxime and azithromycin posted sensitivities of 85.7% and 91.4%, respectively. Low sensitivities (<60% sensitivity to the antibiotics ampicillin, erythromycin, clarithromycin and norfloxacin were observed. Ninety-seven percent similarity to the microbial bank species was noted when the isolates were compared to it. Most hematological indices recorded were within the normal range. In conclusion, exposure to toxic fumes and compounds within fuel products may be a contributing factor to bacterial colonization of the respiratory tract in fuel workers.

  17. Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches.

    Science.gov (United States)

    Rodrigues, P; Santos, C; Venâncio, A; Lima, N

    2011-10-01

    Section Flavi is one of the most significant sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF ICMS) and to evaluate the discriminatory power of the various approaches in species identification.   Aspergillus section Flavi isolates obtained from Portuguese almonds were characterized in terms of macro- and micromorphology, mycotoxin pattern, calmodulin gene sequence and MALDI-TOF protein fingerprint spectra. For each approach, dendrograms were created and results were compared. All data sets divided the isolates into three groups, corresponding to taxa closely related to Aspergillus flavus, Aspergillus parasiticus and Aspergillus tamarii. In the A. flavus clade, molecular and spectral analyses were not able to resolve between aflatoxigenic and nonaflatoxigenic isolates. In the A. parasiticus cluster, two well-resolved clades corresponded to unidentified taxa, corresponding to those isolates with mycotoxin profile different from that expected for A. parasiticus. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  18. Molecular identification of fungal isolates and hatching success of green turtle (Chelonia mydas) nests.

    Science.gov (United States)

    Candan, Esra Deniz

    2018-02-26

    The aim of this study is to investigate the fungal diversity of green turtle nests and to examine phylogenetic relationships among these isolates. During the nesting season, samples of intra-nest sand and failed eggs were collected from 25% of the surviving nests in Sugözü Beaches, which are amongst the most important nesting beaches for endangered green turtles in the Mediterranean. Twenty-three fungi were identified by molecular techniques. Fungal isolates belonged to genera Aspergillus, Emericella, Rhizopus, Actinomucor and Apophysomyces with two undescribed species. Aspergillus variecolor, Aspergillus quadrilinieatus, Aspergillus tubingensis, Rhizopus oryzae, Actinomucor elegans and Apophysomyces variabilis were firstly detected in all sea turtle nests within this study. Our results demonstrate that 36.4% of the nests had fungal contamination. Also hatching success of the nests contaminated by fungi were significantly lower than the uncontaminated nests (P = 0.029). Also, this may represent a threat to marine turtles and a risk for the health of conservation workers. This study is the first molecular phylogenetic study associated with sea turtle nests in the eastern Mediterranean coast and contributes to the wider body of literature on fungal invasion of sea turtle nests with firstly isolated species. These findings are important for improving potential conservation measures for the nest sites.

  19. Microbial quality and molecular identification of cultivable microorganisms isolated from an urban drinking water distribution system (Limassol, Cyprus).

    Science.gov (United States)

    Botsaris, George; Kanetis, Loukas; Slaný, Michal; Parpouna, Christiana; Makris, Konstantinos C

    2015-12-01

    Microorganisms can survive and multiply in aged urban drinking water distribution systems, leading to potential health risks. The objective of this work was to investigate the microbial quality of tap water and molecularly identify its predominant cultivable microorganisms. Tap water samples collected from 24 different households scattered in the urban area of Limassol, Cyprus, were microbiologically tested following standard protocols for coliforms, E. coli, Pseudomonas spp., Enterococcus spp., and total viable count at 22 and 37 °C. Molecular identification was performed on isolated predominant single colonies using 16SrRNA sequencing. Approximately 85% of the household water samples were contaminated with one or more microorganisms belonging to the genera of Pseudomonas, Corynebacterium, Agrobacterium, Staphylococcus, Bacillus, Delftia, Acinetobacter, Enterococcus, Enterobacter, and Aeromonas. However, all samples tested were free from E. coli. This is the first report in Cyprus molecularly confirming specific genera of relevant microbial communities in tap water.

  20. Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon.

    Science.gov (United States)

    Fuerst, J A; Gwilliam, H G; Lindsay, M; Lichanska, A; Belcher, C; Vickers, J E; Hugenholtz, P

    1997-01-01

    Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae. The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented. A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria. Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group. Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species. A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology. PMID:8979353

  1. Identification of morphological and molecular Aspergillus species isolated from patients based on beta-tubulin gene sequencing

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    Mahnaz Kheirkhah

    2017-06-01

    Full Text Available Background: Aspergillus species are opportunistic pathogens among immunocompromised patients. In terms of pathogenesis and mycotoxin production, they are in great value. The aim of the this study was to evaluate of beta-tubulin gene for identification of clinical Aspergillus species by PCR-sequencing method compared to morphological features of clinical isolates (such as conidial shape in direct microscopic examination, colony shape in culture, and physiological tests. Materials and Methods: In this study, 465 patients referred to the Shefa laboratory of Isfahan were evaluated. Morphological and molecular identification of clinical samples were performed using culture on sabouraud agar, malt extract agar, czapekdox agar, direct microscopy, and PCR-sequencing of beta tubulin gene, respectively. Sequences were analyzed in comparison with gene bank data. Results: Thirty nine out of 465 suspected cases (8.4% had aspergillosis. The most prevalent species were Aspergillus flavus (56.4%, A. oryzae (20.5%, and A. fumigatus (10.2%, respectively. Fifty nine percent of patients were females and 49% were males. Conclusion: In comparison with phenotypic tests, sequencing of beta-tubulin gene for identification of Aspergillus species is at great value. Replacement of molecular techniques with conventional tests is recommended for precise identification of microorganism for better management of infection.

  2. Morphological and molecular identification of four Brazilian commercial isolates of Pleurotus spp. and cultivation on corncob

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    Nelson Menolli Junior

    2010-04-01

    Full Text Available The species of Pleurotus have great commercial importance and adaptability for growth and fructification within a wide variety of agro-industrial lignocellulosic wastes. In this study, two substrates prepared from ground corncobs supplemented with rice bran and charcoal were tested for mycelium growth kinetics in test tubes and for the cultivation of four Pleurotus commercial isolates in polypropylene bags. The identification of the isolates was based on the morphology of the basidiomata obtained and on sequencing of the LSU rDNA gene. Three isolates were identified as P. ostreatus, and one was identified as P. djamor. All isolates had better in-depth mycelium development in the charcoal-supplemented substrate. In the cultivation experiment, the isolates reacted differently to the two substrates. One isolate showed particularly high growth on the substrate containing charcoal.Espécies de Pleurotus têm grande importância comercial e adaptabilidade para crescimento e frutificação em uma ampla variedade de resíduos agro-industriais lignocelulósicos. Neste trabalho foram testados dois substratos à base de sabugo de milho triturado, suplementados com farelo de arroz e carvão vegetal, para avaliação da cinética de crescimento micelial em tubos de ensaio e produção em sacos de polipropileno, utilizando quatro isolados comerciais. O estudo taxonômico foi realizado com a análise da morfologia dos basidiomas obtidos em cultivo e pelo seqüenciamento do gene nLSU do DNAr, para certificar a identificação taxonômica. Os isolados tiveram melhor desenvolvimento micelial em profundidade no substrato suplementado com carvão vegetal. Em relação à produção, os isolados reagiram de formas distintas em função dos substratos, sendo significativamente melhor o substrato contendo carvão. Três isolados foram identificados como P. ostreatus e o outro foi identificado como P. djamor.

  3. Isolation, molecular identification and quinolone-susceptibility testing of Arcobacter spp. isolated from fresh vegetables in Spain.

    Science.gov (United States)

    González, Ana; Bayas Morejón, Isidro Favián; Ferrús, María Antonia

    2017-08-01

    Some species of the Arcobacter genus are considered emerging foodborne and waterborne enteropathogens. However, the presence of Arcobacter spp. in vegetables very little is known, because most studies have focused on foods of animal origin. On the other hand, quinolones are considered as first-line drugs for the treatment of infection by campylobacteria in human patients, but few data are currently available about the resistance levels to these antibiotics among Arcobacter species. Therefore, the aim of this study was to investigate the presence and diversity of arcobacters isolated from fresh vegetables such as lettuces, spinaches, chards and cabbages. Resistance to quinolones of the isolates was also investigated. One hundred fresh vegetables samples purchased from seven local retail markets in Valencia (Spain) during eight months were analysed. The study included 41 lettuces, 21 spinaches, 34 chards and 4 cabbages. Samples were analysed by culture and by molecular methods before and after enrichment. By culture, 17 out of 100 analysed samples were Arcobacter positive and twenty-five isolates were obtained from them. Direct detection by PCR was low, with only 4% Arcobacter spp. positive samples. This percentage increased considerably, up 20%, after 48 h enrichment. By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), 17 out of the 25 isolates were identified as A. butzleri and 8 as A. cryaerophilus. Only two A. butzleri isolates showed resistance to levofloxacin and ciprofloxacin. The sequencing of a fragment of the QRDR region of the gyrA gene from the quinolones-resistant isolates revealed the presence of a mutation in position 254 of this gene (C-T transition). This study is the first report about the presence of pathogenic species of Arcobacter spp. in chards and cabbages and confirms that fresh vegetables can act as transmission vehicle to humans. Moreover, the presence of A. butzleri quinolone resistant in vegetables could

  4. Molecular identification of Trichuris vulpis and Trichuris suis isolated from different hosts.

    Science.gov (United States)

    Cutillas, Cristina; de Rojas, Manuel; Ariza, Concepción; Ubeda, José Manuel; Guevara, Diego

    2007-01-01

    Trichuris suis was isolated from the cecum of two different hosts (Sus scrofa domestica -- swine and Sus scrofa scrofa -- wild boar) and Trichuris vulpis from dogs in Sevilla, Spain. Genomic DNA was isolated and internal transcribed spacers (ITS)1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced using polymerase chain reaction techniques. The sequence of T. suis from both hosts was 1,396 bp in length while that of T. vulpis was 1,044 bp. ITS1 of both populations isolated of T. suis was 661 nucleotides in length, while the ITS2 was 534 nucleotides in length. Furthermore, the ITS1 of T. vulpis was 410 nucleotides in length, while the ITS2 was 433 nucleotides in length. One hundred fifty-four nucleotides were observed along the 5.8S gene of T. suis and T. vulpis. Intraindividual and intraspecific variations were detected in the rDNA of both species. The presence of microsatellites was observed in all the individuals assayed. Sequence analysis of the ITSs and the 5.8S gene has demonstrated no sequence differences between T. suis isolated from both hosts (S. scrofa domestica -- swine and S. scrofa scrofa -- wild boar). Nevertheless, clear differences were detected between the ITS1 and ITS2 of T. suis and T. vulpis. Furthermore, a comparative molecular analysis between both species and the previously published ITS1-5.8S-ITS2 sequence data of Trichuris ovis, Trichuris leporis, Trichuris muris, Trichuris arvicolae, and Trichuris skrjabini was carried out. A common homology zone was detected in the ITS1 sequence of all species of trichurids.

  5. Molecular and morphological identification of fungal species isolated from bealmijang meju.

    Science.gov (United States)

    Kim, Ji Yeun; Yeo, Soo-Hwan; Baek, Sung Yeol; Choi, Hye Sun

    2011-12-01

    Bealmijang is a short-term aged paste made from meju, which is a brick of fermented soybeans and other ingredients. Different types of bealmijang are available depending on the geographic region or ingredients used. However, no study has clarified the microbial diversity of these types. We identified 17 and 14 fungal species from black soybean meju (BSM) and buckwheat meju (BWM), respectively, on the basis of morphology, culture characteristics, and internal transcribed spacer and beta-tubulin gene sequencing. In both meju, Aspergillus oryzae, Rhizopus oryzae, Penicillium polonicum, P. steckii, Cladosporium tenuissimum, C. cladosporioides, C. uredinicola, and yeast species Pichia burtonii were commonly found. Moreover, A. flavus, A. niger, P. crustosum, P. citrinum, Eurotium niveoglaucum, Absidia corymbifera, Setomelanomma holmii, Cladosporium spp. and unclassified species were identified from BSM. A. clavatus, Mucor circinelloides, M. racemosus, P. brevicompactum, Davidiella tassiana, and Cladosporium spp. were isolated from BWM. Fast growing Zygomycetous fungi is considered important for the early stage of meju fermentation, and A. oryae and A. niger might play a pivotal role in meju fermentation owing to their excellent enzyme productive activities. It is supposed that Penicillium sp. and Pichia burtonii could contribute to the flavor of the final food products. Identification of this fungal diversity will be useful for understanding the microbiota that participate in meju fermentation, and these fungal isolates can be utilized in the fermented foods and biotechnology industries.

  6. Toxocara nematodes in stray cats from shiraz, southern iran: intensity of infection and molecular identification of the isolates.

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    Fattaneh Mikaeili

    2013-12-01

    Full Text Available Toxocara is a common nematode of cats in different parts of Iran. Despite the close association of cats with human, no attempt has been done so far for molecular identification of this nematode in the country. Therefore, current study was performed on identification of some isolates of Toxocara from stray cats in Shiraz, Fars Province, Southern Iran, based on morphological and molecular approaches, and also determination of intensity of infection.This cross-sectional study was carried out on 30 stray cats trapped from different geographical areas of Shiraz in 2011. Adult male and female worms were recovered from digestive tract after dissection of cats. Morphological features using existing keys and PCR-sequencing of ITS-rDNA region and pcox1 mitochondrial l gene were applied for the delineating the species of the parasites.Eight out of 30 cats (26.7% were found infected with Toxocara nematodes. All the isolates were confirmed as Toxocara cati based on morphological features and the sequence of ribosomal and mitochondrial targets. Intensity of infection ranged from one to a maximum of 39 worms per cat, with a mean of 10.25±12.36, and higher abundance of female nematodes.The most prevalent ascaridoid nematode of stray cats in the study area was T. cati and female nematodes were more abundant than that of males. This issue has important role in spreading of eggs in the environment and impact on human toxocariasis.

  7. Molecular identification and amphotericin B susceptibility testing of clinical isolates of Aspergillus from 11 hospitals in Korea.

    Science.gov (United States)

    Heo, Min Seok; Shin, Jong Hee; Choi, Min Ji; Park, Yeon Joon; Lee, Hye Soo; Koo, Sun Hoe; Lee, Won Gil; Kim, Soo Hyun; Shin, Myung Geun; Suh, Soon Pal; Ryang, Dong Wook

    2015-11-01

    We investigated the species distribution and amphotericin B (AMB) susceptibility of Korean clinical Aspergillus isolates by using two Etests and the CLSI broth microdilution method. A total of 136 Aspergillus isolates obtained from 11 university hospitals were identified by sequencing the internal transcribed spacer (ITS) and β-tubulin genomic regions. Minimal inhibitory concentrations (MICs) of AMB were determined in Etests using Mueller-Hinton agar (Etest-MH) and RPMI agar (Etest-RPG), and categorical agreement with the CLSI method was assessed by using epidemiological cutoff values. ITS sequencing identified the following six Aspergillus species complexes: Aspergillus fumigatus (42.6% of the isolates), A. niger (23.5%), A. flavus (17.6%), A. terreus (11.0%), A. versicolor (4.4%), and A. ustus (0.7%). Cryptic species identifiable by β-tubulin sequencing accounted for 25.7% (35/136) of the isolates. Of all 136 isolates, 36 (26.5%) had AMB MICs of ≥2 μg/mL by the CLSI method. The categorical agreement of Etest-RPG with the CLSI method was 98% for the A. fumigatus, A. niger, and A. versicolor complexes, 87% for the A. terreus complex, and 37.5% for the A. flavus complex. That of Etest-MH was ≤75% for the A. niger, A. flavus, A. terreus, and A. versicolor complexes but was higher for the A. fumigatus complex (98.3%). Aspergillus species other than A. fumigatus constitute about 60% of clinical Aspergillus isolates, and reduced AMB susceptibility is common among clinical isolates of Aspergillus in Korea. Molecular identification and AMB susceptibility testing by Etest-RPG may be useful for characterizing Aspergillus isolates of clinical relevance.

  8. Molecular Identification of Unusual Pathogenic Yeast Isolates by Large Ribosomal Subunit Gene Sequencing: 2 Years of Experience at the United Kingdom Mycology Reference Laboratory▿

    Science.gov (United States)

    Linton, Christopher J.; Borman, Andrew M.; Cheung, Grace; Holmes, Ann D.; Szekely, Adrien; Palmer, Michael D.; Bridge, Paul D.; Campbell, Colin K.; Johnson, Elizabeth M.

    2007-01-01

    Rapid identification of yeast isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. We present here an analysis of the utility of PCR amplification and sequence analysis of the hypervariable D1/D2 region of the 26S rRNA gene for the identification of yeast species submitted to the United Kingdom Mycology Reference Laboratory over a 2-year period. A total of 3,033 clinical isolates were received from 2004 to 2006 encompassing 50 different yeast species. While more than 90% of the isolates, corresponding to the most common Candida species, could be identified by using the AUXACOLOR2 yeast identification kit, 153 isolates (5%), comprised of 47 species, could not be identified by using this system and were subjected to molecular identification via 26S rRNA gene sequencing. These isolates included some common species that exhibited atypical biochemical and phenotypic profiles and also many rarer yeast species that are infrequently encountered in the clinical setting. All 47 species requiring molecular identification were unambiguously identified on the basis of D1/D2 sequences, and the molecular identities correlated well with the observed biochemical profiles of the various organisms. Together, our data underscore the utility of molecular techniques as a reference adjunct to conventional methods of yeast identification. Further, we show that PCR amplification and sequencing of the D1/D2 region reliably identifies more than 45 species of clinically significant yeasts and can also potentially identify new pathogenic yeast species. PMID:17251397

  9. Identification of Mucorales isolates from soil using morphological and molecular methods.

    Science.gov (United States)

    Ziaee, A; Zia, M; Bayat, M; Hashemi, J

    2016-03-01

    Soil is the main habitat of saprophytic and pathogenic fungi. Mucoromycotina constitutes a large group of soil fungi, with certain opportunistic members causing systemic infections in immunocompromised hosts. The majority of human and animal infections are caused by the members of the genera Rhizopus , Mucor , Rhizomucor , Lichtheimia ( Absidia) , Cunninghamella, and Mortierella . Accordingly, in the present study, we aimed to isolate and identify the main genera of the order Mucorales, using molecular assays and morphological features . In total, 340 soil samples were collected from seven public parks throughout the city and sidewalk gardens in 14 municipal districts in Isfahan, Iran. All the samples were cultured on the appropriate media, incubated at 27°C for 2- 4 days, and examined daily for visible fungal growth. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was applied and macroscopic, microscopic, and physiological characteristics were assessed to identify fungal colonies. 400 pure colonies, belonging to the orders Mucorales and Mortierellales, including the genera Lichtheimia , Rhizopus, Rhizomucor, Mucor, Cunninghamella, and Mortierella, were identified . The genus Rhizopus (35.5%) was the most frequent isolate, followed by Mucor (32.25%) and Rhizomucor (27.5%). The results emphasize the importance of opportunistic fungi in public areas and indicate the risk of exposure for immunocompromised individuals.

  10. Identification of Mucorales isolates from soil using morphological and molecular methods

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    Ardeshir Ziaee

    2016-03-01

    Full Text Available Background and Purpose: Soil is the main habitat of saprophytic and pathogenic fungi. Mucormycetes are one of the most parts of soil fungi and certain members are among opportunistic fungi and can cause systemic fungal infections in immunocompromised patients. The majority of human and animal infections are caused by members of the genera Rhizopus, Mucor, Rhizomucor, Lichtheimia (Absidia, Cunninghamella and Mortierella. The objective of this research was to isolate and identify the main genera of Zygomycetes, using molecular assay and morphological features. Materials and Methods: A total of 340 soil samples were collected from different sites of seven public parks and 14 municipality districts in Isfahan. All samples were cultured on appropriate media and incubated at 27° C for 2 to 4 days, and then examined daily for visible fungal growth. PCR-RFLP method and macroscopic, microscopic and physiological characteristics were applied to identify fungal colonies. Results: Four hundred pure colonies belonging to six genera of Zygomycetes including Lichtheimia, Rhizopus, Rhizomucor, Mucor, Cunninghamella and Mortierella were identified. The genus Rhizopus (35.5% was the most frequent isolate, followed by Mucor (32.25% and Rhizomucor (27.5%. Conclusion: These finding may help us to understand about the importance of opportunistic fungi in public areas and the risk of exposure with immunocompromised persons.

  11. Identification and molecular epidemiology of dermatophyte isolates by repetitive-sequence-PCR-based DNA fingerprinting using the DiversiLab system in Turkey.

    Science.gov (United States)

    Koc, A Nedret; Atalay, Mustafa A; Inci, Melek; Sariguzel, Fatma M; Sav, Hafize

    2017-05-01

    Dermatophyte species, isolation and identification in clinical samples are still difficult and take a long time. The identification and molecular epidemiology of dermatophytes commonly isolated in a clinical laboratory in Turkey by repetitive sequence-based PCR (rep-PCR) were assessed by comparing the results with those of reference identification. A total of 44 dermatophytes isolated from various clinical specimens of 20 patients with superficial mycoses in Kayseri and 24 patients in Hatay were studied. The identification of dermatophyte isolates was based on the reference identification and rep-PCR using the DiversiLab System (BioMerieux). The genotyping of dermatophyte isolates from different patients was determined by rep-PCR. In the identification of dermatophyte isolates, agreement between rep-PCR and conventional methods was 87.8 % ( 36 of 41). The dermatophyte strains belonged to four clones (A -D) which were determined by the use of rep-PCR. The dermatophyte strains in Clone B, D showed identical patterns with respect to the region. In conclusion, rep-PCR appears to be useful for evaluation of the identification and clonal relationships between Trichophyton rubrum species complex and Trichophyton mentagrophytes species complex isolates. The similarity and diversity of these isolates may be assessed according to different regions by rep-PCR. © 2017 Blackwell Verlag GmbH.

  12. Molecular identification and characterization of Colletotrichum spp isolates from tahiti lime, tamarillo, and mango

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    Adriana Sanabria

    2010-12-01

    Full Text Available Anthracnose is a very limiting disease affecting production, as well as postharvest quality of numerous fruit crops in Colombia. The current management practices for this disease are partially effective due to limited information about the etiology, the inoculum sources, population structure and variation of the pathogen. A total of 293 Colletotrichum isolates were obtained from symptomatic tissues collected from Tahiti lime, tamarillo and mango orchards. To determine the Colletotrichum species causing the symptoms, amplification, and PCR product analysis for intergenic regions of the ribosomal DNA were conducted. Genetic diversity of the fungal population was assessed with Random Amplified Microsatellites (RAMS. Results of this study indicated that anthracnose in Tahiti lime and tamarillo are caused by Colletotrichun acutatum whereas symptoms on mango were induced by the species Colletotrichum gloeosporioides, which was also fund in few citrus samples. RAMS data analysis indicated the existence of two distinct species groups, with a low similarity index (35%. RAM profiles also showed a clear host differentiation of isolates. The C. acutatum population originated from tamarillo exhibited a narrow and homogeneous genetic base, while the C. acutatum population from Tahiti lime was more heterogeneous and genetically complex, as determined by the analysis of molecular variance (AMOVA and of Ni-Li coefficient. The C. gloeosporioides population originated from mango and Tahiti lime was heterogeneous and highly diverse, with clear host differentiation according to RAM profiles. Collectively, the results from this study provide new insight into the general characteristics of Colletotrichum populations on various hosts; this type of knowledge will prove useful in designing more effective management practices.

  13. Isolation, biochemical and molecular identification of Nocardia species among TB suspects in northeastern, Tanzania; a forgotten or neglected threat?

    Science.gov (United States)

    Hoza, Abubakar S; Mfinanga, Sayoki G S; Moser, Irmgard; König, Brigitte

    2017-06-08

    Pulmonary nocardiosis mimic pulmonary tuberculosis in most clinical and radiological manifestations. In Tanzania, where tuberculosis is one of the major public health threat clinical impact of nocardiosis as the cause of the human disease remains unknown. The objective of the present study was to isolate and identify Nocardia isolates recovered from TB suspects in Northeastern, Tanzania by using biochemical and molecular methods. The study involved 744 sputum samples collected from 372 TB suspects from four periphery diagnostic centers in Northeastern, Tanzania. Twenty patients were diagnosed as having presumptively Nocardia infections based on microscopic, cultural characteristics and biomèrieux ID 32C Yeast Identification system and confirmed using 16S rRNA and hsp65 gene specific primers for Nocardia species and sequencing. Biochemically, the majority of the isolates were N. asteroides (n = 8/20, 40%), N. brasiliensis (n = 4/20, 20%), N. farcinica (n = 3/20, 15%), N. nova (n = 1/20, 5%). Other aerobic actinomycetales included Streptomyces cyanescens (n = 2/20, 10%), Streptomyces griseus, Actinomadura madurae each (n = 1/20, 5%). Results of 16S rRNA and hsp65 sequencing were concordant in 15/17 (88. 2%) isolates and discordant in 2/17 (11.8%) isolates. Majority of the isolates belonged to N. cyriacigeorgica and N. farcinica, four (23.5%) each. Our findings suggest that Nocardia species may be an important cause of pulmonary nocardiosis that is underdiagnosed or ignored. This underscores needs to consider pulmonary nocardiosis as a differential diagnosis when there is a failure of anti-TB therapy and as a possible cause of human infections.

  14. Molecular detection and species identification of Enterocytozoon bieneusi isolated from immunocompetent Orang Asli in Malaysia.

    Science.gov (United States)

    Ashikin, Azah; Al-Mekhlafi, Hesham M; Moktar, Norhayati; Anuar, Tengku Shahrul

    2017-04-01

    Most studies of opportunistic infections focus on immunocompromised patients. However, there is a lack of information on microsporidiosis in healthy people (immunocompetent) worldwide. This study aimed to detect and identify microsporidia species in immunocompetent Orang Asli living in Pahang, Malaysia. Orang Asli is a collective term for a group of indigenous people that usually reside in the interior regions of Peninsular Malaysia. They comprise about 0.7% of the total population in Malaysia and 76% of them lived below the poverty line i.e., poor housing conditions with the lack of access to safe drinking water and adequate sanitation, contaminated environment, high illiteracy rate and unhygienic practices by these people. Stool samples were collected from 209 Orang Asli and analyzed for detecting the presence of Enterocytozoon bieneusi and Encephalitozoon intestinalis by polymerase chain reaction assay targeting small subunit ribosomal RNA gene. E. bieneusi was detected in 8 individuals (3.83%). This infection was commonly found in males than females (5.2% vs. 2.7%). All infected Orang Asli were adults, with a mean age of 44years. Diarrhea and other gastrointestinal symptoms were reported in one case (12.5%) among individuals infected with this species. These findings clearly show that exposure to E. bieneusi may actually be common than reported. The accurate detection and identification of microsporidian species by molecular technique will improve therapy, clinical manifestations and prognosis of this infection, as no antiparasitic therapy has been approved for E. bieneusi. It is hoped that these findings will allow the formulation of better health management and disease prevention advisories, and improvement in the standards of health in similar communities. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Molecular identification of TEM-116 beta-lactamase gene in isolates ...

    African Journals Online (AJOL)

    Purpose: Purpose: To determine TEM-116 beta-lactamase gene prevalence in drug-resistant Pseudomonas aeruginosa isolates from Pakistan. Methods: Sequence analysis of TEM beta-lactamase isolates and their antibiotic susceptibility patterns were carried out. Quantitative bacteriostatic concentrations for commonly ...

  16. Candida albicans isolated from urine: Phenotypic and molecular identification, virulence factors and antifungal susceptibility

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    Laura Wiebusch

    2017-07-01

    Conclusions: C. albicans isolates from urine have a high capacity for virulence and can be associated with infectious processes. Furthermore, the high percentage of isolates resistant to itraconazole is important because this antifungal agent is commonly used to treat fungal infections in the hospital environment.

  17. Morphological and molecular identification of filamentous fungi isolated from cosmetic powders

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    Flavia Cristina Jastale Pinto

    2012-12-01

    Full Text Available Seven fungi were isolated from 50 samples of cosmetic powders. Morphological analyses and ribosomal DNA Internal Transcribed Spacers sequencing were performed which allowed the discrimination of the isolated fungi as Aspergillus fumigatus, Penicillium sp., and Cladosporium sp. which could have, among their species, potentially pathogenic microorganisms.

  18. Molecular identification of candida species isolated from women with vulvovaginal candidiasis: brief report

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    Maryam Khanmohamadi

    2017-10-01

    Conclusion: Regarding to the results of this study, C. albicans was the most common Candida species, isolated from patients with vulvovaginal candidiasis and approximately 30% of this infection causing by non-albicans species of Candida.

  19. Molecular identification of TEM-116 beta-lactamase gene in isolates ...

    African Journals Online (AJOL)

    Keywords: P. aeruginosa, Clinical isolates, Sequencing, TEM-116, Antibiotic susceptibility. Tropical Journal of ... organisms produce a wide range of beta ... TEM-1 is the most commonly encountered beta-lactamase in Gram-negative bacteria.

  20. Identification and molecular characterization of Roseomonas genomospecies 5 isolated from Umbilical Cord Blood Unit

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    J.M. Bello-López

    2017-01-01

    Conclusions: This is the first report on the isolation of Roseomonas genomospecies 5 in a UCBU for transplantation, an unusual bacteria isolated from umbilical cord blood, associated with a possible immunosuppression in the donor. Its presence in UCBU can be fatal in immunocompromised patients if it were used for transplantation of Hematopoietic Stem Cells (HSC, due to the potential virulence of the strains and the resistance to antimicrobials commonly used.

  1. Identification of lignionolytic fungi isolated in Mexico using its like molecular marker

    International Nuclear Information System (INIS)

    Rivera-Rios, J. M.; Cruz Ramirez, M. G.; Cruz Madrid, L. C.; Medina Moreno, S. A.; Tellez-Jurado, A.; Arana-Cuenca, A.; Maqueda Galvez, A. P.

    2009-01-01

    Huejutla de Reyes is a place with a warm-humid climate and counts on an annual average temperature of 30 degree centigrade. We collected fungi that growth in wood or trees with the purpose of isolation this ligninolytic fungi in two seasons (one is spring, before raining station and another one in autumn, during raining station). The experimental work was done in the laboratory of the university, where the fungi were isolated. (Author)

  2. MOLECULAR CHARACTERIZATION AND IDENTIFICATION OF BIOCONTROL ISOLATES OF TRICHODERMA HARZIANUM FROM EMBU DISTRICT, KENYA

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    Elizabeth M Siameto

    2010-10-01

    Full Text Available Species in the genus Trichoderma are important as commercial source of several enzymes, biofungicides, and growth promoters. The most common biological control agents of the genus are strains of T. harzianum, T .viride and T. viriens. In this study, sixteen selected isolates of T. harzianum from different land use types in Embu, Kenya were tested for anatogonistic action against five soil borne phytopathogenic fungi (Rhizoctonia solani, Pythium sp, Fusarium graminearum, F. oxysporum f. sp phaseoli and F. oxysporum f. sp Lycopersici using dual culture assay and through production of non-volatile inhibitors. Seven isolates were further characterized using RAPD-PCR procedure to determine genetic variability. All T. harzianum isolates had considerable antagonistic effect on mycelial growth of the pathogens in dual cultures compared to the controls. Maximum inhibition occurred in Pythium sp-055E interaction (73%.The culture filtrates obtained from Czapek’s liquid medium reduced the dry weight (mg of the mycelia significantly while those from the potato dextrose broth showed minimum inhibition growth. Pythium sp was inhibited the most compared to other pathogens. Genetic similarities generated using Jaccard’s coefficient of similarity ranged from 0.231 between isolates 055E and 011E to 0.857 between isolates 010E and 015E. The technique of RAPD was efficient in demonstrating the DNA polymorphism in the isolates of T. harzianum tested showing intraspecific genetic variability. Since all T. harzianum isolates evaluated were effective in controlling colony growth of the soil borne pathogens both in dual cultures and in culture filtrates they should be tried as a broad spectrum biological control agent in the greenhouse and under field conditions.

  3. Isolation and identification of burn wound superbugs by molecular technique and their susceptibility to silver nanoparticles

    Science.gov (United States)

    Mala, R.; Celsia, A. S. Ruby

    2018-02-01

    Burn wound is a global problem affecting millions of people. It is the major cause of mortality and morbidity. This study was aimed to isolate and identify the wound isolates by 16S rRNA and to assess their susceptibility to antibiotics and silver nanoparticles. Silver nanoparticles were synthesized using aqueous extract of A.indica. The silver nanoparticles were characterized by FESEM, XRD, FTIR and DSC. Antibacterial susceptibility of the isolates was assessed by well diffusion method. The wound isolates were identified as S.aureus and E.coli. Both isolates were resistant to β lactum antibiotics, aminoglycoside, quinolones and macrolides. The inhibition zone exhibited by all antibiotics against both organisms was less than 5 mm. The size of silver nanoparticles were recorded as 55 nm. XRD confirmed the crystalline nature of the nanoparticles. TGA and DSC of silver nanoparticles showed the loss of weight and the melting point of silver nanoparticles was recorded at 871.3°C. Silver nano particles inhibited S.aureus and E.coli with an inhibition zone of 27 mm and 32 mm respectively. Therefore the study demonstrated that only silver containing dressings can be used in burn wounds infected by multi drug resistant super bugs.

  4. MOLECULAR IDENTIFICATION AND ANTIMICROBIAL RESISTANCE PATTERN OF SEVEN CLINICAL ISOLATES OF Nocardia spp. IN BRAZIL

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    Larissa Anuska Zeni CONDAS

    2015-06-01

    Full Text Available Nocardia is a ubiquitous microorganism related to pyogranulomatous infection, which is difficult to treat in humans and animals. The occurrence of the disease is on the rise in many countries due to an increase in immunosuppressive diseases and treatments. This report of cases from Brazil presents the genotypic characterization and the antimicrobial susceptibility pattern using the disk-diffusion method and inhibitory minimal concentration with E-test® strips. In summary, this report focuses on infections in young adult men, of which three cases were cutaneous, two pulmonary, one neurological and one systemic. The pulmonary, neurological and systemic cases were attributed to immunosuppressive diseases or treatments. Sequencing analysis of the 16S rRNA segments (1491 bp identified four isolates of Nocardia farcinica, two isolates of Nocardia nova and one isolate of Nocardia asiatica. N. farcinica was involved in two cutaneous, one systemic and other pulmonary cases; N. nova was involved in one neurological and one pulmonary case; and Nocardia asiatica in one cutaneous case. The disk-diffusion antimicrobial susceptibility test showed that the most effective antimicrobials were amikacin (100%, amoxicillin/clavulanate (100%, cephalexin (100% and ceftiofur (100%, while isolates had presented most resistance to gentamicin (43%, sulfamethoxazole/trimethoprim (43% and ampicillin (29%. However, on the inhibitory minimal concentration test (MIC test, only one of the four isolates of Nocardia farcinica was resistant to sulfamethoxazole/trimethoprim.

  5. Molecular identification of t4 and t5 genotypes in isolates from acanthamoeba keratitis patients.

    Science.gov (United States)

    Ledee, D R; Iovieno, A; Miller, D; Mandal, N; Diaz, M; Fell, J; Fini, M E; Alfonso, E C

    2009-05-01

    Acanthamoeba keratitis (AK) is a rare but sight-threatening ocular infection. Outbreaks have been associated with contaminated water and contact lens wear. The epidemiology and pathology may be associated with unique genotypes. We determined the Rns genotype for 37 clinical isolates from 23 patients presenting at the University of Miami Bascom Palmer Eye Institute with confirmed AK infections in 2006 to 2008. The genus-specific ASA.S1 amplicon allowed for rapid genotyping of the nonaxenic cultures. Of the 37 isolates, 36 were of the T4 genotype. Within this group, 13 unique diagnostic fragment 3 sequences were identified, 3 of which were not in GenBank. The 37th isolate was a T5, the first in the United States and second worldwide to be found in AK. For five patients with isolates from the cornea and contact lens/case, identical sequences within each patient cluster were observed, confirming the link between contact lens contamination and AK infection. Genotyping is an important tool in the epidemiological study of AK. In this study, it allowed for the detection of new strains and provided an etiological link between source and infection. Additionally, it can allow for accurate categorizing of physiological differences, such as strain virulence, between isolates and clades.

  6. Molecular identification and characterization of Colletotrichum sp. isolates from Tahiti lime, tamarillo, and mango

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    Sanabria Adriana

    2010-11-01

    mango were induced by the species Colletotrichum gloeosporioides, which was also fund in few

    citrus samples. RAMS data analysis indicated the existence of two distinct species groups, with a low similarity index (35%. RAM profiles also showed a clear host differentiation of isolates. The C. acutatum population originated from tamarillo exhibited a narrow and homogeneous genetic base, while the C. acutatum population from Tahiti lime was more heterogeneous and  genetically complex, as determined by the analysis of molecular variance (AMOVA and of Ni-Li coefficient. The C. gloeosporioides

  7. Optimization and molecular identification of novel cellulose degrading bacteria isolated from Egyptian environment

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    Azhar A. Hussain

    2017-06-01

    Full Text Available Cellulase producing bacteria were isolated from both soil and ward poultry, using CMC (carboxymethylcellulose agar medium and screened by iodine method. Cellulase activity of the isolated bacteria was determined by DNS (dinitrosalicylic acid method. The highly cellulolytic isolates (BTN7A, BTN7B, BMS4 and SA5 were identified on the basis of Gram staining, morphological cultural characteristics, and biochemical tests. They were also identified with 16S rDNA analysis. The phylogenetic analysis of their 16S rDNA sequence data showed that BTN7B has 99% similarity with Anoxybacillus flavithermus, BMS4 has 99% similarity with Bacillus megaterium, SA5 has 99% homology with Bacillus amyloliquefaciens and BTN7A was 99% similar with Bacillus subtilis. Cellulase production by these strains was optimized by controlling different environmental and nutritional factors such as pH, temperature, incubation period, different volumes of media, aeration rate and carbon source. The cellulase specific activity was calculated in each case. In conclusion four highly cellulolytic bacterial strains were isolated and identified and the optimum conditions for each one for cellulase production were determined. These strains could be used for converting plant waste to more useful compounds.

  8. Isolation, screening and molecular identification of novel bacterial strain removing methylene blue from water solutions

    Science.gov (United States)

    Kilany, Mona

    2017-11-01

    The potentially deleterious effects of methylene blue (MB) on human health drove the interest in its removal promptly. Bioremediation is an effective and eco friendly for removing MB. Soil bacteria were isolated and examined for their potential to remove MB. The most potent bacterial candidate was characterized and identified using 16S rRNA sequence technique. The evolutionary history of the isolate was conducted by maximum likelihood method. Some physiochemical parameters were optimized for maximum decolorization. Decolorization mechanism and microbial toxicity study of MB (100 mg/l) and by-products were investigated. Participation of heat killed bacteria in color adsorption have been investigated too. The bacterial isolate was identified as Stenotrophomonas maltophilia strain Kilany_MB 16S ribosomal RNA gene with 99% sequence similarity. The sequence was submitted to NCBI (Accession number = KU533726). Phylogeny depicted the phylogenetic relationships between 16S ribosomal RNA gene, partial sequence (1442 bp), of the isolated strain and other strains related to Stenotrophomonas maltophilia in the GenBank database. The optimal conditions were investigated to be pH 5 at 30 °C, after 24 h using 5 mg/l MB showing optimum decolorization percentage (61.3%). Microbial toxicity study demonstrated relative reduction in the toxicity of MB decolorized products on test bacteria. Mechanism of color removal was proved by both biosorption and biodegradation, where heat-killed and live cells showed 43 and 52% of decolorization, respectively, as a maximum value after 24-h incubation. It was demonstrated that the mechanism of color removal is by adsorption. Therefore, good performance of S maltophilia in MB color removal reinforces the exploitation of these bacteria in environmental clean-up and restoration of the ecosystem.

  9. Identification of carbapenemase-mediated resistance among Enterobacteriaceae bloodstream isolates: A molecular study from India

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    Srujana Mohanty

    2017-01-01

    Full Text Available Acquired resistance in carbapenem-resistant Enterobacteriaceae (CRE conferred by carbapenemases is a major concern worldwide. Consecutive, non-duplicate isolates of Escherichia coli (EC and Klebsiella pneumoniae from clinically diagnosed bloodstream infections were screened for the presence of carbapenem resistance by standard disk-diffusion method and minimum inhibitory concentration breakpoints using the Clinical and Laboratory Standards Institute guidelines. Carbapenemase-encoding genes were amplified by polymerase chain reaction. Of 387 isolates (214 K. pneumoniae, 173 EC tested, 93 (24.03% were found to be CRE. Of these, 71 (76.3% were positive for at least one tested carbapenemase gene. The frequency of carbapenemase genes was New Delhi metallo-β-lactamse-1 (65.6%, oxacillinase (OXA-48 (24.7%, OXA-181 (23.6%, Verona integron-encoded metallo-β-lactamase (6.4% and K. pneumoniae carbapenemase (2.1%. Our study identified presence of carbapenemases in a large proportion of CRE isolates. Delineation of resistance mechanisms is important in view of future therapeutics concerned with the treatment of CRE and for aiding control efforts by surveillance and infection control interventions.

  10. Molecular Identification of Ancylostoma caninum Isolated from Cats in Southern China Based on Complete ITS Sequence

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    Yuanjia Liu

    2013-01-01

    Full Text Available Ancylostoma caninum is a blood-feeding parasitic intestinal nematode which infects dogs, cats, and other mammals throughout the world. A highly sensitive and species-specific PCR-RFLP technique was utilised to detect the prevalence of A. caninum in cats in Guangzhou, southern China. Of the 102 fecal samples examined, the prevalence of A. caninum in cats was 95.1% and 83.3% using PCR-RFLP and microscopy, respectively. Among them, the prevalence of single hookworm infection with A. caninum was 54.90%, while mixed infections with both A. caninum and A. ceylanicum were 40.20%. Comparative analysis of three complete ITS sequences obtained from cat-derived A. caninum showed the same length (738 bp as that of dog-derived A. caninum. However, the sequence variation range was 98.6%–100%, where only one cat isolate (M63 showed 100% sequence similarity in comparison with two dog-derived A. caninum isolates (AM850106, EU159416 in the same studied area. The phylogenetic tree revealed A. caninum derived from both cats and dogs in single cluster. Results suggest that cats could be the main host of A. caninum in China, which may cause cross-infection between dogs and cats in the same area.

  11. Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA

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    Aboubaker M. Garbaj

    2016-11-01

    Full Text Available Aim: The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. Materials and Methods: A total of 108 samples of raw milk (cow, she-camel, and goat and locally made dairy products (fermented cow’s milk, Maasora, Ricotta and ice cream were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA. Results: Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey that include 3 isolates from cow’s milk (11%, 3 isolates from she-camel’s milk (11%, two isolates from goat’s milk (7.4% and 7 isolates from fermented raw milk samples (26%, isolates from fresh locally made soft cheeses (Maasora and Ricotta were 9 (33% and 3 (11%, respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain. Conclusion: These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya.

  12. Molecular identification and potential of an isolate of white rot fungi in bioremediation of petroleum contaminated soils

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    Maryam Mohammadi-sichani

    2017-06-01

    Full Text Available Introduction:Elimination or reduction of petroleum hydrocarbons from natural resources such as water and soil is a serious problem of countries, particularly oil-rich countries of the world. Using white rotting fungi compost for bioremediation of soils contaminated by petroleum hydrocarbons is effective. The aim of this study is molecular identification and potential of anisolate of white rot fungi in bioremediation of petroleum contaminated soils. Materials and methods: Spent compost of white rotting fungi was inoculated with petroleum contaminated soil into 3%, 5% and 10% (w/w. Treatments were incubated at 25-23 °C for 3 months. Reduction of petroleum hydrocarbons in treated soil was determined by gas chromatography. Ecotoxicity of soil was evaluated by seed germination test. Results: Based on the genome sequence of 18s rRNA, it is revealed that this isolate is Ganoderma lucidum and this isolate is deposited as accession KX525204 in the Gene Bank database. Reduction of petroleum hydrocarbons in soil treated with compost (3, 5 and 10% ranged from 42% to 71%. The germination index (% in ecotoxicity tests ranged from 20.8% to 70.8%. Gas chromatography results also showed a decrease in soil Hydrocarbons compounds. Discussion and conclusion: The compost of Ganoderma lucidum, a white rot fungus, has a potential ability to remove petroleum hydrocarbons in contaminated soil. Removal of hydrocarbons was increased with increase in compost mixed with contaminated soil. Petroleum contaminated soil amended with spent compost of G.lucidum 10% during three months is appropriate to remove this pollutant.

  13. Prevalence and molecular identification of Cryptosporidium isolates from pet lizards and snakes in Italy

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    Rinaldi L.

    2012-11-01

    Full Text Available In order to acquire prevalence and genetic data on Cryptosporidium infections in captive lizards and snakes kept as pets, a survey was conducted on 150 individual reptiles from southern Italy. Fecal samples were preserved in 5% formalin and analyzed using a commercial immunofluorescence assay (IFA for the detection of Cryptosporidium oocysts and Giardia cysts. IFA revealed the presence of Cryptosporidium oocysts in nine of the 150 samples examined (6.0%, precisely in 6/125 snakes (4.8% and in 3/25 lizards (12.0%; all fecal samples tested negative for the presence of Giardia cysts. Molecular characterization based on nested PCR amplification and sequencing of the SSU-rRNA gene, revealed the presence of Cryptosporidium serpentis in three samples from snakes (Boa constrictor constrictor, Elapheguttata guttata guttata and Python molurus.

  14. Molecular identification and antifungal susceptibility profile of Candida species isolated from patients with vulvovaginitis in Tehran, Iran.

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    Sharifynia, Somayeh; Falahati, Mehraban; Akhlaghi, Lame; Foroumadi, Alireza; Fateh, Roohollah

    2017-01-01

    Rapid and accurate identification and evaluation of antifungal susceptibility pattern of Candida isolates are crucial to determine suitable antifungal drugs for the treatment of patients with vulvovaginitis candidiasis. Vaginal samples were collected from 150 women with suspicious vaginal candidiasis, and then cultured on Sabouraoud's Dextrose Agar with chloramphenicol to isolate Candida species. After identification of Candida isolates using polymerase chain reaction-restriction fragment length polymorphism technique, antifungal susceptibility testing of four azolic antifungal drugs was carried out using broth microdilution method according to the CLSI M27-A3. Candida species were isolated from eighty suspected patients (61.79%). The most common pathogen was Candida albicans (63.75%). Resistance to fluconazole and ketoconazole was observed in 27.5% and 23.75% of Candida isolates, respectively, and only 2% of Candida isolates were resistant to miconazole. Interestingly, resistance to fluconazole in C. albicans was more than other Candida species. The results indicated that therapy should be selected according to the antifungal susceptibility tests for the prevention of treatment failure and miconazole therapy can be considered as the best therapeutic choice in the management of vulvovaginitis.

  15. Molecular identification and antifungal susceptibility profile of Candida species isolated from patients with vulvovaginitis in Tehran, Iran

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    Somayeh Sharifynia

    2017-01-01

    Full Text Available Background: Rapid and accurate identification and evaluation of antifungal susceptibility pattern of Candida isolates are crucial to determine suitable antifungal drugs for the treatment of patients with vulvovaginitis candidiasis. Materials and Methods: Vaginal samples were collected from 150 women with suspicious vaginal candidiasis, and then cultured on Sabouraoud's Dextrose Agar with chloramphenicol to isolate Candida species. After identification of Candida isolates using polymerase chain reaction-restriction fragment length polymorphism technique, antifungal susceptibility testing of four azolic antifungal drugs was carried out using broth microdilution method according to the CLSI M27-A3. Results: Candida species were isolated from eighty suspected patients (61.79%. The most common pathogen was Candida albicans (63.75%. Resistance to fluconazole and ketoconazole was observed in 27.5% and 23.75% of Candida isolates, respectively, and only 2% of Candida isolates were resistant to miconazole. Interestingly, resistance to fluconazole in C. albicans was more than other Candida species. Conclusion: The results indicated that therapy should be selected according to the antifungal susceptibility tests for the prevention of treatment failure and miconazole therapy can be considered as the best therapeutic choice in the management of vulvovaginitis.

  16. Molecular Identification of Isolated Fungi from Unopened Containers of Greek Yogurt by DNA Sequencing of Internal Transcribed Spacer Region

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    Irshad M. Sulaiman

    2014-06-01

    Full Text Available In our previous study, we described the development of an internal transcribed spacer (ITS1 sequencing method, and used this protocol in species-identification of isolated fungi collected from the manufacturing areas of a compounding company known to have caused the multistate fungal meningitis outbreak in the United States. In this follow-up study, we have analyzed the unopened vials of Greek yogurt from the recalled batch to determine the possible cause of microbial contamination in the product. A total of 15 unopened vials of Greek yogurt belonging to the recalled batch were examined for the detection of fungi in these samples known to cause foodborne illness following conventional microbiological protocols. Fungi were isolated from all of the 15 Greek yogurt samples analyzed. The isolated fungi were genetically typed by DNA sequencing of PCR-amplified ITS1 region of rRNA gene. Analysis of data confirmed all of the isolated fungal isolates from the Greek yogurt to be Rhizomucor variabilis. The generated ITS1 sequences matched 100% with the published sequences available in GenBank. In addition, these yogurt samples were also tested for the presence of five types of bacteria (Salmonella, Listeria, Staphylococcus, Bacillus and Escherichia coli causing foodborne disease in humans, and found negative for all of them.

  17. Design of two molecular methodologies for the rapid identification of Colombian community-acquired methicillin-resistant Staphylococcus aureus isolates

    OpenAIRE

    Escobar, Javier Antonio; Gómez, Ingrid Tatiana; Murillo, Martha Johanna; Castro, Betsy Esperanza; Chavarro, Bibiana; Márquez, Ricaurte Alejandro; Vanegas, Natasha

    2012-01-01

    Introduction. Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are found with increasing the frequency, both in healthy individuals in the community and in hospitalized patients. In Colombia and the Andean region, CA-MRSA isolates have a genetic background that is related to the pandemic USA300 clone. Objective. Two molecular methods are designed and standardized for the rapid differentiation of Colombian community-acquired and hospital-acquired methicillin-...

  18. Molecular identification and in-vitro antifungal susceptibility testing of Candida species isolated from patients with onychomycosis

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    Keyvan Pakshir

    2015-03-01

    Full Text Available Background and Purpose: Candida species are the most opportunistic fungi affecting the nails and resulting in onychomycosis. In this study, we identified and evaluated in-vitro susceptibility of the recovered isolates against fluconazole (FLC, voriconazole (VRC, and clotrimazole (CLT using the Clinical and Laboratory Standards Institute (CLSI M27-A3 document. Materials and Methods: From patients with either clinically or mycologically proven onychomycosis, 97 isolates comprising of seven Candida species were isolated, which were identified by both conventional and molecular techniques such as polymerase chain reaction-restriction fragment length polymorphism. In addition, Candida dubliniensis was confirmed by restriction endonuclease analysis. Antifungal susceptibility of each isolate against the three azoles applied in this study was determined using the CLSI microdilution reference method M27-A3. Results: Candida parapsilosis (C. parapsilosis was the most frequently isolated species (n=44, followed by C. albicans (n=23, C. tropicalis (n=13, C. glabrata (n=7, C. krusei (n=6, C. guilliermondii (n=3, and C. dubliniensis (n=1. All the isolates were susceptible to CLT. VRC had lower minimum inhibitory concentration (MIC values for the isolates compared to FLC. Geometric mean MIC values of VRC, FLC, and CLT for C. parapsilosis isolates were 0.07 µg/ml, 0.8 µg/ml, and 0.35 µg/ml, respectively. Collectively, all species exhibited greater susceptibility to VRC in comparison to C. albicans (P≤0.001. Conclusion: This study showed that non-albicans Candida species were the most common etiologic agents of non-dermatophyte onychomycosis. The major antifungal agents used in clinics to empirically treat yeast onychomycosis are FLC and CLT. Our data suggested that CLT is a better choice for the treatment of Candida onychomycosis, especially in drug resistant cases.

  19. Isolation and molecular identification of a UV-resistant strain of Dietzia maris and antioxidant activity of pigment

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    Narges Zamanian

    2016-09-01

    Full Text Available Introduction: The ability of radioresistant bacteria to survive high levels of UV radiation has been linked to their strong DNA repair systems and ability to produce primary and secondary metabolic products. The biosynthesis of pigments provides an opportunity for bacteria to live in radiation-rich environment. Recent radiation-responsive pigments are used commercially as food colorants, anticancer drugs, as well as antibiotics and for cosmetic purposes. Materials and methods: Soil sample of Omidiyeh city was collected during the spring of 2014 and UV-resistant strain was isolated after primary and secondary screening. Then it was identified by molecular methods (16S rRNA gene sequencing. Antioxidant activity of pigment was evaluated by 2,2 -diphenyl-1-picryl hydrazyl (DPPH and the reducing power of pigments were analyzed by ferric chloride. Results: In this present study, new UV-resistant strain NM2 was isolated and by comparison of these 16S rRNA gene sequences to public database using the BLAST, the genus and species of the isolate was identified as Dietzia maris with 99% similarity. Extraction of pigment from isolated strain was carried out by methanol and acetone as solvents. The spectrum is characterized by maximum peak at 473 nm for pigment of NM2 strain. Antioxidant activity and the reducing ability of pigments increased by increasing their concentrations. NM2 strain pigment showed EC50 concentration of 3.30 mg/ml for DPPH free radical scavenging activity, and EC50 concentration of 28.46 µg/ml for reducing power. Discussion and conclusion: Isolation of natural resources of pigment is very important with high anti-oxidant activity. In the current study, pigment of UV-resistant bacteria demonstrated a strong antioxidant activity in vitro and pigment of these bacteria could play an important role in UV tolerance. Pigment of UV-resistant bacteria may be an appropriate source for antioxidative-related functional foods and the pharmaceutical

  20. ISOLATION AND MOLECULAR IDENTIFICATION OF POTENTIALLY PATHOGENIC Escherichia coli AND Campylobacter jejuni IN FERAL PIGEONS FROM AN URBAN AREA IN THE CITY OF LIMA, PERU

    Science.gov (United States)

    CABALLERO, Moisés; RIVERA, Isabel; JARA, Luis M.; ULLOA-STANOJLOVIC, Francisco M.; SHIVA, Carlos

    2015-01-01

    SUMMARY Feral pigeons (Columbia livia) live in close contact with humans and other animals. They can transmit potentially pathogenic and zoonotic agents. The objective of this study was to isolate and detect strains of diarrheagenic Escherichia coli and Campylobacter jejuni of urban feral pigeons from an area of Lima, Peru. Fresh dropping samples from urban parks were collected for microbiological isolation of E. coli strains in selective agar, and Campylobacter by filtration method. Molecular identification of diarrheagenic pathotypes of E.coli and Campylobacter jejuni was performed by PCR. Twenty-two parks were sampled and 16 colonies of Campylobacter spp. were isolated. The 100% of isolates were identified as Campylobacter jejuni. Furthermore, 102 colonies of E. coliwere isolated and the 5.88% resulted as Enteropathogenic (EPEC) type and 0.98% as Shiga toxin-producing E. coli (STEC). The urban feral pigeons of Lima in Peru can act as a reservoir or carriers of zoonotic potentially pathogenic enteric agents. PMID:26603225

  1. Molecular identification and polymorphism determination of cutaneous and visceral leishmaniasis agents isolated from human and animal hosts in Iran.

    Science.gov (United States)

    Hajjaran, Homa; Mohebali, Mehdi; Mamishi, Setareh; Vasigheh, Farzaneh; Oshaghi, Mohammad Ali; Naddaf, Saied Reza; Teimouri, Aref; Edrissian, Gholam Hossein; Zarei, Zabiholah

    2013-01-01

    Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA) gene followed by RFLP analysis and sequencing was used to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL) in humans and animal reservoir hosts from various geographical areas in Iran. We also used random amplified polymorphic DNA (RAPD-PCR) to obtain polymorphisms among isolates of Leishmania spp. Totally, 362 suspected human and animal cases including 173 CL, 49 VL, 60 rodents, and 80 domestic dogs were examined for Leishmania infection. From 112 culture-positive samples prepared from CL cases, 75 (67%) were infected with L. major and 37 (33%) with L. tropica. Of the 60 rodents examined, 25 (41.6%) harbored the Leishmania infection; 21 were infected with L. major and 4 with L. turanica. From 49 suspected VL, 29 were positive by direct agglutination test (DAT), whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients. Two VL patients were infected with L. tropica and 26 with L. infantum. Of the 80 domestic dogs, 56 showed anti-Leishmania antibodies with DAT. Of these, 55 were positive by both microscopy and culture. Molecular identity, obtained only for 47 samples, revealed L. infantum in 43 and L. tropica in 4 dogs. The polymorphisms among L. tropica and L. major isolates were 3.6% and 7.3%; the rate among human and canine VL isolates was 2.8% and 9.8%, respectively. Our results showed that at least four different Leishmania species with various polymorphisms circulate among humans and animal hosts in Iran.

  2. Molecular Identification and Polymorphism Determination of Cutaneous and Visceral Leishmaniasis Agents Isolated from Human and Animal Hosts in Iran

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    Homa Hajjaran

    2013-01-01

    Full Text Available Amplification of internal transcript spacer 1 of ribosomal RNA (ITS1-RNA gene followed by RFLP analysis and sequencing was used to identify the causing agents of cutaneous and visceral leishmaniasis (CL and VL in humans and animal reservoir hosts from various geographical areas in Iran. We also used random amplified polymorphic DNA (RAPD-PCR to obtain polymorphisms among isolates of Leishmania spp. Totally, 362 suspected human and animal cases including 173 CL, 49 VL, 60 rodents, and 80 domestic dogs were examined for Leishmania infection. From 112 culture-positive samples prepared from CL cases, 75 (67% were infected with L. major and 37 (33% with L. tropica. Of the 60 rodents examined, 25 (41.6% harbored the Leishmania infection; 21 were infected with L. major and 4 with L. turanica. From 49 suspected VL, 29 were positive by direct agglutination test (DAT, whereas microscopy detected parasite in bone marrow of 25 and culture in 28 of the patients. Two VL patients were infected with L. tropica and 26 with L. infantum. Of the 80 domestic dogs, 56 showed anti-Leishmania antibodies with DAT. Of these, 55 were positive by both microscopy and culture. Molecular identity, obtained only for 47 samples, revealed L. infantum in 43 and L. tropica in 4 dogs. The polymorphisms among L. tropica and L. major isolates were 3.6% and 7.3%; the rate among human and canine VL isolates was 2.8% and 9.8%, respectively. Our results showed that at least four different Leishmania species with various polymorphisms circulate among humans and animal hosts in Iran.

  3. Isolation and molecular identification of Mycobacterium from commercially available pasteurized milk and raw milk samples collected from two infected cattle farms in Alborz Province, Iran.

    Science.gov (United States)

    Eftekhari, Mohsen; Mosavari, Nader

    2016-12-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is an etiological agent of Johne's disease in ruminant including cattle, sheep and goats. This disease is considered an economically important disease in cattle. Animals with paratuberculosis shed viable MAP, particularly in their milk and feces. MAP may be involved in the development of Crohn's disease in humans through the consumption of contaminated milk and dairy products. Common methods of pasteurization are not enough to kill all MAP present in the milk and the bacterium has been isolated from raw milk, pasteurized milk and cheese samples. The purpose of this study was to evaluate two different methods for detecting MAP in milk and milk products. We analyzed the commonly used methods such as culture and molecular biology for identification of MAP. For this study, 50 milk samples from cows with suspected Johne's disease located in two dairy farms and 10 commercially available pasteurized milk and cheese samples from the market in Karaj city, Iran were selected. Following Ziehl-Neelsen staining of milk samples, direct microscopic detection of MAP was performed. All milk samples were centrifuged, and the concentrated samples were decontaminated using hexadecyl pyridinium chloride. The decontaminated milk suspensions were washed three times by centrifuging, and the collected filtrates were cultivated on Herrold's egg yolk medium enriched by Mycobactin J. Finally, identification and confirmation of isolates to MAP was performed using IS900-nested polymerase chain reaction (PCR). According to the obtained results by culture and PCR methods, none of the pasteurized milk and cheese samples showed the presence of MAP. However, 10% of the tested raw milk samples collected from suspected cattle showed the presence of MAP by both culture and PCR methods. Culture and PCR methods are reliable for identification of MAP from milk samples. Copyright © 2016.

  4. Morphological and molecular identification of Phytophthora species isolated from the rhizosphere of declining oak trees in Krotoszyn Plateau

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    Tkaczyk Miłosz

    2017-01-01

    Full Text Available The following paper presents the results on the determination of the diversity of species from the Phytophthora genus occurring in the declining oak stands in Krotoszyn Plateau in Poland. From the 50s of the last century, significant deterioration of oak health was observed in these stands, and Phytophthora species were suggested as one of the factors of the decline. In order to determine the presence of pathogenic organisms from the Phytophthora genus in these stands, 180 rhizosphere soil samples from three forest districts throughout the Krotoszyn Plateau were collected and subjected to the isolation method. Phytophthora species were consistently isolated from all the sampled stands, and 194 isolates from 111 positive samples were obtained. However, 150 (77% and 44 (23% isolates originated from the samples taken under the symptomatic and asymptomatic trees, respectively. All the obtained isolates were morphologically classified using the light and scanning electron microscopy and divided into morphological groups. Genomic DNA was isolated from selected isolates representing each group, ITS regions were amplified and sequence analyses were performed. In total, four different Phytophthora species were detected, including P. cactorum, P. plurivora, P. quercina and P. europaea. The most often isolated species were P. cactorum and P. plurivora. This is the first report of P. europaea in oak stands in Poland.

  5. Characterization of Phosphate Solubilizing Bacteria in Sediments from a Shallow Eutrophic Lake and a Wetland: Isolation, Molecular Identification and Phosphorus Release Ability Determination

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    Jie Tang

    2010-11-01

    Full Text Available The transformation of phosphorus (P is a major factor of lake eutrophication, and phosphate releasing bacteria play an important role in the release process. Experiments were conducted to investigate P content and characterize phosphate solubilizing bacterial composition at the molecular level in a shallow eutrophic lake and a wetland. Results showed that P concentrations were relatively high and derived from agricultural runoff and domestic or industrial pollution. Enumeration and molecular identification of these strains indicated that these bacterial groups were abundant in the ecosystem and various kinds of bacteria participated in the phosphorus release process. Twelve phosphate solubilizing bacteria, including eight organic P-solubilizing bacteria (OPBs and four inorganic P-solubilizing bacteria (IPBs, which belonged to three different families, were isolated and identified. Cupriavidus basilensis was found for the first time to have the ability to mineralize organic P (OP. Laboratory tests on P release ability revealed that IPBs were more effective at releasing P than OPBs. The most efficient IPB strain could accumulate over 170 mg·L-1 orthophosphate, while the equivalent OPB strain only liberated less than 4 mg·L-1 orthophosphate in liquid culture. The results obtained from this investigation should help clarify the roles of microorganisms in aquatic systems and the mechanisms of eutrophication.

  6. Evaluation of molecular markers for Phytophthora ramorum detection and identification: Testing for specificity using a standardized library of isolates

    Science.gov (United States)

    F.N. Martin; M.D. Coffey; K. Zeller; R.C. Hamelin; P. Tooley; M. Garbelotto; K.J.D. Hughes; T. Kubisiak; G.J. Bilodeau; L. Levy; C. Blomquist; P.H. Berger

    2009-01-01

    Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World...

  7. First report of Streptococcus agalactiae isolated from Oreochromis niloticus in Piura, Peru: Molecular identification and histopathological lesions

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    Yessica Ortega Asencios

    2016-11-01

    Full Text Available The aim of this study was to identify the bacterium Streptococcus agalactiae isolated in farmed Nile tilapia (Oreochromis niloticus from Piura, Peru and to characterize the histopathological lesions caused by this pathogen. Sixteen tilapias were sampled with clinic signs of the disease such as erratic swimming, exophthalmia and haemorrhages on the body and fins. Qualitative PCR in real time and histopathological analysis were performed. Nine fishes positives to S. agalactiae were found. The main histopathological findings were fibrinosuppurative epicarditis, periesplenitis, meninigitis and panophtaltmitis with predominance of mononuclear infiltration in all tissues. The correlation between qPCR and histopathological findings demonstrated nine fish (prevalence of 56.25% with Cq lower than 30, associated to high degree of tissue injuries. This study reports the first isolation of S. agalactiae by PCR in real time in tilapia farmed in Peru and characterizes the major histopathological changes caused by this bacterium.

  8. Isolation and identification of Salmonella spp. in drinking water, streams, and swine wastewater by molecular techniques in Taiwan

    Science.gov (United States)

    Kuo, C.; Hsu, B.; Shen, T.; Tseng, S.; Tsai, J.; Huang, K.; Kao, P.; Chen, J.

    2013-12-01

    Salmonella spp. is a common water-borne pathogens and its genus comprises more than 2,500 serotypes. Major pathogenic genotypes which cause typhoid fever, enteritis and other intestinal-type diseases are S. Typhimurium, S. Enteritidis, S. Stanley, S. Agona, S.Albany, S. Schwarzengrund, S. Newport, S. Choleraesuis, and S. Derby. Hence, the identification of the serotypes of Salmonella spp. is important. In the present study, the analytical procedures include direct concentration method, non-selective pre-enrichment method and selective enrichment method of Salmonella spp.. Both selective enrichment method and cultured bacteria were detected with specific primers of Salmonella spp. by polymerase chain reaction (PCR). At last, the serotypes of Salmonella were confirmed by using MLST (multilocus sequence typing) with aroC, dnaN, hemD, hisD, purE, sucA, thrA housekeeping genes to identify the strains of positive samples. This study contains 121 samples from three different types of water sources including the drinking water (51), streams (45), and swine wastewater (25). Thirteen samples with positive invA gene are separated from culture method. The strains of these positive samples which identified from MLST method are S. Albany, S. Typhimurium, S. Newport, S. Bareilly, and S. Derby. Some of the serotypes, S. Albany, S. Typhimurium and S. Newport, are highly pathogenic which correlated to human diarrhea. In our results, MLST is a useful method to identify the strains of Salmonella spp.. Keywords: Salmonella, PCR, MLST.

  9. Molecular Identification and Susceptibility of Trichosporon Species Isolated from Clinical Specimens in Qatar : Isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp nov.

    NARCIS (Netherlands)

    Taj-Aldeen, Saad J.; Al-Ansari, Nasser; El Shafei, Sittana; Meis, Jacques F.; Curfs-Breuker, Ilse; Theelen, Bart; Boekhout, Teun

    Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid

  10. Molecular identification and susceptibility of Trichosporon species isolated from clinical specimens in Qatar: isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov

    NARCIS (Netherlands)

    Taj-Aldeen, S.J.; Al-Ansari, N.; El Shafei, S.; Meis, J.F.; Curfs-Breuker, I.; Theelen, B.J.F.; Boekhout, T.

    2009-01-01

    Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid

  11. Identification of three new isolates of Tomato spotted wilt virus from different hosts in China: molecular diversity, phylogenetic and recombination analyses.

    Science.gov (United States)

    Zhang, Zhenjia; Wang, Deya; Yu, Chengming; Wang, Zenghui; Dong, Jiahong; Shi, Kerong; Yuan, Xuefeng

    2016-01-14

    Destructive diseases caused by Tomato spotted wilt virus (TSWV) have been reported associated with many important plants worldwide. Recently, TSWV was reported to infect different hosts in China. It is of value to clone TSWV isolates from different hosts and examine diversity and evolution among different TSWV isolates in China as well as worldwide. RT-PCR was used to clone the full-length genome (L, M and S segments) of three new isolates of TSWV that infected different hosts (tobacco, red pepper and green pepper) in China. Identity of nucleotide and amino acid sequences among TSWV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. Whole-genome sequences of three new TSWV isolates in China were determined. Together with other available isolates, 29 RNA L, 62 RNA M and 66 RNA S of TSWV isolates were analyzed for molecular diversity, phylogenetic and recombination events. This analysis revealed that the entire TSWV genome, especially the M and S RNAs, had major variations in genomic size that mainly involve the A-U rich intergenic region (IGR). Phylogenetic analyses on TSWV isolates worldwide revealed evidence for frequent reassortments in the evolution of tripartite negative-sense RNA genome. Significant numbers of recombination events with apparent 5' regional preference were detected among TSWV isolates worldwide. Moreover, TSWV isolates with similar recombination events usually had closer relationships in phylogenetic trees. All five Chinese TSWV isolates including three TSWV isolates of this study and previously reported two isolates can be divided into two groups with different origins based on molecular diversity and phylogenetic analysis. During their evolution, both reassortment and recombination played roles. These results suggest that recombination could be an important mechanism in the evolution of multipartite RNA viruses, even negative

  12. Molecular Identification of Hookworm Isolates in Humans, Dogs and Soil in a Tribal Area in Tamil Nadu, India.

    Directory of Open Access Journals (Sweden)

    Santosh George

    2016-08-01

    Full Text Available Hookworms (Necator americanus and Ancylostoma duodenale remain a major public health problem worldwide. Infections with hookworms (e.g., A. caninum, A. ceylanicum and A. braziliense are also prevalent in dogs, but the role of dogs as a reservoir for zoonotic hookworm infections in humans needs to be further explored.As part of an open-label community based cluster-randomized trial in a tribal area in Tamil Nadu (India; 2013-2015, a total of 143 isolates of hookworm eggs from human stool were speciated based on a previously described PCR-RFLP methodology. The presence of hookworm DNA was confirmed in 119 of 143 human samples. N. americanus (100% was the most prevalent species, followed by A. caninum (16.8% and A. duodenale (8.4%. Because of the high prevalence of A. caninum in humans, dog samples were also collected to assess the prevalence of A. caninum in dogs. In 68 out of 77 canine stool samples the presence of hookworms was confirmed using PCR-RFLP. In dogs, both A. caninum (76.4% and A. ceylanicum (27.9% were identified. Additionally, to determine the contamination of soil with zoonotic hookworm larvae, topsoil was collected from defecating areas. Hookworm DNA was detected in 72 out of 78 soil samples that revealed presence of hookworm-like nematode larvae. In soil, different hookworm species were identified, with animal hookworms being more prevalent (A. ceylanicum: 60.2%, A. caninum: 29.4%, A. duodenale: 16.6%, N. americanus: 1.4%, A. braziliense: 1.4%.In our study we regularly detected the presence of A. caninum DNA in the stool of humans. Whether this is the result of infection is currently unknown but it does warrant a closer look at dogs as a potential reservoir.

  13. Molecular Identification of Hookworm Isolates in Humans, Dogs and Soil in a Tribal Area in Tamil Nadu, India.

    Science.gov (United States)

    George, Santosh; Levecke, Bruno; Kattula, Deepthi; Velusamy, Vasanthakumar; Roy, Sheela; Geldhof, Peter; Sarkar, Rajiv; Kang, Gagandeep

    2016-08-01

    Hookworms (Necator americanus and Ancylostoma duodenale) remain a major public health problem worldwide. Infections with hookworms (e.g., A. caninum, A. ceylanicum and A. braziliense) are also prevalent in dogs, but the role of dogs as a reservoir for zoonotic hookworm infections in humans needs to be further explored. As part of an open-label community based cluster-randomized trial in a tribal area in Tamil Nadu (India; 2013-2015), a total of 143 isolates of hookworm eggs from human stool were speciated based on a previously described PCR-RFLP methodology. The presence of hookworm DNA was confirmed in 119 of 143 human samples. N. americanus (100%) was the most prevalent species, followed by A. caninum (16.8%) and A. duodenale (8.4%). Because of the high prevalence of A. caninum in humans, dog samples were also collected to assess the prevalence of A. caninum in dogs. In 68 out of 77 canine stool samples the presence of hookworms was confirmed using PCR-RFLP. In dogs, both A. caninum (76.4%) and A. ceylanicum (27.9%) were identified. Additionally, to determine the contamination of soil with zoonotic hookworm larvae, topsoil was collected from defecating areas. Hookworm DNA was detected in 72 out of 78 soil samples that revealed presence of hookworm-like nematode larvae. In soil, different hookworm species were identified, with animal hookworms being more prevalent (A. ceylanicum: 60.2%, A. caninum: 29.4%, A. duodenale: 16.6%, N. americanus: 1.4%, A. braziliense: 1.4%). In our study we regularly detected the presence of A. caninum DNA in the stool of humans. Whether this is the result of infection is currently unknown but it does warrant a closer look at dogs as a potential reservoir.

  14. Visual Analysis of DNA Microarray Data for Accurate Molecular Identification of Non-albicans Candida Isolates from Patients with Candidemia Episodes

    OpenAIRE

    De Luca Ferrari, Michela; Ribeiro Resende, Mariângela; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Gonoi, Tohru; Mikami, Yuzuru; Tominaga, Kenichiro; Kamei, Katsuhiko; Zaninelli Schreiber, Angelica; Trabasso, Plinio; Moretti, Maria Luiza

    2013-01-01

    The performance of a visual slide-based DNA microarray for the identification of non-albicans Candida spp. was evaluated. Among 167 isolates that had previously been identified by Vitek 2, the agreement between DNA microarray and sequencing results was 97.6%. This DNA microarray platform showed excellent performance.

  15. Molecular identification of Candida species isolated from cases of neonatal candidemia using polymerase chain reaction-restriction fragment length polymorphism in a tertiary care hospital

    Directory of Open Access Journals (Sweden)

    Akeela Fatima

    2017-01-01

    Full Text Available Context: Candida spp. is an emerging cause of bloodstream infections worldwide. Delay in speciation of Candida isolates by conventional methods and resistance to antifungal drugs in various Candida species are responsible for the increase in morbidity and mortality due to candidemia. Hence, the rapid identification of Candida isolates is very important for the proper management of patients with candidemia. Aims: The aim was to re-evaluate the identification of various Candida spp. by polymerase chain reaction (PCR-restriction fragment length polymorphism (RFLP and to evaluate the accuracy, speed, and cost of phenotypic methodology versus PCR-RFLP. Settings and Design: Hospital-based cross-sectional study. Materials and Methods: Ninety consecutive clinical isolates of seven Candida species, isolated from blood of neonates and identified by routine phenotypic methods, were re-evaluated using universal primers internal transcribed spacer 1 (ITS1 and ITS4 for PCR amplification and Msp I restriction enzyme for RFLP. Statistical Analysis Used: Kappa test for agreement. Results: The results of PCR-RFLP were 100% in agreement with those obtained using conventional phenotypic methods. Identification could be achieved within 3 work days by both the methods. Our routine methods proved to be cost effective than PCR-RFLP. Conclusions: We can continue with our routine phenotypic methods and PCR-RFLP can be used for periodic quality control or when conventional methods fail to identify a species.

  16. Molecular identification of nontuberculous mycobacteria isolated from pyogenic bovine tissues in South Darfur State and Alsabalouga slaughterhouse at Omdurman area, Sudan

    Directory of Open Access Journals (Sweden)

    A.E. El Tigani-Asil

    2014-02-01

    Full Text Available This study identified nontuberculous mycobacteria (NTM recovered from bovine pyogenic affections obtained at necropsy using the molecular target 16S-23S rDNA internal transcribed spacer region. Postmortem inspection of cattle was conducted at South Darfur State abattoirs and Alsabalouga Slaughterhouse at Omdurman area during 2007-2009. Specimens were examined for the presence of acid fast bacteria (AFB using microscopic and standard culturing techniques. AFB were identified phenotypically and confirmed by 16S-23S rDNA ITS. Fifty nine NTM were recovered and confirmed as acid fast filaments out of 165 positive AFB specimens, of which 52 isolates were identified as bovine farcy causative agents, while 7 cultures were excluded due to drying. 16S-23S rDNA ITS of NTM revealed three different amplicons 500 bp. (32 isolates, 550 bp. (2 isolates and 600 bp. (14 isolates. Four isolates were contaminated.

  17. Molecular identification of nontuberculous mycobacteria isolated from pyogenic bovine tissues in South Darfur State and Alsabalouga slaughterhouse at Omdurman area, Sudan.

    Science.gov (United States)

    Tigani-Asil, A E El; Sanousi, S M El; Aljameel, M A; Beir, H El; Adam, A; Abdallatif, M M; Hamid, M E

    2014-01-01

    This study identified nontuberculous mycobacteria (NTM) recovered from bovine pyogenic affections obtained at necropsy using the molecular target 16S-23S rDNA internal transcribed spacer region. Postmortem inspection of cattle was conducted at South Darfur State abattoirs and Alsabalouga Slaughterhouse at Omdurman area during 2007-2009. Specimens were examined for the presence of acid fast bacteria (AFB) using microscopic and standard culturing techniques. AFB were identified phenotypically and confirmed by 16S-23S rDNA ITS. Fifty nine NTM were recovered and confirmed as acid fast filaments out of 165 positive AFB specimens, of which 52 isolates were identified as bovine farcy causative agents, while 7 cultures were excluded due to drying. 16S-23S rDNA ITS of NTM revealed three different amplicons 500 bp. (32) isolates, 550 bp. (2) isolates and 600 bp. (14) isolates. Four isolates were contaminated.

  18. ISOLATION AND IDENTIFICATION OF FUNGI RESPONSIBLE FOR ...

    African Journals Online (AJOL)

    User

    technique. The diseased plant leaves were taken to the laboratory for culture, isolation, and identification ... The tree is native to Asia particularly eastern India, Burma, and the Andaman ..... in Southeastern Nigeria and Biological. Control with ...

  19. Molecular identification of Aspergillus spp. isolated from coffee beans Identificação molecular de Aspergillus spp. isolados de grãos de café

    Directory of Open Access Journals (Sweden)

    Marciane Magnani

    2005-01-01

    Full Text Available Some species belonging to the genus Aspergillus are potential producers of ochratoxin A (OA, a mycotoxin with nephrotoxic, immunosuppressive, teratogenic and carcinogenic effects. The aim of the present study was to identify the species of Aspergillus that contaminate the inside of coffee beans collected in the stage of maturation and drying, from 16 producing areas located in the northern region of the State of Paraná, in the South of Brazil. A total of 108 isolates of Aspergillus spp. was identified at the species level, by sequencing the internal transcribed spacer (ITS1-5.8S-ITS2 of ribosomal DNA (rDNA. The results revealed the presence of potentially ochratoxigenic species in 82% of the geographic regions studied, among which Aspergillus niger was the species most frequently detected, followed by A. ochraceus and A. carbonarius. The presence of A. carbonarius in immature coffee fruits harvested from trees is reported for the first time.Algumas espécies pertencentes ao gênero Aspergillus possuem potencial para produção de Ocratoxina A (OA, uma micotoxina de efeitos nefrotóxicos, imunossupressivos, teratogênicos e carcinogênicos. Com o objetivo de identificar as espécies de Aspergillus que contaminam o interior de grãos de café, foram coletadas amostras em diferentes estádios de maturação do produto, em 16 propriedades produtoras do norte do estado do Paraná. Um total de 108 isolados de Aspergillus spp. foram identificados ao nível de espécie, pelo sequenciamento dos espaços internos transcritos (ITS1-5,8S-ITS2 do DNA ribossomal (rDNA. Os resultados revelaram a presença de espécies potencialmente ocratoxigênicas em 82% das regiões analisadas, sendo dentre estas, Aspergillus niger a espécie mais freqüentemente detectada,seguida por A. ochraceus, e A. carbonarius. É relatada pela primeira vez a presença de A. carbonarius em frutos de café coletados na árvore.

  20. Site identification presentation: Basalt Waste Isolation Project

    International Nuclear Information System (INIS)

    1979-11-01

    The final step in the site identification process for the Basalt Waste Isolation Project is described. The candidate sites are identified. The site identification methodology is presented. The general objectives which must be met in selecting the final site are listed. Considerations used in the screening process are also listed. Summary tables of the guidelines used are included

  1. Evaluation of matrix-assisted laser desorption/ionization time-of-fight mass spectrometry for identification of 345 clinical isolates of Aspergillus species from 11 Korean hospitals: comparison with molecular identification.

    Science.gov (United States)

    Park, Ju Heon; Shin, Jong Hee; Choi, Min Ji; Choi, Jin Un; Park, Yeon-Joon; Jang, Sook Jin; Won, Eun Jeong; Kim, Soo Hyun; Kee, Seung Jung; Shin, Myung Geun; Suh, Soon Pal

    2017-01-01

    We evaluated the ability of the Filamentous Fungi Library 1.0 of the MALDI-TOF MS Biotyper system to identify 345 clinical Aspergillus isolates from 11 Korean hospitals. Compared with results of the internal transcribed spacer region sequencing, the frequencies of correct identification at the species-complex level were 94.5% and 98.8% with cutoff values of 2.0 and 1.7, respectively. Compared with results of β-tubulin gene sequencing, the frequencies of correct identification at the species level were 96.0% (cutoff 2.0) and 100% (cutoff 1.7) for 303 Aspergillus isolates of five common, non-cryptic species, but only 4.8% (cutoff 1.7) and 0% (cutoff 2.0) for 42 Aspergillus isolates of six cryptic species (identifiable by β-tubulin or calmodulin sequencing). These results show that the MALDI Biotyper using the Filamentous Fungi Library version 1.0 enables reliable identification of the majority of common clinical Aspergillus isolates, although the database should be expanded to facilitate identification of cryptic species. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya

    Science.gov (United States)

    Azwai, S.M.; Alfallani, E.A.; Abolghait, S.K.; Garbaj, A.M.; Naas, H.T.; Moawad, A.A.; Gammoudi, F.T.; Rayes, H.M.; Barbieri, I.; Eldaghayes, I.M.

    2016-01-01

    The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk). Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6 % of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 ×104 CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 ×104 CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products. PMID:27004169

  3. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya

    Directory of Open Access Journals (Sweden)

    S.M. Azwai

    2016-03-01

    Full Text Available The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk. Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6% yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS with culture characteristics of Vibrio spp. More than half (n=27 of processed seafood samples (n=46 yielded colonies on TCBS, while only 44.6% of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 х104 CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 х104 CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9% were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products.

  4. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya.

    Science.gov (United States)

    Azwai, S M; Alfallani, E A; Abolghait, S K; Garbaj, A M; Naas, H T; Moawad, A A; Gammoudi, F T; Rayes, H M; Barbieri, I; Eldaghayes, I M

    2016-01-01

    The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk). Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6 % of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 ×10(4) CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 ×10(4) CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products.

  5. PCR-based isolation and identification of full-length low-molecular-weight glutenin subunit genes in bread wheat (Triticum aestivum L.).

    Science.gov (United States)

    Zhang, Xiaofei; Liu, Dongcheng; Jiang, Wei; Guo, Xiaoli; Yang, Wenlong; Sun, Jiazhu; Ling, Hongqing; Zhang, Aimin

    2011-12-01

    Low-molecular-weight glutenin subunits (LMW-GSs) are encoded by a multi-gene family and are essential for determining the quality of wheat flour products, such as bread and noodles. However, the exact role or contribution of individual LMW-GS genes to wheat quality remains unclear. This is, at least in part, due to the difficulty in characterizing complete sequences of all LMW-GS gene family members in bread wheat. To identify full-length LMW-GS genes, a polymerase chain reaction (PCR)-based method was established, consisting of newly designed conserved primers and the previously developed LMW-GS gene molecular marker system. Using the PCR-based method, 17 LMW-GS genes were identified and characterized in Xiaoyan 54, of which 12 contained full-length sequences. Sequence alignments showed that 13 LMW-GS genes were identical to those found in Xiaoyan 54 using the genomic DNA library screening, and the other four full-length LMW-GS genes were first isolated from Xiaoyan 54. In Chinese Spring, 16 unique LMW-GS genes were isolated, and 13 of them contained full-length coding sequences. Additionally, 16 and 17 LMW-GS genes in Dongnong 101 and Lvhan 328 (chosen from the micro-core collections of Chinese germplasm), respectively, were also identified. Sequence alignments revealed that at least 15 LMW-GS genes were common in the four wheat varieties, and allelic variants of each gene shared high sequence identities (>95%) but exhibited length polymorphism in repetitive regions. This study provides a PCR-based method for efficiently identifying LMW-GS genes in bread wheat, which will improve the characterization of complex members of the LMW-GS gene family and facilitate the understanding of their contributions to wheat quality.

  6. Molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa.

    Science.gov (United States)

    Visintin, Simonetta; Alessandria, Valentina; Valente, Antonio; Dolci, Paola; Cocolin, Luca

    2016-01-04

    Yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) populations, isolated from cocoa bean heap and box fermentations in West Africa, have been investigated. The fermentation dynamicswere determined by viable counts, and 106 yeasts, 105 LAB and 82 AAB isolateswere identified by means of rep-PCR grouping and sequencing of the rRNA genes. During the box fermentations, the most abundant species were Saccharomyces cerevisiae, Candida ethanolica, Lactobacillus fermentum, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii, while S. cerevisiae, Schizosaccharomyces pombe, Hanseniaspora guilliermondii, Pichia manshurica, C. ethanolica, Hanseniaspora uvarum, Lb. fermentum, Lb. plantarum, A. pasteurianus and Acetobacter lovaniensis were identified in the heap fermentations. Furthermore, the most abundant species were molecularly characterized by analyzing the rep-PCR profiles. Strains grouped according to the type of fermentations and their progression during the transformation process were also highlighted. The yeast, LAB and AAB isolates were physiologically characterized to determine their ability to grow at different temperatures, as well as at different pH, and ethanol concentrations, tolerance to osmotic stress, and lactic acid and acetic acid inhibition. Temperatures of 45 °C, a pH of 2.5 to 3.5, 12% (v/v) ethanol and high concentrations of lactic and acetic acid have a significant influence on the growth of yeasts, LAB and AAB. Finally, the yeastswere screened for enzymatic activity, and the S. cerevisiae, H. guilliermondii, H. uvarumand C. ethanolica species were shown to possess several enzymes that may impact the quality of the final product.

  7. Three new insertion sequence elements ISLdl2, ISLdl3, and ISLdl4 in Lactobacillus delbrueckii: isolation, molecular characterization, and potential use for strain identification.

    Science.gov (United States)

    Ravin, Victor; Alatossava, Tapani

    2003-05-01

    A group of new insertion sequence (IS) elements, ISLdl2, ISLdl3, and ISLdl4, from Lactobacillus delbrueckii subsp. lactis ATCC 15808 was isolated, characterized, and used for strain identification together with ISLdl1, recently characterized as an L. delbrueckii IS element belonging to the ISL3 family. ISLdl2 was 1367 bp in size and had a 24 bp IR and an 8 bp DR. The single ORF of ISLdl2 encoded a protein of 392 aa similar to transposases of the IS256 family. ISLdl3 had a single ORF encoding a protein of 343 aa similar to transposases of the IS30 family. Finally, ISLdl4 had a single ORF encoding a protein of 406 aa and displayed homology to the transposases of the IS110 family. ISLdl4 was only slight different from ISL4 (Accession No. AY040213). ISLdl1, ISLdl2, and ISLdl4 were present in all of the 10 L. delbrueckii subsp. lactis and subsp. delbrueckii strains tested, as well as in three of the 11 L. delbrueckii subsp. bulgaricus strains tested. ISLdl3 was present only in four closely related strains of L. delbrueckii subsp. lactis. These IS elements were not observed in Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, or Lactobacillus plantarum. A cluster of IS elements, ISLdl1, ISLdl2, ISLdl3, ISLdl4, and ISL6, was observed in L. delbrueckii subsp. lactis strain ATCC 15808. Within this cluster, ISLdl4 was inserted into ISLdl1 between the left IR and the start codon of ORF455, encoding a putative transposase. Most of the integration sites of the IS elements were strain-specific. We have observed that IS elements can migrate from one strain to another as integral parts of bacterial DNA by using phage LL-H as a vehicle. We demonstrate for the first time that inverse PCR and vectorette PCR methods with primers based on sequences of the IS elements could be used for identification of L. delbrueckii strains.

  8. Isolement et identification des levures.

    Directory of Open Access Journals (Sweden)

    S. DOMERCQ

    1967-06-01

    Full Text Available La fermentation du moût de raisin est assurée par les levures qui se trouvent sur la pellicule du raisin, sur les rafles, et aussi, par celles qui envahissent le matériel de vendange et de vinification dès que la récolte est commencée. L'apparition deLa fermentation du moût de raisin est assurée par les levures qui se trouvent sur la pellicule du raisin, sur les rafles, et aussi, par celles qui envahissent le matériel de vendange et de vinification dès que la récolte est commencée. L'apparition des signes de la fermentation est spontanée. La fermentation est rapide dans la majorité des cas, si elle devient trop violente et que la température s'élève dangeureusement, il faut la « canaliser », la calmer. Des accidents peuvent se produire; des arrêts de fermentation laissant des sucres non fermentés pouvant être attaqués par d'autres microorganismes, des développements de levures formant des substances nuisibles à la qualité; par la suite, il s'agit d'obtenir la stabilité du vin, en particulier, éviter les refermentations des vins doux. L'étude de ces problèmes nécessite une étude préliminaire, celle des levures responsables de la fermentation alcoolique du jus de raisin, qui ne peut être faite que sur des cultures pures, isolées au préalable à l'aide de prélèvements soigneusement réalisés.s signes de la fermentation est spontanée. La fermentation est rapide dans la majorité des cas, si elle devient trop violente et que la température s'élève dangeureusement, il faut la « canaliser », la calmer. Des accidents peuvent se produire; des arrêts de fermentation laissant des sucres non fermentés pouvant être attaqués par d'autres microorganismes, des développements de levures formant des substances nuisibles à la qualité; par la suite, il s'agit d'obtenir la stabilité du vin, en particulier, éviter les refermentations des vins doux. L'étude de ces problèmes nécessite une étude pr

  9. Isolation and identification of Pseudomonas azotoformans for induced calcite precipitation.

    Science.gov (United States)

    Heidari Nonakaran, Siamak; Pazhouhandeh, Maghsoud; Keyvani, Abdullah; Abdollahipour, Fatemeh Zahra; Shirzad, Akbar

    2015-12-01

    Biomineralization is a process by which living organisms produce minerals. The extracellular production of these biominerals by microbes has potential for various bioengineering applications. For example, crack remediation and improvement of durability of concrete is an important goal for engineers and biomineral-producing microbes could be a useful tool in achieving this goal. Here we report the isolation, biochemical characterization and molecular identification of Pseudomonas azotoformans, a microbe that produces calcite and which potentially be used to repair cracks in concrete structures. Initially, 38 bacterial isolates were isolated from soil and cements. As a first test, the isolates were screened using a urease assay followed by biochemical tests for the rate of urea hydrolysis, calcite production and the insolubility of calcite. Molecular amplification and sequencing of a 16S rRNA fragment of selected isolates permitted us to identify P. azotoformans as a good candidate for preparation of biotechnological concrete. This species was isolated from soil and the results show that among the tested isolates it had the highest rate of urea hydrolysis, produced the highest amount of calcite, which, furthermore was the most adhesive and insoluble. This species is thus of interest as an agent with the potential ability to repair cracks in concrete.

  10. Phenotypic and Molecular Identification of Bacteria Involved in Decubitus Ulcers

    Directory of Open Access Journals (Sweden)

    Mehran Dolati

    2017-07-01

    Full Text Available Background:    Bacterial secondary infection of pressure ulcers (bedsores, so called as decubitus ulcers, leads to ulcer development and it interferes with the healing process. Thus, such infections can be lethal due to the sepsis if no constructive medicinal measures regarded. Drug resistance of bacteria in pressure ulcers leads to healing inhibition. Molecular identification of bacteria involved in such infections seem necessary as culture and phenotypic approaches may result in misidentification. . The purpose of this study was to isolate and identify aerobic bacteria detected in bedsores in three Hospitals: Rasool-e-Akram, Imam Hossein and Tajrish Shohada Hospitals, Tehran, Iran.Methods:    To this end, decubitus ulcer samples of 49 patients were obtained using sterile swabs. After direct microscopic examination, the swabs were used to streak BHI agar plates supplemented with %5 defibrinated sheep blood for enrichment of probable aerobic cultures. Bacterial isolates diagnosed by biochemical tests. Antibiotic susceptibility of the isolates determined based on CLSI guideline. For molecular identification, PCR amplification of the 16S rRNA gene performed using Eubacterial universal primers. Then, the PCR products were sequenced and the nucleotide sequences of the PCR products were analyzed by BLASTN similarity search program available at NCBI. Results:   Among the isolates, Pseudomonas aeruginosa (36% had the highest frequency, followed by Staphylococcus aureus (32% and Escherichia coli (30%. The frequencies of Klebsiella pneumonia and Proteus spp. were 10% and 8%, respectively. Most of the isolated bacteria showed a widespread antibiotic resistance. Molecular identification of the bacterial isolates resulted in 6 isolates of Escherichia coli, two isolates of each of Proteus mirabilis and Shigella spp., 4 isolates of Enterobacter cloacae, and 1 isolate of each of Cronobacter sakazakii and Morganella morganii.Conclusion:

  11. Isolation and identification of Staphylococcus sp. in powdered infant milk

    Science.gov (United States)

    Palilu, Prayolga Toban; Budiarso, Tri Yahya

    2017-05-01

    Staphylococcus sp. is one of the most dangerous bacteria that could cause food poisoning. It is a pathogenic bacterium which is able to produce enterotoxin in foods. Milk is an ideal growth medium for Staphylococcus sp., that may cause problem if it is to be consumed, especially by infant. It is the objective of this research to detect the presence of Staphylococcus sp. in powdered infant milk. As many as 14 samples obtained from market were used as samples for bacterial isolation. The isolation were done by employing enrichment step on BHI-broth, continued with Baird-Parker Agar which will produce a typical colony. It is then picked and grown on Mannitol Salt Agar, and gram staining, coagulase assay, and fermentation tests. The confirmation step was done by using API-Staph which gives the identification of Staphylococcus hemoliticus, Staphylococcus aureus and Staphylococcus epidermidis, with a percentage of identity ranging from 65.9-97.7%. Two isolates with the highest identification similarity values were then picked for molecular detection. A PCR primer pair targeting gene coding for enterotoxin A was used, and it gives positive result for the two isolates being tested. It is then concluded that the two isolates belong to Staphylococcus sp., and further research need to be done to correctly identify these isolates.

  12. Molecular Identification of Methicillin-Resistant Staphylococcus ...

    African Journals Online (AJOL)

    We use the molecular techniques of PCR and PFGE to identify MRSA from clinical isolates of Staphylococcus aureus causing infections among hospitalized patients in Benin-City, Nigeria. A total of 36 isolates were obtained from the University of Benin Teaching Hospital between July-September, 2007. The MRSA strains ...

  13. Molecular characterization of antimicrobial susceptibility of Salmonella isolates: First identification of a plasmid carrying qnrD or oqxAB in Taiwan

    Directory of Open Access Journals (Sweden)

    Cheng-Yen Kao

    2017-04-01

    Conclusion: GyrA mutations are the major mechanisms associated with quinolone-resistant Salmonella isolates in Taiwan. Overproduction of efflux pump genes and the presence of qnr and oqxAB play additional roles in reduced susceptibility to quinolones.

  14. Molecular application for identification of polycyclic aromatic hydrocarbons degrading bacteria (PAHD) species isolated from oil polluted soil in Dammam, Saud Arabia.

    Science.gov (United States)

    Ibrahim, Mohamed M; Al-Turki, Ameena; Al-Sewedi, Dona; Arif, Ibrahim A; El-Gaaly, Gehan A

    2015-09-01

    Soil contamination with petroleum hydrocarbon products such as diesel and engine oil is becoming one of the major environmental problems. This study describes hydrocarbons degrading bacteria (PHAD) isolated from long-standing petrol polluted soil from the eastern region, Dammam, Saudi Arabia. The isolated strains were firstly categorized by accessible shape detection, physiological and biochemistry tests. Thereafter, a technique established on the sequence analysis of a 16S rDNA gene was used. Isolation of DNA from the bacterial strains was performed, on which the PCR reaction was carried out. Strains were identified based on 16S rDNA sequence analysis, As follows amplified samples were spontaneously sequenced automatically and the attained results were matched to open databases. Among the isolated bacterial strains, S1 was identified as Staphylococcus aureus and strain S1 as Corynebacterium amycolatum.

  15. Isolation and molecular characterization of phytase producing ...

    African Journals Online (AJOL)

    Isolation and molecular characterization of phytase producing bacteria from Malaysia hot springs. ... The strains were further analyzed in broth culture using sodium phytate as substrate. Among them, strain L3 was selected as the best producer (0.16 U/ml after 72 h of culture). This phytase showed optimal activity at 37 °C ...

  16. Molecular cloning of cellulase genes from indigenous bacterial isolates

    International Nuclear Information System (INIS)

    Jong Bor Chyan; Pauline Liew Woan Ying; Mat Rasol Awang

    2006-01-01

    Indigenous cellulolytic bacterial isolates having high activities in degrading carboxymethyl cellulose (CMC) were isolated from local environments. Identification of these isolates were performed by molecular techniques. By using polymerase chain reaction (PCR) techniques, PCR products encoding cellulase gene were amplified from the total genomic DNAs. Purified PCR product was successfully cloned and expressed in Escherichia coli host system. The complete nucleotide sequences of the cellulase genes determined. The analysis of amino acid sequences deduced from the genes indicated that the cloned DNA fragments show high homology to those of endoglucanase genes of family GH5. All cloned genes consist of an N-terminal signal peptide, a catalytic domain of family 5 glycosyl hydrolase and a cellulose-binding domain of family III. (Author)

  17. Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya

    OpenAIRE

    S.M. Azwai; E.A. Alfallani; S.K. Abolghait; A.M. Garbaj; H.T. Naas; A.A. Moawad; F.T. Gammoudi; H.M. Rayes; I. Barbieri; I.M. Eldaghayes

    2016-01-01

    The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localitie...

  18. Molecular identification and biodegradation of 3-chloropropionic acid (3CP by filamentous fungi-Mucor and Trichoderma species isolated from UTM agricultural land

    Directory of Open Access Journals (Sweden)

    Parvizpour, S.

    2013-01-01

    Full Text Available Aims: This study was carried out to further characterize fungal species that could degrade 3-chloropropionic acid (3CPas sole source of carbon and energy. Methodology and Results: Both fungi were able to grow on 3CP after 10 days on solid minimal media. Based on sequencing of its segment of 18S rRNA these isolates were identified as Mucor sp. SP1 and Trichoderma sp. SP2. The isolated strains were not able to grow on media plates containing 10 mM of 2,2-dichloropropionate (2,2DCP as sole source of carbon. 3CP degradation was observed in liquid minimal medium containing 10 mM 3CP after 18 days cultureperiod. The chloride ion released was detected in both growth medium containing Mucor sp. SP1 and Trichoderma sp. SP2. At least 80% of 10 mM 3CP was utilized in the growth medium. Conclusion, significance and impact of study: Dehalogenase enzyme that can degrade α-chloro-substituted haloalkanoic acids for example 2,2DCP is well studied up to protein crystallization. Very few reports on the degradationof β-chloro-substituted haloalkanoic acids such as 3CP and none from fungi. This study is considered important because it can be compared to that of well-documented α-chloro-substituted haloalkanoic acids degradation. This is the first study to indicate fungal growth on 3CP as sole carbon and energy sources.

  19. Isolation and molecular identification of lactic acid bacteria and Bifidobacterium spp. from faeces of the blue-fronted Amazon parrot in Brazil.

    Science.gov (United States)

    Allegretti, L; Revolledo, L; Astolfi-Ferreira, C S; Chacón, J L; Martins, L M; Seixas, G H F; Ferreira, A J P

    2014-12-01

    In Brazil, the blue-fronted Amazon parrot (Amazona aestiva) is a common pet. The faecal microbiota of these birds include a wide variety of bacterial species, the majority of which belong to the Gram-positive lactic acid bacteria (LAB) clade. The aim of this study was to investigate differences in the diversity and abundance of LAB and Bifidobacterium spp. in the cloacae between wild and captive birds and to select, identify and characterise LAB for consideration as a parrot probiotic. Cloacal swabs were collected from 26 wild and 26 captive birds. Bacterial DNA was extracted, and the 16S rRNA genes were amplified. The numbers of PCR-positive Enterococcus, Pediococcus, and Lactobacillus species isolated from wild and captive birds were significantly different (PLactobacillus, Lactococcus and Bifidobacterium. Enterococcus faecium, Pediococcus pentosaceus, Lactococcus lactis, Lactobacillus coryniformis, Lactobacillus sanfranciscensis and Bifidobacterium bifidum were the most frequently isolated species from all birds. This study increases our understanding of the faecal microbiota, and may help to improve the nutrition and habitat management of captive and wild parrots. The bacterial population identified in the faecal microbiota of clinically healthy wild and captive parrots can serve as a database to analyse variations in the gut microbiota of pathogen-infected parrots and to develop probiotics specific to these genera.

  20. Molecular DNA Analysis in Forensic Identification.

    Science.gov (United States)

    Dumache, Raluca; Ciocan, Veronica; Muresan, Camelia; Enache, Alexandra

    2016-01-01

    Serological and biochemical identification methods used in forensics have several major disadvantages, such as: long time in processing biological sample and lack of sensitivity and specificity. In the last 30 years, DNA molecular analysis has become an important tool in forensic investigations. DNA profiling is based on the short tandem repeats (STR) and aids in human identification from biological samples. Forensic genetics, can provide information on the events which occurred at the crime scene or to supplement other methods of forensic identification. Currently, the methods used in identification are based on polymerase chain reaction (PCR) analyses. This method analyses the autosomal STRs, the Y-chromosome, and the mitochondrial DNA. Correlation of biological samples present at the crime scene with identification, selection, and the probative value factor is therefore the first aspect to be taken into consideration in the forensic genetic analysis. In the last decade, because of the advances in the field of molecular biology, new biomarkers such as: microRNAs (miR), messenger RNA (mRNA), and DNA methylation have been studied and proposed to be used in the forensic identifications of body fluids.

  1. Molecular identification and technological characterization of lactic acid bacteria isolated from fermented kidney beans flours (Phaseolus vulgaris L. and P. coccineus) in northwestern Argentina.

    Science.gov (United States)

    Sáez, Gabriel D; Hébert, Elvira M; Saavedra, Lucila; Zárate, Gabriela

    2017-12-01

    Legumes are an important protein source in developing countries and their flours represent an attractive alternative for the manufacture of gluten free products. In the present study, 4 kidney bean varieties (Alubia, Pallar, Black and Red beans) commonly cultivated in northwestern Argentina, were milled and spontaneously fermented in order to isolate and select autochthonous lactic acid bacteria (LAB) with relevant technological and functional properties for usage as starter cultures. Twelve doughs were fermented with daily back-slopping at 37°C for 6days and evolution of total mesophiles, lactic acid bacteria, and yeasts and molds populations were followed by plate counting. A combination of phenotypic and genotypic methods including (GTG) 5 -based PCR fingerprinting and 16S rRNA gene sequencing were used to differentiate and identify the isolated LAB to species level. LAB counts ranged from around 0.89±0.81 to 8.74±0.03logcfu/g with a pH decline from 6.4 to 3.9 throughout fermentation. Four genera and nine species of LAB: Enterococcus durans, E. faecium, E. mundtii, E. casseliflavus; Lactobacillus rhamnosus, Lactococcus garvieae, Weissella cibaria and W. paramesenteroides were found on kidney beans. Twenty five LAB strains were assessed for their abilities to grow on kidney bean extracts, acidifying capacities (pH and acidification rates), amylolytic, proteolytic, tannase and gallate decarboxylase activities as well as pathogens inhibition by antimicrobials. Based on these properties E. durans CRL 2178 and W. paramesenteroides CRL 2182 were inoculated singly and combined in Alubia kidney bean flour and fermented for 24h at 37°C. LAB strains were beneficial for removing trypsin inhibitors and tannins from sourdoughs and for improving amino acids and phenolics contents, increasing the antioxidant activities of kidney bean matrices. Selected strains have potential as starter cultures for obtaining fermented bean products with high nutritional and functional

  2. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Hana Šuranská

    2016-03-01

    Full Text Available Abstract In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines.

  3. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase

    Directory of Open Access Journals (Sweden)

    Biplab Kumar Dash

    2015-01-01

    Full Text Available A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25% as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37°C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L and sodium lauryl sulfate (0.2 g/L resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50°C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time.

  4. Molecular Identification of a Newly Isolated Bacillus subtilis BI19 and Optimization of Production Conditions for Enhanced Production of Extracellular Amylase.

    Science.gov (United States)

    Dash, Biplab Kumar; Rahman, M Mizanur; Sarker, Palash Kumar

    2015-01-01

    A study was carried out with a newly isolated bacterial strain yielding extracellular amylase. The phylogenetic tree constructed on the basis of 16S rDNA gene sequences revealed this strain as clustered with the closest members of Bacillus sp. and identified as Bacillus subtilis BI19. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Rice flour (1.25%) as a cheap natural carbon source was found to induce amylase production mostly. A combination of peptone and tryptone as organic and ammonium sulfate as inorganic nitrogen sources gave highest yield. Maximum production was obtained after 24 h of incubation at 37 °C with an initial medium pH 8.0. Addition of surfactants like Tween 80 (0.25 g/L) and sodium lauryl sulfate (0.2 g/L) resulted in 28% and 15% increase in enzyme production, respectively. Amylase production was 3.06 times higher when optimized production conditions were used. Optimum reaction temperature and pH for crude amylase activity were 50 °C and 6.0, respectively. The crude enzyme showed activity and stability over a fair range of temperature and pH. These results suggest that B. subtilis BI19 could be exploited for production of amylase at relatively low cost and time.

  5. Extracellular polymeric substances (EPS) producing bacterial strains of municipal wastewater sludge: isolation, molecular identification, EPS characterization and performance for sludge settling and dewatering.

    Science.gov (United States)

    Bala Subramanian, S; Yan, S; Tyagi, R D; Surampalli, R Y

    2010-04-01

    Wastewater treatment plants often face the problems of sludge settling mainly due to sludge bulking. Generally, synthetic organic polymer and/or inorganic coagulants (ferric chloride, alum and quick lime) are used for sludge settling. These chemicals are very expensive and further pollute the environment. Whereas, the bioflocculants are environment friendly and may be used to flocculate the sludge. Extracellular polymeric substances (EPS) produced by sludge microorganisms play a definite role in sludge flocculation. In this study, 25 EPS producing strains were isolated from municipal wastewater treatment plant. Microorganisms were selected based on EPS production properties on solid agar medium. Three types of EPS (slime, capsular and bacterial broth mixture of both slime and capsular) were harvested and their characteristics were studied. EPS concentration (dry weight), viscosity and their charge (using a Zetaphoremeter) were also measured. Bioflocculability of obtained EPS was evaluated by measuring the kaolin clay flocculation activity. Six bacterial strains (BS2, BS8, BS9, BS11, BS15 and BS25) were selected based on the kaolin clay flocculation. The slime EPS was better for bioflocculation than capsular EPS and bacterial broth. Therefore, extracted slime EPS (partially purified) from six bacterial strains was studied in terms of sludge settling [sludge volume index (SVI)] and dewatering [capillary suction time (CST)]. Biopolymers produced by individual strains substantially improved dewaterability. The extracted slime EPS from six different strains were partially characterized. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  6. Isolation, identification and characterization of Bacillus amyloliquefaciens BZ-6, a bacterial isolate for enhancing oil recovery from oily sludge.

    Science.gov (United States)

    Liu, Wuxing; Wang, Xiaobing; Wu, Longhua; Chen, Mengfang; Tu, Chen; Luo, Yongming; Christie, Peter

    2012-06-01

    Over 100 biosurfactant-producing microorganisms were isolated from oily sludge and petroleum-contaminated soil from Shengli oil field in north China. Sixteen of the bacterial isolates produced biosurfactants and reduced the surface tension of the growth medium from 71 to treat oily sludge and the recovery efficiencies of oil from oily sludge were determined. The oil recovery efficiencies of different isolates ranged from 39% to 88%. Bacterial isolate BZ-6 was found to be the most efficient strain and the three phases (oil, water and sediment) were separated automatically after the sludge was treated with the culture medium of BZ-6. Based on morphological, physiological characteristics and molecular identification, isolate BZ-6 was identified as Bacillus amyloliquefaciens. The biosurfactant produced by isolate BZ-6 was purified and analyzed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry. There were four ion peaks representing four different fengycin A homologues. Copyright © 2012. Published by Elsevier Ltd.

  7. Isolation, characterization and identification of actinomycetes from ...

    African Journals Online (AJOL)

    A total of 62 isolates of actinomycetes were isolated from 7 soil samples collected from Agriculture Research Center Semongok, Sarawak. All 62 isolates exhibited a range of colony colours (dark grey, grey, dark brown, brownish, whitish and yellowish white). All the isolates were later purified and subjected to a few ...

  8. Molecular identification of the Sporothrix schenckii complex.

    Science.gov (United States)

    Oliveira, Manoel Marques Evangelista; Almeida-Paes, Rodrigo; Gutierrez-Galhardo, Maria Clara; Zancope-Oliveira, Rosely M

    2014-01-01

    Sporothrix schenckii, an ascomycetous dimorphic organism that for over a century was recognized as the sole agent of sporotrichosis, a subcutaneous mycosis with a worldwide distribution. However, it has been proposed, based on physiologic and molecular aspects, that S. schenckii is a complex of distinct species: Sporothrix brasiliensis, Sporothrix mexicana, Sporothrix globosa, S. schenckii sensu strictu, Sporothrix luriei, and Sporothrix albicans (formerly Sporothrix pallida). Human disease has a broad range of clinical manifestations and can be classified into fixed cutaneous, lymphocutaneous, disseminated cutaneous, and extracutaneous sporotrichosis. The gold standard for the diagnosis of sporotrichosis is the culture; however, serologic, histopathologic and molecular approaches have been recently adopted for the diagnosis of this mycosis. Few molecular methods have been applied to the diagnosis of sporotrichosis to detect S. schenckii DNA from clinical specimens, and to identify Sporothrix spp. in culture. Until now, Sporothrix is the unique clinically relevant dimorphic fungus without an elucidated genome sequence, thus limiting molecular knowledge about the cryptic species of this complex, and the sexual form of all S. schenckii complex species. In this review we shall focus on the current diagnosis of the sporotrichosis, and discuss the current molecular tools applied to the diagnosis and identification of the Sporothrix complex species. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  9. Toward the identification of molecular cogs.

    Science.gov (United States)

    Dziubiński, Maciej; Lesyng, Bogdan

    2016-04-05

    Computer simulations of molecular systems allow determination of microscopic interactions between individual atoms or groups of atoms, as well as studies of intramolecular motions. Nevertheless, description of structural transformations at the mezoscopic level and identification of causal relations associated with these transformations is very difficult. Structural and functional properties are related to free energy changes. Therefore, to better understand structural and functional properties of molecular systems, it is required to deepen our knowledge of free energy contributions arising from molecular subsystems in the course of structural transformations. The method presented in this work quantifies the energetic contribution of each pair of atoms to the total free energy change along a given collective variable. Next, with the help of a genetic clustering algorithm, the method proposes a division of the system into two groups of atoms referred to as molecular cogs. Atoms which cooperate to push the system forward along a collective variable are referred to as forward cogs, and those which work in the opposite direction as reverse cogs. The procedure was tested on several small molecules for which the genetic clustering algorithm successfully found optimal partitionings into molecular cogs. The primary result of the method is a plot depicting the energetic contributions of the identified molecular cogs to the total Potential of Mean Force (PMF) change. Case-studies presented in this work should help better understand the implications of our approach, and were intended to pave the way to a future, publicly available implementation. © 2015 Wiley Periodicals, Inc.

  10. Isolation and identification of radiation resistant yeasts from sea water

    International Nuclear Information System (INIS)

    Park, Jong Cheon; Jeong, Yong Uk; Kim, Du Hong; Jo, Eun A

    2011-12-01

    This study was conducted to isolate radiation-resistant yeasts from sea water for development of application technology of radiation-resistant microorganism. · Isolation of 656 yeasts from sea water and selection of 2 radiation-resistant yeasts (D 10 value >3) · Identification of isolated yeasts as Filobasidium elegans sharing 99% sequence similarity · Characterization of isolated yeast with ability to repair of the DNA damage and membrane integrity to irradiation

  11. Isolation and identification of Metarhizium anisopliae from Chilo ...

    African Journals Online (AJOL)

    *

    2012-04-12

    Apr 12, 2012 ... 1Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences (Key Laboratory of ... Keywords: Metarhizium anisopliae, isolation, identification, Chilo venosatus, culture medium, biological control. .... with a Leica microscope and average values were compared for all.

  12. Isolation and identification of flavonoids from anticancer and ...

    African Journals Online (AJOL)

    Isolation and identification of flavonoids from anticancer and neuroprotective extracts of Trigonella foenum graecum. Shabina Ishtiaq Ahmed, Muhammad Qasim Hayat, Saadia Zahid, Muhammad Tahir, Qaisar Mansoor, Muhammad Ismail, Kristen Keck, Robert Bates ...

  13. Isolation and identification of two galangin metabolites from rat urine ...

    African Journals Online (AJOL)

    Isolation and identification of two galangin metabolites from rat urine and determination of their in vitro hypolipidemic activity. Xuguang Zhang, Shouqian Cheng, Hailong Li, Xiaopo Zhang, Feng Chen, Youbin Li, Junqing Zhang, Yinfeng Tan ...

  14. Enumeration, isolation and identification of bacteria and fungi from ...

    African Journals Online (AJOL)

    Enumeration, isolation and identification of bacteria and fungi from soil contaminated with petroleum products ... dropping can be useful in the bioremediation of soil contaminated with petroleum products and possibly other oil polluted sites.

  15. Isolation, cloning and molecular characterization of a thermotolerant ...

    African Journals Online (AJOL)

    Isolation, cloning and molecular characterization of a thermotolerant xylanase from Streptomyces sp. THW31. Thayat Sriyapai, Peechapack Somyoonsap, Supatra Areekit, Paisarn Khawsak, Arda Pakpitcharoen, Kosum Chansiri ...

  16. MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF Fusarium SPECIES AND THEIR PATHOGENICITY FOR WHEAT

    Directory of Open Access Journals (Sweden)

    Jelena Poštić

    2012-12-01

    Full Text Available From the root and lower stem parts of weeds and plant debris of maize, wheat, oat and sunflower we isolated 300 isolates of Fusarium spp. and performed morphological and molecular identification. With molecular identification using AFLP method we determined 14 Fusarium species: F. acuminatum, F. avenaceum, F. concolor, F. crookwellense, F. equiseti, F. graminearum, F. oxysporum, F. proliferatum, F. semitectum, F. solani, F. sporotrichioides, F. subglutinans, F. venenatum and F. verticillioides.By comparing results of morphological and molecular identification we found out that determination of 16,7% isolates was incorrect. Out of 300 isolates identified with molecular methods, 50 did not belong to the species determined with morphological determination.With pathogenicity tests of 30 chosen Fusarium isolates we determined that many of them were pathogenic to wheat and maize seedlings and to wheat heads. The most pathogenic were isolates of F. graminearum from A. retroflexus, A. theophrasti and C. album, F. venenatum from maize debris and and A. theophrasti, F. crookwellense from A. lappa. Antifungal influence of 11 essential oils on mycelia growth and sporulation of chosen Fusarium isolates determined that essential oils of T. vulgaris, P. anisum and E. caryophyllus had the strongest effect on mycelial growth. Influence of essential oils on sporulation was not statistically significant.

  17. Molecular characterization of lactobacilli isolated from fermented idli batter

    Directory of Open Access Journals (Sweden)

    Perumal Jayaprabha Agaliya

    2013-12-01

    Full Text Available Lactic acid bacteria are non pathogenic organism widely distributed in nature typically involved in a large number of spontaneous food fermentation. The purpose of this study was to characterize the bacteriocinogenic lactobacilli from fermented idli batter which can find application in biopreservation and biomedicine. Eight most promising lactobacilli were chosen from twenty two isolates based on their spectrum of activity against other lactic acid bacteria and pathogens. The eight lactobacilli were characterized based on the various classical phenotypic tests, physiological tests and biochemical tests including various carbohydrate utilization profiles. All isolates were homo fermentative, catalase, and gelatin negative. Molecular characterization was performed by RAPD, 16S rRNA analysis, 16S ARDRA, and Multiplex PCR for species identification. RAPD was carried out using the primer R2 and M13. Five different clusters were obtained based on RAPD indicating strain level variation. 16S rRNA analysis showed 99 to 100% homology towards Lactobacillus plantarum. The restriction digestion pattern was similar for all the isolates with the restriction enzyme AluI. The subspecies were identified by performing Multiplex PCR using species specific primer. Among the five clusters, three clusters were clearly identified as Lactobacillus plantarum subsp. plantarum, Lactobacillus pentosus, and Lactobacillus plantarum subsp. argentoratensis.

  18. Occurrence, isolation and DNA identification of Streptococcus ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... Streptococcus thermophilus involved in Algerian ... among reference, and wild strains of S. thermophilus and for their differentiation from Enterococcus spp. ..... Isolation and characterization of Lactobacillus delbrueckii ssp.

  19. Antifungal activity and molecular identification of endophytic fungi ...

    African Journals Online (AJOL)

    Antifungal activity and molecular identification of endophytic fungi from the angiosperm Rhodomyrtus tomentosa. Juthatip Jeenkeawpieam, Souwalak Phongpaichit, Vatcharin Rukachaisirikul, Jariya Sakayaroj ...

  20. Molecular Genetic Identification Of Some Flax Mutants

    International Nuclear Information System (INIS)

    AMER, I.M.; MOUSTAFA, H.A.M.

    2009-01-01

    Five flax genotypes (Linum usitatissimum L.) i.e., commercial cultivar Sakha 2, the mother variety Giza 4 and three mutant types induced by gamma rays, were screened for their salinity tolerance in field experiments (salinity concentration was 8600 and 8300 ppm for soil and irrigation water, respectively). Mutation 6 was the most salt tolerant as compared to the other four genotypes.RAPD technique was used to detect some molecular markers associated with salt tolerance in flax (Mut 6), RAPD-PCR results using 12 random primers exhibited 149 amplified fragments; 91.9% of them were polymorphic and twelve molecular markers (8.1%) for salt tolerant (mutant 6) were identified with molecular size ranged from 191 to 4159 bp and only eight primers successes to amplify these specific markers. Concerning the other mutants, Mut 15 and Mut 25 exhibited 4.3% and 16.2% specific markers, respectively. The induced mutants exhibited genetic similarity to the parent variety were about 51%, 58.3% and 61.1% for Mut 25, Mut 6 and Mut 15, respectively. These specific markers (SM) are used for identification of the induced mutations and it is important for new variety registration.

  1. Identificación Molecular de Bacterias Productoras de Polihidroxialcanoatos en Subproductos de Lácteos y Caña de Azúcar / Molecular Identification of Polyhydroxyalkanoate-Producing Bacteria Isolated from Dairy and Sugarcane Residues

    Directory of Open Access Journals (Sweden)

    Ana Carolina Cardona Echavarría

    2013-12-01

    . In this work, the presence of PHAs-producing bacteria in whey, sugarcane molasses, cachaza and bagasse was investigated. Bacteria were isolated using a minimal medium supplemented with 2% glucoseand 1 μL mL-1 Nile red (0.1%. Colonies exhibitingfluorescence at 340 nm were further analyzed by fluorescence microscopy with Nileblue. When positive for both tests, bacterial isolates were classified as PHAs producers and their 16S rDNA sequenced. For selected isolates, the presence of the phaC gene was confirmed by PCR. A total of 38 strains, grouped into 18 different morphotypes, were identified as PHA producers. Bacteria belonging to Lactococcus, Klebsiella, Pseudomonas, Enterobacter and Enterococcus genera were isolated from whey and Bacillus, Enterobacter, Pantoea, Klebsiella and Gluconobacter from sugar cane residues. The phaC gene was detected by PCR in 16 bacteria withtype I and IV arrangements. This work opens up the possibility of using the isolated bacteria as an environmentally sustainablealternative for the disposal of agro-industrial residues as well as an additional income source.

  2. Isolation and Identification of the Chitinolytic Bacteria from Rumen Ecosystem

    Directory of Open Access Journals (Sweden)

    Sri Rahayu

    2003-05-01

    Full Text Available Rumen is an interesting ecosystem for microbial exploration and their products. Isolation of the chitinolytic bacteria from the rumen ecosystem found 109 colonies that produced clear zone, 84 colonies (86% anaerobic and 17 colonies (14% aerobic. Clear zone appeared in the third and fourth days incubation. Four potential isolates were chosen for identification purposes. Results showed that the bacteria were sticky, gram-positive, motile, endospore-forming, mesophilic and aerobic. It was supposed to Bacillus spp. the optimal pH and temperature to produce chitinase from isolate 18 are pH 6.0 and temperature of 35-40ºC. Divalent cations Mg, Ca, Zn, and Mn increase chitinase activity, while Cu and Co inhibit enzyme activity. When isolate 18 was grown on shrimp waste meal, it showed aptimal activity on the fifth days incubation. (Animal Production 5(2: 73-78 (2003   Key Words : Isolation, Identification, Chitinolytic Bacteria, Rumen

  3. Molecular prey identification in Central European piscivores.

    Science.gov (United States)

    Thalinger, Bettina; Oehm, Johannes; Mayr, Hannes; Obwexer, Armin; Zeisler, Christiane; Traugott, Michael

    2016-01-01

    Diet analysis is an important aspect when investigating the ecology of fish-eating animals and essential for assessing their functional role in food webs across aquatic and terrestrial ecosystems. The identification of fish remains in dietary samples, however, can be time-consuming and unsatisfying using conventional morphological analysis of prey remains. Here, we present a two-step multiplex PCR system, comprised of six assays, allowing for rapid, sensitive and specific detection of fish DNA in dietary samples. This approach encompasses 78 fish and lamprey species native to Central European freshwaters and enables the identification of 31 species, six genera, two families, two orders and two fish family clusters. All targeted taxa were successfully amplified from 25 template molecules, and each assay was specific when tested against a wide range of invertebrates and vertebrates inhabiting aquatic environments. The applicability of the multiplex PCR system was evaluated in a feeding trial, wherein it outperformed morphological prey analysis regarding species-specific prey identification in faeces of Eurasian otters. Additionally, a wide spectrum of fish species was detected in field-collected faecal samples and regurgitated pellets of Common Kingfishers and Great Cormorants, demonstrating the broad applicability of the approach. In conclusion, this multiplex PCR system provides an efficient, easy to use and cost-effective tool for assessing the trophic ecology of piscivores in Central Europe. Furthermore, the multiplex PCRs and the primers described therein will be applicable wherever DNA of the targeted fish species needs to be detected at high sensitivity and specificity. © 2015 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

  4. Testing of DNA isolation for the identification of hemp

    Directory of Open Access Journals (Sweden)

    Tomáš Vyhnánek

    2015-12-01

    Full Text Available Hemp is diploid organism (2n = 2x = 20, genome size 534 Mb with nine pairs of autosomes plus XX (♀ or XY (♂ chromosomes. Cannabis sativa L. is an important economic plant for the production of food, fibre, oils, and intoxicants. Genotypes (varieties or chemovar of hemp with low Δ9-tetrahydrocannabinol content are used for industrial applications. Varieties with high Δ9-tetrahydrocannabinol or high cannabidiol content are used for medicinal applications. Biochemical and molecular methods can be used for identification and classification. An important step for molecular biology methods is to obtain the matrix of the native and sufficiently pure DNA. We tested two different experimental variant of samples (20 mg and 100 mg of seeds, oilcake and dried flowers for analysis of the Italian variety Carmagnola for analysis (harvested in 2014, Hempoint Ltd., Czech Republic. The DNeasy® Plant Mini Kit (Qiagen, GE was used to isolate the DNA. The DNA concentration and purity was assessed by agarose electrophoresis and via a spectrophotometer. Samples of lower weight yielded lower values of DNA concentration (average 16.30 - 38.90 ng.µL-1, but with better purity than samples of higher weight (ratio A260nm/A280nm for low-weight samples was near 1.80. To test the applicability of DNA analysis, we used two SSR markers (CAN1347 and CAN2913. PCR products were separated on 1% agarose and on 8% polyacrylamide electrophoresis. DNA samples obtained from samples of higher weight exhibited less PCR amplification than samples of lower weight. We found no effect of sample weight on the formation of non-specific amplification products during the PCR reaction. Based on our results we can be recommended for practical isolation procedure using DNeasy® Plant Mini Kit with lower of sample weight (20 mg. In future work the procedure for DNA isolating from wheat-cannabis products, e. g. breads, rolls or pasta, will be optimized.

  5. Isolation, Characterization and Identification of Environmental Bacterial Isolates with Screening for Antagonism Against Three Bacterial Targets

    Science.gov (United States)

    2017-04-01

    ISOLATES WITH SCREENING FOR ANTAGONISM AGAINST THREE BACTERIAL TARGETS 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Identification of environmental isolates followed the flowchart from “Bergey’s Manual of Determinative Bacteriology” (Holt et al. 1994), which

  6. Molecular and pathological identification of feline coronavirus type I

    African Journals Online (AJOL)

    DR. NJ TONUKARI

    2012-06-05

    Jun 5, 2012 ... In this study, we described the isolation and molecular characterization of .... fecv2b) designed in the regions of S-protein gene were used to differentiate ..... The molecular dynamics of feline coronaviruses. Vet. Microbiol.

  7. Isolation and identification of Profenofos degrading bacteria

    Directory of Open Access Journals (Sweden)

    Saadatullah Malghani

    2009-12-01

    Full Text Available An enrichment culture technique was used to isolate bacterial strains responsible for the biodegradation of profenofos in a soil from Hubei province of central China. Two pure bacterial cultures, named W and Y, were isolated and subsequently characterized by sequencing of 16S rRNA genes and biochemical tests. Isolate W showed 96% similarity to the 16S rRNA gene of a Pseudomonas putida unlike Y which showed 99% similarity to the 16S rRNA gene of Burkholderia gladioli. Both strains grew well at pH 5.5-7.2 with a broad temperature profile ranging from 28º to 36 ºC. Bioremediation of profenofos-contaminated soil was examined using soil treated with 200 ug g-1; profenofos resulted in a higher degradation rate than control soils without inoculation. In a mineral salt medium (FTW reduction in profenofos concentration was 90% within 96 hours of incubation. A literature survey revealed that no data is available regarding the role of Burkholderia gladioli on pesticide biodegradation as well as on profenofos.

  8. Isolation, identification and molecular characterisation of an isolate ...

    African Journals Online (AJOL)

    On the basis of host reactions, electron microscopy, serology, and size of the coat protein, a potyvirus infecting cucumber, squash, pumpkin and melon samples collected from farms around KZN was positively identified as ZYMV. Mechanical inoculation of test plants showed that the virus host range was limited to the ...

  9. Limitations of the Current Microbial Identification System for Identification of Clinical Yeast Isolates

    Science.gov (United States)

    Kellogg, James A.; Bankert, David A.; Chaturvedi, Vishnu

    1998-01-01

    The ability of the rapid, computerized Microbial Identification System (MIS; Microbial ID, Inc.) to identify a variety of clinical isolates of yeast species was compared to the abilities of a combination of tests including the Yeast Biochemical Card (bioMerieux Vitek), determination of microscopic morphology on cornmeal agar with Tween 80, and when necessary, conventional biochemical tests and/or the API 20C Aux system (bioMerieux Vitek) to identify the same yeast isolates. The MIS chromatographically analyzes cellular fatty acids and compares the results with the fatty acid profiles in its database. Yeast isolates were subcultured onto Sabouraud dextrose agar and were incubated at 28°C for 24 h. The resulting colonies were saponified, methylated, extracted, and chromatographically analyzed (by version 3.8 of the MIS YSTCLN database) according to the manufacturer’s instructions. Of 477 isolates of 23 species tested, 448 (94%) were given species names by the MIS and 29 (6%) were unidentified (specified as “no match” by the MIS). Of the 448 isolates given names by the MIS, only 335 (75%) of the identifications were correct to the species level. While the MIS correctly identified only 102 (82%) of 124 isolates of Candida glabrata, the predictive value of an MIS identification of unknown isolates as C. glabrata was 100% (102 of 102) because no isolates of other species were misidentified as C. glabrata. In contrast, while the MIS correctly identified 100% (15 of 15) of the isolates of Saccharomyces cerevisiae, the predictive value of an MIS identification of unknown isolates as S. cerevisiae was only 47% (15 of 32), because 17 isolates of C. glabrata were misidentified as S. cerevisiae. The low predictive values for accuracy associated with MIS identifications for most of the remaining yeast species indicate that the procedure and/or database for the system need to be improved. PMID:9574676

  10. Isolation and identification of Mycoplasma mycoides cluster strains from goats in Chongqing, China

    Directory of Open Access Journals (Sweden)

    Wang Haoju

    2014-03-01

    Full Text Available In order to evaluate the prevalence of the Mycoplasma mycoides cluster in goats in Chongqing, China, an epidemiological survey in this area was carried out. A total of 68 samples were subjected to bacteria isolation on Hartley’s medium. Four isolates (three from lung tissue and one from nasal discharges were recovered from the samples and identified as the Mycoplasma species by their morphological and biochemical characteristics. They were further confirmed by PCR using 16S rRNA specific primer pairs and by restriction enzyme analysis. In vitro antimicrobial susceptibility of the isolates indicated that some strains had developed resistance to the antibiotics tested. This is the first report on the isolation, identification, and molecular characterisation of Mycoplasma species isolated from goats in Chongqing. This study also revealed a prevalence of Mycoplasma species infection in goats in this area.

  11. Isolement et identification de microorganismes indigènes de ...

    African Journals Online (AJOL)

    Isolement et identification de microorganismes indigènes de cacaoyères en côte d'ivoire et mise en évidence de leurs effets antagonistes vis-àvis de Phytophthora palmivora , agent de la pourriture brune des cabosses.

  12. Identification of Bacitracin Produced by Local Isolate of Bacillus ...

    African Journals Online (AJOL)

    Bacillus licheniformis was isolated from soil of different house gardens. Diagnosis was performed according to Gram stain, motility, shape forming, aerobic condition and other tests. Bacitracin was primary identified after its activity was tested against some species of Gram positive and Gram negative bacteria. Identification ...

  13. Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates.

    Directory of Open Access Journals (Sweden)

    Francesco Celandroni

    Full Text Available The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.

  14. Identification of listeria species isolated in Tunisia by Microarray based assay : results of a preliminary study

    International Nuclear Information System (INIS)

    Hmaied, Fatma; Helel, Salma; Barkallah, Insaf; Leberre, V.; Francois, J.M.; Kechrid, A.

    2008-01-01

    Microarray-based assay is a new molecular approach for genetic screening and identification of microorganisms. We have developed a rapid microarray-based assay for the reliable detection and discrimination of Listeria spp. in food and clinical isolates from Tunisia. The method used in the present study is based on the PCR amplification of a virulence factor gene (iap gene). the PCR mixture contained cyanine Cy5labeled dCTP. Therefore, The PCR products were fluorescently labeled. The presence of multiple species-specific sequences within the iap gene enabled us to design different oligoprobes per species. The species-specific sequences of the iap gene used in this study were obtained from genBank and then aligned for phylogenetic analysis in order to identify and retrieve the sequences of homologues of the amplified iap gene analysed. 20 probes were used for detection and identification of 22 food isolates and clinical isolates of Listeria spp (L. monocytogenes, L. ivanovi), L. welshimeri, L. seeligeri, and L. grayi). Each bacterial gene was identified by hybridization to oligoprobes specific for each Listeria species and immobilized on a glass surface. The microarray analysis showed that 5 clinical isolates and 2 food isolates were identified listeria monocytogenes. Concerning the remaining 15 food isolates; 13 were identified listeria innocua and 2 isolates could not be identified by microarray based assay. Further phylogenetic and molecular analysis are required to design more species-specific probes for the identification of Listeria spp. Microarray-based assay is a simple and rapid method used for Listeria species discrimination

  15. Isolation and molecular characterisation of Mycobacterium bovis ...

    African Journals Online (AJOL)

    : Three hundred and six milk samples collected from 102 infected cows in different Tunisian regions were analysed. M. bovis isolates were further characterized by spoligotyping and variable number tandem repeat typing. Results: A total of ...

  16. Partial Molecular Characterization Of Cowpea Stunt Isolates Of ...

    African Journals Online (AJOL)

    Partial molecular characterization of the coat protein of the cowpea stunt-causing isolates of Cucumber Mosaic Virus (CMV) from Arkansas and Georgia revealed that both isolates of CMV belong to CMV subgroup I and differ at eight nucleotides positions, resulting in two amino acids difference. There was only one amino ...

  17. Isolation and molecular characterization of Cryptococcus species isolated from pigeon nests and Eucalyptus trees.

    Science.gov (United States)

    Kamari, A; Sepahvand, A; Mohammadi, R

    2017-06-01

    Cryptococcus species are pathogenic and non-pathogenic basidiomycete yeasts that are found widely in the environment. Based on phenotypic methods, this genus has many species; however, its taxonomy is presently being re-evaluated by modern techniques. The Cryptococcus species complex includes two sibling taxa of Cryptococcus neoformans and Cryptococcus gattii . We aimed to investigate the possible distribution of Cryptococcus species in pigeon nests and Eucalyptus trees in Ilam, Iran, using molecular techniques. Two hundred and seventy-four specimens were collected from pigeon nests and Eucalyptus trees during 2016-2017. All the specimens were sub-cultured on Sabouraud Glucose Agar with chloramphenicol and bird seed agar. For molecular identification, the ITS15.8SITS2 rDNA region was amplified using the first and fourth internal transcribed spacer (ITS1 and ITS4, respectively) primers. The purified products were applied for cycle sequencing reactions in forward direction with ITS1 primer. The obtained results were analyzed with Chromas 2.3. Thirty-three out of 186 cultures (17.7%) and 11 out of 88 cultures (12.5%) were positive among pigeon nest and Eucalyptus tree specimens, respectively. Cryptococcus albidus (17.2%), C. albidus var. kuetzingii (3.4%), C. adeliensis (3.4%), C. uzbekistanensis (3.4%), and C. neoformans var. grubii (3.4%) were isolated from pigeon nests, and Cryptococcus adeliensis (25%) was the only Cryptococcus species isolated from Eucalyptus trees. The presence of pigeons and Eucalyptus trees in the vicinity of some particular places such as rest homes and hospitals should be considered as a risk factor for the immunocompromised population.

  18. Molecular identification of Coccidioides spp. in soil samples from Brazil.

    Science.gov (United States)

    de Macêdo, Regina C L; Rosado, Alexandre S; da Mota, Fabio F; Cavalcante, Maria A S; Eulálio, Kelsen D; Filho, Antônio D; Martins, Liline M S; Lazéra, Márcia S; Wanke, Bodo

    2011-05-16

    Since 1991 several outbreaks of acute coccidioidomycosis (CM) were diagnosed in the semi-arid Northeast of Brazil, mainly related to disturbance of armadillo burrows caused by hunters while digging them for the capture of these animals. This activity causes dust contaminated with arthroconidia of Coccidioides posadasii, which, once inhaled, cause the mycosis. We report on the identification of C. posadasii in soil samples related to outbreaks of CM. Twenty four soil samples had their DNA extracted and subsequently submitted to a semi-nested PCR technique using specific primers. While only 6 (25%) soil samples were positive for C. posadasii by mice inoculation, all (100%) were positive by the molecular tool. This methodology represents a simple, sensitive and specific molecular technique to determine the environmental distribution of Coccidioides spp. in endemic areas, but cannot distinguish the species. Moreover, it may be useful to identify culture isolates. Key-words: 1. Coccidioidomycosis. 2. Coccidioides spp. 3. C. posadasii. 4. Semi-arid. 5. Semi-nested PCR.

  19. Molecular identification of Coccidioides spp. in soil samples from Brazil

    Directory of Open Access Journals (Sweden)

    Filho Antônio D

    2011-05-01

    Full Text Available Abstract Background Since 1991 several outbreaks of acute coccidioidomycosis (CM were diagnosed in the semi-arid Northeast of Brazil, mainly related to disturbance of armadillo burrows caused by hunters while digging them for the capture of these animals. This activity causes dust contaminated with arthroconidia of Coccidioides posadasii, which, once inhaled, cause the mycosis. We report on the identification of C. posadasii in soil samples related to outbreaks of CM. Results Twenty four soil samples had their DNA extracted and subsequently submitted to a semi-nested PCR technique using specific primers. While only 6 (25% soil samples were positive for C. posadasii by mice inoculation, all (100% were positive by the molecular tool. Conclusion This methodology represents a simple, sensitive and specific molecular technique to determine the environmental distribution of Coccidioides spp. in endemic areas, but cannot distinguish the species. Moreover, it may be useful to identify culture isolates. Key-words: 1. Coccidioidomycosis. 2. Coccidioides spp. 3. C. posadasii. 4. Semi-arid. 5. Semi-nested PCR

  20. Molecular characterization of Cryptosporidium isolates from humans in Equatorial Guinea.

    Science.gov (United States)

    Blanco, María Alejandra; Iborra, Asunción; Vargas, Antonio; Nsie, Eugenia; Mbá, Luciano; Fuentes, Isabel

    2009-12-01

    The aim of the study was to perform a molecular characterization of clinical isolates of Cryptosporidium species from Equatorial Guinea. Standard laboratory methods were used to identify 35 cryptosporidiosis cases among 185 patients. PCR-RFLP successfully identified 34 Cryptosporidium species from these 35 cases, comprising C. parvum (52.9%), C. hominis (44.1%) and C. meleagridis (2.9%); over 90% of the species were isolated from HIV-positive patients. This is the first report of the molecular characterization of Cryptosporidium species isolated from humans in Equatorial Guinea and shows that zoonotic and anthroponotic transmission is present in this country.

  1. Molecular identification of microorganisms associated to the rhizosphere of vanilla plants in Colombia

    International Nuclear Information System (INIS)

    Alvarez Lopez, Claudia Lucia; Osorio Vega, Nelson Walter; Marin Montoya, Mauricio Alejandro

    2013-01-01

    The cultivation of vanilla (Vanilla planifolia) is highly promising in Colombia, but more research is needed on its agronomical management and beneficial microorganisms that grow associated to its rhizosphere, on which the plant depends for its nutrition and growth. This study involved the identification of microorganisms associated to the rhizosphere of vanilla plants in a crop located in Sopetran, Colombia. The microbes were isolated in selective media for functional groups such as cellulolytic, proteolytic, inorganic and organic phosphate (phytate) solubilizers, and asymbiotic nitrogen fixing bacteria. After isolation and purification, 109 microbial isolates were obtained. DNA was extracted from 52 selected isolates for molecular identification based on its and 16s RDNA sequencing, for fungi and bacteria, respectively. The diversity of rhizosphere microorganisms found was significant. Bacteria such as Bacillus Megaterium, Pseudomonas koreensis and Acinetobacter sp., and the Fungus Plectosphaerella sp., may have a high potential to be used as biofertilizers to improve vanilla plant nutrition and growth.

  2. Molecular characterization of Danish Cryptosporidium parvum isolates

    DEFF Research Database (Denmark)

    Enemark, Heidi L.; Ahrens, Peter; Juel, Cynthia Dawn

    2002-01-01

    The genetic polymorphism among 271 Danish Cryptosporidium isolates of human and animal origin was studied by partial amplification and sequencing of the Cryptosporidium oocyst wall protein (COWP) gene, the 18S rDNA, and a microsatellite locus.dagger Furthermore, the microsatellite locus was studi...

  3. Isolation, characterization and molecular weight determination of ...

    African Journals Online (AJOL)

    Shanmugam

    2013-01-30

    Jan 30, 2013 ... an attempt was made to isolate and characterize collagen from the marine sponge, Spirastrella inconstans. The total ... major constituent of many connective tissues. It is distributed in skin, bones, cartilage, tendons, ligaments, blood vessels, teeth ..... sponge fiber skeleton: a biomimetic scaffold for human.

  4. Isolation and identification of two marine-derived Streptomyces from ...

    African Journals Online (AJOL)

    Administrator

    2011-09-26

    Sep 26, 2011 ... isolated from marine actinomycetes in the 21th century is more than twice of the last century. Research ... South China Sea is a huge natural biological treasure. 11856 Afr. J. Biotechnol. trove with an abundant ... Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4.0 ...

  5. Identification and characterization of Daldinia eschscholtzii isolated from skin scrapings, nails, and blood

    Directory of Open Access Journals (Sweden)

    Kee Peng Ng

    2016-12-01

    Full Text Available Background Daldinia eschscholtzii is a filamentous wood-inhabiting endophyte commonly found in woody plants. Here, we report the identification and characterization of nine D. eschscholtzii isolates from skin scrapings, nail clippings, and blood. Methods The nine isolates were identified based on colony morphology, light microscopy, and internal transcribed spacer (ITS-based phylogeny. In vitro antifungal susceptibility of the fungal isolates was evaluated by the Etest to determine the minimum inhibitory concentration (MIC. Results The nine isolates examined were confirmed as D. eschscholtzii. They exhibited typical features of Daldinia sp. on Sabouraud Dextrose Agar, with white felty colonies and black-gray coloration on the reverse side. Septate hyphae, branching conidiophore with conidiogenous cells budding from its terminus, and nodulisporium-like conidiophores were observed under the microscope. Phylogenetic analysis revealed that the nine isolates were clustered within the D. eschscholtzii species complex. All the isolates exhibited low MICs against azole agents (voriconazole, posaconazole, itraconazole, and ketoconazole, as well as amphotericin B, with MIC of less than 1 µg/ml. Discussion Early and definitive identification of D. eschscholtzii is vital to reducing misuse of antimicrobial agents. Detailed morphological and molecular characterization as well as antifungal profiling of D. eschscholtzii provide the basis for future studies on its biology, pathogenicity, and medicinal potential.

  6. Molecular identification of polydorid polychaetes (Annelida ...

    African Journals Online (AJOL)

    The early detection and correct identification of polydorid polychaete species is essential as they are often encountered as invasive alien pests in aquaculture facilities or the intertidal where they may modify the ecosystem. Accurate identification is, however, often hampered by high levels of morphological similarity among ...

  7. Molecular Identification, Enzyme Assay, and Metabolic Profiling of Trichoderma spp.

    Science.gov (United States)

    Bae, Soo-Jung; Park, Young-Hwan; Bae, Hyeun-Jong; Jeon, Junhyun; Bae, Hanhong

    2017-06-28

    The goal of this study was to identify and characterize selected Trichoderma isolates by metabolic profiling and enzyme assay for evaluation of their potential as biocontrol agents against plant pathogens. Trichoderma isolates were obtained from the Rural Development Administration Genebank Information Center (Wanju, Republic of Korea). Eleven Trichoderma isolates were re-identified using ribosomal DNA internal transcribed spacer (ITS) regions. ITS sequence results showed new identification of Trichoderma isolates. In addition, metabolic profiling of the ethyl acetate extracts of the liquid cultures of five Trichoderma isolates that showed the best anti- Phytophthora activities was conducted using gas chromatography-mass spectrometry. Metabolic profiling revealed that Trichoderma isolates shared common metabolites with well-known antifungal activities. Enzyme assays indicated strong cell walldegrading enzyme activities of Trichoderma isolates. Overall, our results indicated that the selected Trichoderma isolates have great potential for use as biocontrol agents against plant pathogens.

  8. Isolation and identification of fungi from a meju contaminated with aflatoxins.

    Science.gov (United States)

    Jung, Yu Jung; Chung, Soo Hyun; Lee, Hyo Ku; Chun, Hyang Sook; Hong, Seung Beom

    2012-12-01

    A home-made meju sample contaminated naturally with aflatoxins was used for isolation of fungal strains. Overall, 230 fungal isolates were obtained on dichloran rosebengal chloramphenicol (DRBC) and dichloran 18% glycerol (DG18) agar plates. Morphological characteristics and molecular analysis of a partial beta-tubulin gene and the internal transcribed spacer (ITS) of rDNA were used for the identification of the isolates. The fungal isolates were divided into 7 genera: Aspergillus, Eurotium, Penicillium, Eupenicillium, Mucor, Lichtheimia, and Curvularia. Three strains from 56 isolates of the A. oryzae/flavus group were found to be aflatoxigenic A. flavus, by the presence of the aflatoxin biosynthesis genes and confirmatory aflatoxin production by high-performance liquid chromatography (HPLC). The predominant isolate from DRBC plates was A. oryzae (42 strains, 36.2%), whereas that from DG18 was A. candidus (61 strains, 53.5%). Out of the 230 isolates, the most common species was A. candidus (34.3%) followed by A. oryzae (22.2%), Mucor circinelloides (13.0%), P. polonicum (10.0%), A. tubingensis (4.8%), and L. ramosa (3.5%). A. flavus and E. chevalieri presented occurrence levels of 2.2%, respectively. The remaining isolates of A. unguis, P. oxalicum, Eupenicillium cinnamopurpureum, A. acidus, E. rubrum, P. chrysogenum, M. racemosus, and C. inaequalis had lower occurrence levels of < 2.0%.

  9. Molecular and Pathotype Identification of Potato Cyst Nematodes

    Directory of Open Access Journals (Sweden)

    Mulyadi Mulyadi

    2014-07-01

    Full Text Available In Indonesia, potato cyst nematode (PCN was first reported in Bumiaji, Kota Batu, East Java by PT Syngenta and was identified as Globodera rostochiensis. Based on the surveillances, G. rostochiensis were also found in Batur, Banjarnegara, and Kejajar, Wonosobo, and Pangalengan, Bandung. In addition, in Batur, Banjarnegara, another species which was identified as G. pallida was found. The aim of this research were to identify the species of PCN using molecular method, pathotype identification, and to study the distributions of PCN especially in Java. The PCN are collected from potato planting areas in Kota Batu, East Java; Wonosobo and Banjarnegara, Central Java; and Pangalengan, Bandung, West Java. PCN were extracted and isolated from soil, and then identified by  morphological and molecular analysis. PCN were found in potato planting areas in Kota Batu, East Java; Wonosobo and Banjarnegara, Central Java; and Pangalengan, West Java. Based on the morphological characters, molecular method, and the differential host test, the PCN identified as G. rostochiensis are amplified an approximately 434 bp with pathotype Ro2.   Di Indonesia, nematoda sista kentang (NSK pertama dilaporkan di Bumiaji, Kota Batu, Jawa Timur oleh PT Syngenta yang diidentifikasi sebagai Globodera rostochiensis. Berdasarkan hasil survei, NSK ditemukan di Batur, Banjarnegara dan Kejajar, Wonosobo, Pangalengan. Spesies G. pallida juga ditemukan Batur, Banjarnegara. Penelitian ini bertujuan untuk mengidentifikasi spesies NSK menggunakan metode molekuler, identifikasi patotipe NSK, dan untuk mengetahui penyebaran NSK khususnya di Pulau Jawa. Sampel NSK dikumpulkan dari lahan pertanaman kentang di Bumiaji, Kota Batu, Jawa Timur; Wonosobo dan Banjarnegara, Jawa Tengah; serta Pangalengan, Bandung, Jawa Tengah. NSK diekstraksi dan diisolasi dari tanah yang selanjutnya diidentifikasi secara morfologi dan analisis molekuler. NSK yang terdapat pada lahan pertanaman kentang ditemukan di

  10. Isolation and identification of marine fish tumour (odontoma associated bacteria

    Directory of Open Access Journals (Sweden)

    Ramalingam Vijayakumar

    2015-09-01

    Full Text Available Objective: To identify fish tumour associated bacteria. Methods: The marine fish Sphyraena jello with odontoma was collected from in Tamil Nadu (Southeast India, and tumour associated bacteria were isolated. Then the isolated bacteria were identified based on molecular characters. Results: A total of 4 different bacterial species were isolated from tumour tissue. The bacterial species were Bacillus sp., Pontibacter sp., Burkholderia sp. and Macrococcus sp., and the sequences were submitted in DNA Data Bank of Japan with accession numbers of AB859240, AB859241, AB859242 and AB859243 respectively. Conclusions: Four different bacterial species were isolated from Sphyraena jello, but the role of bacteria within tumour needs to be further investigated.

  11. Isolation, Characterization and Molecular weight determination of ...

    African Journals Online (AJOL)

    Enzyme purification to homogeneity was carried out by anion exchange chromatography on DEAE-Sepharose. SDS-PAGE revealed molecular mass of 87 kDa. Maximal activity of the enzymes was observed at 50°C at pH 4 and was stimulated by Ca2+, Co2+, Mg2+ (test at 10 Mm each) and inhibited by Fe2+. Ethanol at an ...

  12. Molecular identification of the first SIFamide receptor

    DEFF Research Database (Denmark)

    Jørgensen, Lars M; Hauser, Frank; Cazzamali, Giuseppe

    2006-01-01

    , an impressive sequence conservation (67-77% amino acid sequence identities between the seven-transmembrane areas; 82-87% sequence similarities). The identification of well-conserved SIFamide receptor orthologues in all other insects with a sequenced genome, suggests that the SIFamide/receptor couple must have...... an essential function in arthropods. This paper is the first report on the identification of a SIFamide receptor....

  13. Isolation, Identification, and Evaluation of Novel Probiotic Strains Isolated from Feces of Breast-Fed Infants.

    Science.gov (United States)

    Panya, Marutpong; Lulitanond, Viraphong; Rattanachaikunsopon, Pongsak; Srivoramas, Thanyakarn; Chaiwong, Tarinee

    2016-01-01

    To isolate, identify, and evaluate the probiotic properties of lactic acid bacteria (LAB) isolated from the feces of breast-fed infants. The probiotic tests included investigation of hemolysis activity, survival in simulated gastrointestinal tract conditions (acid and bile salt tolerance), susceptibility to antibiotics, and ability to inhibit selected bacterial pathogens (Escherichia coli O157:H7, Vibrio cholerae and Salmonella enterica subsp enterica serovar Typhimurium). The bacterial species identification was performed by both carbohydrate utilization and partial 16S ribosomal RNA sequencing. Five of fifty LAB isolates (UBU-03, UBU-06, UBU-09, UBU-34, and UBU-37) showed good probiotic properties. These five isolates showed non-hemolysis type (gamma-hemolysis), susceptibility to all antibiotics tested except for vancomycin, ability to survive in the simulated gastrointestinal conditions of both acid and bile salt solution, and ability to inhibit growth of E. coli O157: H7 and V. cholerae. Bacterial species identification revealed that all five isolates were firmly identified as Lactobacillus rhamnosus species. The L. rhamnosus strains that were isolated and characterized in this study could be considered as probiotic strains, and then used for further probiotic characterization in human cell cultures or animal models.

  14. Molecular and antimicrobial susceptibility characterization of Globicatella sulfidifaciens isolated from sow's urinary tract infection.

    Science.gov (United States)

    Matajira, Carlos E C; Moreno, Luisa Z; Gomes, Vasco T M; Silva, Ana Paula S; Mesquita, Renan E; Amigo, Cristina R; Christ, Ana Paula G; Sato, Maria Inês Z; Moreno, Andrea M

    2017-12-01

    The Globicatella genus comprises Gram-positive, facultative anaerobic, α-hemolytic and catalase negative cocci morphologically and phenotypically very similar to Streptococcus and Aerococcus genus which can lead to misidentification and underestimation of this pathogen. Globicatella species have already been isolated from human and animals with heart and brain disorders. Their clinical relevance in animals, and its zoonotic potential, remains unknown due to the difficulty in their identification. To present the isolation, phenotypic and molecular characterization of G. sulfidifaciens from urinary tract infection in sows. Urine samples from 140 sows of two swine herds located in São Paulo State (Brazil) yielded the isolation of three presumptive G. sulfidifaciens strains. Identification and species confirmation were done by MALDI-TOF MS and 16S rRNA sequencing. Strains were further characterized by single enzyme amplified fragments length polymorphism (SE-AFLP) and broth microdilution techniques. All three isolates were confirmed as G. sulfidifaciens. The SE-AFLP genotyping resulted in distinct fingerprint patterns for each strain. All isolates presented high MIC values to tetracycline, sulphonamides, aminoglycosides and tylosin tartrate, which present high usage in human and animal medicine. Globicatella sulfidifaciens could be related to sporadic urinary tract infections in swine and appear to present alarming antimicrobial susceptibility profile. It is necessary to differentiate Streptococcus-like microorganisms in routine laboratory diagnostics for the correct identification of underestimated species potentially pathogenic to animals.

  15. Biochemical and molecular genetic studies on some cyanobacterial isolates

    International Nuclear Information System (INIS)

    Kamal, E.A.R.; Ebrahim, S.A.A.

    2011-01-01

    In the present study, the isolation and purification of a set of Cyanobacteria strains belonging to genus Oscillatoria was undertaken, followed by the analyses of phylogenetic relationships using different biochemical and molecular genetic techniques (SOS-PAGE and RAPO-PCR). A total of 45 protein bands were observed within the studied Osci/latoria isolates by SOS-PAGE (only three unique bands, eight monomorphic bands and 37 polymorphic bands). On the other hand, extracted ONA from isolates was used to identify the molecular fingerprints. A sum of 94 polymorphic bands was generated by these primers in the Ocsi/laloria genotypes under study. A total of 20 unique bands were identified out of the polymorphic ones. These unique bands were used to discriminate among the studied Ocsi/latoria isolates. Most isolates of Ocsi/latoria genotypes were discriminated by one or more unique bands. Numerical taxonomic using 45 protein attributes of 19 isolates and RAPO markers on five isolates. Two methods -Clustering (UPGMA) and Principal Component Analysis (PCA) were used for these analyses. The similarities and clusters produced between the studied isolates were discussed.

  16. Biochemical and molecular genetic studies on some cyanobacterial isolates

    Energy Technology Data Exchange (ETDEWEB)

    Kamal, E A.R. [Umm Al-Qura University, Makkah (Saudi Arabia). Dept. of Biology; Ebrahim, S A.A. [Ain Sham University, Cairo (Egypt). Dept. of Cytogenetic

    2011-11-15

    In the present study, the isolation and purification of a set of Cyanobacteria strains belonging to genus Oscillatoria was undertaken, followed by the analyses of phylogenetic relationships using different biochemical and molecular genetic techniques (SOS-PAGE and RAPO-PCR). A total of 45 protein bands were observed within the studied Osci/latoria isolates by SOS-PAGE (only three unique bands, eight monomorphic bands and 37 polymorphic bands). On the other hand, extracted ONA from isolates was used to identify the molecular fingerprints. A sum of 94 polymorphic bands was generated by these primers in the Ocsi/laloria genotypes under study. A total of 20 unique bands were identified out of the polymorphic ones. These unique bands were used to discriminate among the studied Ocsi/latoria isolates. Most isolates of Ocsi/latoria genotypes were discriminated by one or more unique bands. Numerical taxonomic using 45 protein attributes of 19 isolates and RAPO markers on five isolates. Two methods -Clustering (UPGMA) and Principal Component Analysis (PCA) were used for these analyses. The similarities and clusters produced between the studied isolates were discussed.

  17. Morphological and molecular identification of phytophthora species from maple trees in Serbia

    Directory of Open Access Journals (Sweden)

    Milenković Ivan

    2014-01-01

    Full Text Available The paper presents the results of the study performed with aims to determine the presence and diversity of Phytophthora species on maple trees in Serbia. Due to high aggressiveness and their multicyclic nature, presence of these pathogens is posing significant threat to forestry and biodiversity. In total, 29 samples of water, soil and tissues were taken from 10 different localities, and six different maple hosts were tested. After the isolation tests, 17 samples from five different maple hosts were positive for the presence of Phytophthora spp., and 31 isolates were obtained. After the detailed morphological and physiological classification, four distinct groups of isolates were separated. DNA was extracted from selected representative isolates and molecular identification with sequencing of ITS region was performed. Used ITS4 and ITS6 primers successfully amplified the genomic DNA of chosen isolates and morphological identification of obtained isolates was confirmed after the sequencing. Four different Phytophthora species were detected, including P. cactorum, P. gonapodyides, P. plurivora and P. lacustris. The most common isolated species was homothallic, and with very variable and semipapillate sporangia, P. plurivora with 22 obtained isolates. This is the first report of P. plurivora and P. gonapodyides on A. campestre, P. plurivora and P. lacustris on Acer heldreichii and first report of P. lacustris on A. pseudoplatanus and A. tataricum in Serbia. [Projekat Ministarstva nauke Republike Srbije, br. TR 37008

  18. [Phenotypic and genotypic identification of Candida strains isolated as nosocomial pathogens].

    Science.gov (United States)

    Sahiner, Fatih; Ergünay, Koray; Ozyurt, Mustafa; Ardıç, Nurittin; Hoşbul, Tuğrul; Haznedaroğlu, Tunçer

    2011-07-01

    in which ITS1-ITS4 products cut by Msp I ve Bln I restriction enzymes] for the identification of Candida species revealed that CHROMagar Candida medium combined with API ID 32C kit yielded the same results (100% compatible) as molecular techniques for the species identification of Candida isolates. Since these phenotypic methods were simple and cost effective when compared to molecular techniques, they should be considered in the identification of Candida species.

  19. Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

    Science.gov (United States)

    Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

    2015-04-01

    Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Isolation and identification of iron ore-solubilising fungus

    Directory of Open Access Journals (Sweden)

    Damase Khasa

    2010-09-01

    Full Text Available Potential mineral-solubilising fungi were successfully isolated from the surfaces of iron ore minerals. Four isolates were obtained and identified by molecular and phylogenetic methods as close relatives of three different genera, namely Penicillium (for isolate FO, Alternaria (for isolates SFC2 and KFC1 and Epicoccum (for isolate SFC2B. The use of tricalcium phosphate (Ca3(PO42in phosphate-solubilising experiments confirmed isolate FO as the only phosphate solubiliser among the isolated fungi. The bioleaching capabilities of both the fungus and its spent liquid medium were tested and compared using two types of iron ore materials, conglomerate and shale, from the Sishen Iron Ore Mine as sources of potassium (K and phosphorus (P. The spent liquid medium removed more K (a maximum of 32.94% removal, from conglomerate, than the fungus (a maximum of 21.36% removal, from shale. However, the fungus removed more P (a maximum of 58.33% removal, from conglomerate than the spent liquid medium (a maximum of 29.25% removal, from conglomerate. The results also indicated a potential relationship between the removal of K or P and the production of organic acids by the fungus. A high production of gluconic acid could be related to the ability of the fungus to reduce K and P. Acetic, citric and maleic acids were also produced by the fungus, but in lower quantities. In addition, particle size and iron ore type were also shown to have significant effects on the removal of potassium and phosphorus from the iron ore minerals. We therefore conclude that the spent liquid medium from the fungal isolate FO can potentially be used for biobeneficiation of iron ore minerals.

  1. Molecular Typing of Salmonella Isolates in Poultry by Pulsed-Field Gel Electrophoresis in Iran

    Directory of Open Access Journals (Sweden)

    Narges Golab

    2014-11-01

    Full Text Available Background: Salmonella is one of the most widespread zoonotic enter pathogenic microorganisms found in the global food chain. Poultryand Poultry products have been identified as one of the important foodborne sources of Salmonella. Pulsed-Field Gel Electrophoresis (PFGE is a gold standard typing method for identification of Salmonella isolates during outbreaks and epidemiological investigations. Objectives: The aim of this study was to carry out molecular typing of Salmonella enterica spp. by PFGE technique. Materials and Methods: All 47 Salmonella isolates were serotyped and then subjected to PFGE. Total isolates were analyzed by means of the molecular technique XbaI PFGE. Results: In the current work, PFGE and serotyping were used to subtype 47 Salmonella isolates belonging to 22 different serotypes and derived from poultry. Thirty-nine PFGE patterns out of 47 isolates were obtained. The Discrimination Index (DI by serotyping (0.93 was lower than PFGE (DI = 0.99. Conclusions: In conclusion, molecular methods such as PFGE can be used for epidemiological characterization of Salmonella serotypes.

  2. Molecular identification of phosphate solubilizing bacterium ...

    African Journals Online (AJOL)

    A phosphate solubilizing bacterium was isolated from the rhizosphere soil of upland rice and identified by 16S rRNA gene sequencing. The gene sequence showed 99% homology with Alcaligenes faecalis. Based on the gene sequence homology, it was identified as A. faecalis. Interaction effect of this bacterium on growth ...

  3. Molecular Identification of Methicillin-Resistant Staphylococcus ...

    African Journals Online (AJOL)

    Antimicrobial resistance has become a great public health problem worldwide and multi-drug resistant Staphylococcus aureus has been widely reported. Methods: The presence or absence of methicillin resistance gene (mecA) in 48 clinical wound isolates of S. aureus was examined by the polymerase chain reaction ...

  4. Molecular screening and isolation of Newcastle disease virus from ...

    African Journals Online (AJOL)

    Molecular screening and isolation of Newcastle disease virus from live poultry markets and chickens from commercial poultry farms in Zaria, Kaduna state, Nigeria. ... The PDF file you selected should load here if your Web browser has a PDF reader plug-in installed (for example, a recent version of Adobe Acrobat Reader).

  5. Molecular prey identification in Central European piscivores

    OpenAIRE

    Thalinger, Bettina; Oehm, Johannes; Mayr, Hannes; Obwexer, Armin; Zeisler, Christiane; Traugott, Michael

    2015-01-01

    Abstract Diet analysis is an important aspect when investigating the ecology of fish?eating animals and essential for assessing their functional role in food webs across aquatic and terrestrial ecosystems. The identification of fish remains in dietary samples, however, can be time?consuming and unsatisfying using conventional morphological analysis of prey remains. Here, we present a two?step multiplex PCR system, comprised of six assays, allowing for rapid, sensitive and specific detection o...

  6. Molecular characterisation of Aspergillus flavus isolates from peanut fields in India using AFLP

    Directory of Open Access Journals (Sweden)

    Diwakar Singh

    2015-09-01

    Full Text Available Aflatoxin contamination of peanut, due to infection by Aspergillus flavus, is a major problem of rain-fed agriculture in India. In the present study, molecular characterisation of 187 Aspergillus flavus isolates, which were sampled from the peanut fields of Gujarat state in India, was performed using AFLP markers. On a pooled cluster analysis, the markers could successfully discriminate among the ‘A’, ‘B’ and ‘G’ group A. flavus isolates. PCoA analysis also showed equivalent results to the cluster analysis. Most of the isolates from one district could be clustered together, which indicated genetic similarity among the isolates. Further, a lot of genetic variability was observed within a district and within a group. The results of AMOVA test revealed that the variance within a population (84% was more than that between two populations (16%. The isolates, when tested by indirect competitive ELISA, showed about 68.5% of them to be atoxigenic. Composite analysis between the aflatoxin production and AFLP data was found to be ineffective in separating the isolate types by aflatoxigenicity. Certain unique fragments, with respect to individual isolates, were also identified that may be used for development of SCAR marker to aid in rapid and precise identification of isolates.

  7. Molecular species identification and population genetics of ...

    African Journals Online (AJOL)

    Molecular genetic techniques, such as DNA barcoding and genotyping, are increasingly being used to assist with the conservation and management of chondrichthyans worldwide. Southern Africa is a shark biodiversity hotspot, with a large number of endemic species. According to the IUCN Red List, a quarter of South ...

  8. Molecular characterization and antimicrobial susceptibility of nasal Staphylococcus aureus isolates from a Chinese medical college campus.

    Directory of Open Access Journals (Sweden)

    Jimei Du

    Full Text Available Staphylococcus aureus colonization and infection occur more commonly among persons living or working in crowded conditions, but characterization of S. aureus colonization within medical communities in China is lacking. A total of 144 (15.4%, 144/935 S. aureus isolates, including 28 (3.0%, 28/935 MRSA isolates, were recovered from the nares of 935 healthy human volunteers residing on a Chinese medical college campus. All S. aureus isolates were susceptible to vancomycin, quinupristin/dalfopristin and linezolid but the majority were resistant to penicillin (96.5%, ampicillin/sulbactam (83.3% and trimethoprim/sulfamethoxazole (93.1%. 82%, (23/28 of the MRSA isolates and 66% (77/116 of the MSSA isolates were resistant to multiple antibiotics, and 3 MRSA isolates were resistant to mupirocin--an agent commonly used for nasal decolonization. 16 different sequence types (STs, as well as SCCmec genes II, III, IVd, and V, were represented among MRSA isolates. We also identified, for the first time, two novel STs (ST1778 and ST1779 and 5 novel spa types for MRSA. MRSA isolates were distributed in different sporadic clones, and ST59-MRSA-VId- t437 was found within 3 MRSA isolates. Moreover, one isolate with multidrug resistance belonging to ST398-MRSA-V- t571 associated with animal infections was identified, and 3 isolates distributed in three different clones harbored PVL genes. Collectively, these data indicate a high prevalence of nasal MRSA carriage and molecular heterogeneity of S. aureus isolates among persons residing on a Chinese medical college campus. Identification of epidemic MRSA clones associated with community infection supports the need for more effective infection control measures to reduce nasal carriage and prevent dissemination of MRSA to hospitalized patients and health care workers in this community.

  9. Systematic Internal Transcribed Spacer Sequence Analysis for Identification of Clinical Mold Isolates in Diagnostic Mycology: a 5-Year Study▿ †

    Science.gov (United States)

    Ciardo, Diana E.; Lucke, Katja; Imhof, Alex; Bloemberg, Guido V.; Böttger, Erik C.

    2010-01-01

    The implementation of internal transcribed spacer (ITS) sequencing for routine identification of molds in the diagnostic mycology laboratory was analyzed in a 5-year study. All mold isolates (n = 6,900) recovered in our laboratory from 2005 to 2009 were included in this study. According to a defined work flow, which in addition to troublesome phenotypic identification takes clinical relevance into account, 233 isolates were subjected to ITS sequence analysis. Sequencing resulted in successful identification for 78.6% of the analyzed isolates (57.1% at species level, 21.5% at genus level). In comparison, extended in-depth phenotypic characterization of the isolates subjected to sequencing achieved taxonomic assignment for 47.6% of these, with a mere 13.3% at species level. Optimization of DNA extraction further improved the efficacy of molecular identification. This study is the first of its kind to testify to the systematic implementation of sequence-based identification procedures in the routine workup of mold isolates in the diagnostic mycology laboratory. PMID:20573873

  10. Systematic internal transcribed spacer sequence analysis for identification of clinical mold isolates in diagnostic mycology: a 5-year study.

    Science.gov (United States)

    Ciardo, Diana E; Lucke, Katja; Imhof, Alex; Bloemberg, Guido V; Böttger, Erik C

    2010-08-01

    The implementation of internal transcribed spacer (ITS) sequencing for routine identification of molds in the diagnostic mycology laboratory was analyzed in a 5-year study. All mold isolates (n = 6,900) recovered in our laboratory from 2005 to 2009 were included in this study. According to a defined work flow, which in addition to troublesome phenotypic identification takes clinical relevance into account, 233 isolates were subjected to ITS sequence analysis. Sequencing resulted in successful identification for 78.6% of the analyzed isolates (57.1% at species level, 21.5% at genus level). In comparison, extended in-depth phenotypic characterization of the isolates subjected to sequencing achieved taxonomic assignment for 47.6% of these, with a mere 13.3% at species level. Optimization of DNA extraction further improved the efficacy of molecular identification. This study is the first of its kind to testify to the systematic implementation of sequence-based identification procedures in the routine workup of mold isolates in the diagnostic mycology laboratory.

  11. Virulence and molecular polymorphism of Prunus necrotic ringspot virus isolates.

    Science.gov (United States)

    Hammond, R W; Crosslin, J M

    1998-07-01

    Prunus necrotic ringspot virus (PNRSV) occurs as numerous strains or isolates that vary widely in their pathogenic, biophysical and serological properties. Prior attempts to distinguish pathotypes based upon physical properties have not been successful; our approach was to examine the molecular properties that may distinguish these isolates. The nucleic acid sequence was determined from 1.65 kbp RT-PCR products derived from RNA 3 of seven distinct isolates of PNRSV that differ serologically and in pathology on sweet cherry. Sequence comparisons of ORF 3a (putative movement protein) and ORF 3b (coat protein) revealed single nucleotide and amino acid differences with strong correlations to serology and symptom types (pathotypes). Sequence differences between serotypes and pathotypes were also reflected in the overall phylogenetic relationships between the isolates.

  12. Selection, isolation, and identification of fungi for bioherbicide production

    Directory of Open Access Journals (Sweden)

    Angélica Rossana Castro de Souza

    Full Text Available Abstract Production of a bioherbicide for biological control of weeds requires a series of steps, from selection of a suitable microbial strain to final formulation. Thus, this study aimed to select fungi for production of secondary metabolites with herbicidal activity using biological resources of the Brazilian Pampa biome. Phytopathogenic fungi were isolated from infected tissues of weeds in the Pampa biome. A liquid synthetic culture medium was used for production of metabolites. The phytotoxicity of fungal metabolites was assessed via biological tests using the plant Cucumis sativus L., and the most promising strain was identified by molecular analysis. Thirty-nine fungi were isolated, and 28 presented some phytotoxic symptoms against the target plant. Fungus VP51 belonging to the genus Diaporthe showed the most pronounced herbicidal activity. The Brazilian Pampa biome is a potential resource for the development of new and sustainable chemical compounds for modern agriculture.

  13. Molecular Characterization and Analysis of 16S Ribosomal DNA in Some Isolates of Demodex folicullorum.

    Science.gov (United States)

    Daneshparvar, Afrooz; Mowlavi, Gholamreza; Mirjalali, Hamed; Hajjaran, Homa; Mobedi, Iraj; Naddaf, Saeed Reza; Shidfar, Mohammadreza; Sadat Makki, Mahsa

    2017-01-01

    Demodicosis is one of the most prevalent skin diseases resulting from infestation by Demodex mites. This parasite usually inhabits in follicular infundibulum or sebaceous duct and transmits through close contact with an infested host. This study was carried from September 2014 to January 2016 at Tehran University of Medical Sciences, Tehran, Iran. DNA extraction and amplification of 16S ribosomal RNA was performed on four isolates, already obtained from four different patients and identified morphologically though clearing with 10% Potassium hydroxide (KOH) and microscopical examination. Amplified fragments from the isolates were compared with GeneBank database and phylogenetic analysis was carried out using MEGA6 software. A 390 bp fragment of 16S rDNA was obtained in all isolates and analysis of generated sequences showed high similarity with those submitted to GenBank, previously. Intra-species similarity and distance also showed 99.983% and 0.017, respectively, for the studied isolates. Multiple alignments of the isolates showed Single Nucleotide Polymorphisms (SNPs) in 16S rRNA fragment. Phylogenetic analysis revealed that all 4 isolates clustered with other D. folliculorum, recovered from GenBank database. Our accession numbers KF875587 and KF875589 showed more similarity together in comparison with two other studied isolates. Mitochondrial 16S rDNA is one of the most suitable molecular barcodes for identification D. folliculorum and this fragment can use for intra-species characterization of the most human-infected mites.

  14. Molecular Characterization and Analysis of 16S Ribosomal DNA in some Isolates of Demodex folliculorum

    Directory of Open Access Journals (Sweden)

    Afrooz DANESHPARVAR

    2017-06-01

    Full Text Available Background: Demodicosis is one of the most prevalent skin diseases resulting from infestation by Demodex mites. This parasite usually inhabits in follicular infundibulum or sebaceous duct transmitted through close contact with an infested host.Methods: This study was carried from September 2014 to January 2016 at Tehran University of Medical Sciences, Tehran, Iran. DNA extraction and amplification of 16S ribosomal RNA was performed on four isolates, obtained from four patients and identified morphologically through clearing with 10% Potassium hydroxide (KOH and microscopical examination. Amplified fragments from the isolates were compared with GenBank database and phylogenetic analysis was carried out using MEGA6 software.Results: A 390 bp fragment of 16S rDNA was obtained in all isolates and analysis of generated sequences showed high similarity with those submitted to GenBank, previously. Intra-species similarity and distance also showed 99.983% and 0.017, respectively, for the studied isolates. Multiple alignments of the isolates showed Single Nucleotide Polymorphisms (SNPs in 16S rRNA fragment. Phylogenetic analysis revealed that all 4 isolates clustered with other D. folliculorum, recovered from GenBank database. Our accession numbers KF875587 and KF875589 showed more similarity together in comparison with two other studied isolates. Conclusion: Mitochondrial 16S rDNA is one of the most suitable molecular barcodes for identification D. folliculorum and this fragment can use for intra-species characterization of the most human-infected mites.

  15. [Isolation and identification of rumen bacteria for cellulolytic enzyme production].

    Science.gov (United States)

    Aihemaiti, Maierhaba; Zhen, Fan; Li, Yuezhong; Aibaidoula, Gulisimayi; Yimit, Wusiman

    2013-05-04

    We screened aerobic bacteria with cellulolytic activity from ruminal fluid of sheep, cattle and camel in Xinjiang. Fresh ruminal fluid was inoculated on sterilized sodium carboxymethylcellulose agar plates. Highly cellulolytic aerobic bacteria were screened out by using Congo red staining and liquid secondary screening culture media. The combination of morphological and biochemical test with 16SrDNA sequence analysis were used to classify the strains. Enzymatic activities of four strains with strong cellulose-decomposing abilities were studied under different culture conditions. Out 84 isolated cellulolytic strains, 40 exhibited strong abilities in decomposing cellulose. They are including 37 Gram-negative isolates and 3 Gram-positive strains. Identification of these 40 strains shows that they belong to 11 species of 6 genera, 16 strains in Stenotrophomonas maltophilia, 10 Ochrobactrum, 5 Sphingobacterium, 3 Microbacterium, 3 Paracoccus and 2 Pseudomonas. The results of the enzymatic studies of four strains with strong cellulolytic abilities indicates that the strains have the best enzyme producing property when straw powder was chosen as the carbon source; the pH at 5.5 -6.0 and temperature at 37 degrees C. The strains with highly cellulolytic abilities isolated from ruminal fluid show strong abilities in cellulose decomposition.

  16. Isolation and identification of aerobic polychlorinated biphenyls degrading bacteria

    Directory of Open Access Journals (Sweden)

    Bibi Fatemeh Nabavi

    2013-01-01

    Full Text Available Aims: The purpose of this study was to isolate and identify aerobic polychlorinated biphenyls (PCBs degrading bacteria. Materials and Methods: This study was performed in lab scale aerobic sequencing batch biofilm reactor. Polyurethane foams were used as bio-carrier and synthetic wastewater was prepared with PCBs in transformer oil as the main substrate (20-700 μg/l and acetone as a solvent for PCBs as well as microelements. After achieving to adequate microbial population and acclimation of microorganisms to PCB compounds with high efficiency of PCB removal, identification of degrading microbial species was performed by 16s rRNA gene sequencing of isolated bacteria. Results: Gene sequencing results of the isolated bacteria showed that Rhodococcus spp., Pseudomonas spp., Pseudoxanthomonas spp., Agromyces spp., and Brevibacillus spp. were dominant PCB-degrading bacteria. Conclusion: PCB compounds can be degraded by some microorganisms under aerobic or anaerobic conditions or at least be reduced to low chlorinated congeners, despite their chemical stability and toxicity. Based on the results of the study, five bacterial species capable of degrading PCBs in transformer oil have been identified.

  17. Molecular identification and genotyping of Microsporidia in selected hosts

    Czech Academy of Sciences Publication Activity Database

    Valenčáková, A.; Balent, P.; Ravaszová, P.; Horák, Aleš; Oborník, Miroslav; Halanová, M.; Malčeková, B.; Novotný, F.; Goldová, M.

    2012-01-01

    Roč. 110, č. 2 (2012), s. 689-693 ISSN 0932-0113 Institutional research plan: CEZ:AV0Z60220518 Keywords : ENCEPHALITOZOON-CUNICULI * RIBOSOMAL-RNA * SPECIES IDENTIFICATION * AIDS PATIENTS * PET RABBITS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.852, year: 2012

  18. Seasonal abundance and molecular identification of West Nile virus ...

    African Journals Online (AJOL)

    Seasonal abundance and molecular identification of West Nile virus vectors, Culex pipens and Culex ... Background: West Nile virus (WNV) infection, is an arbovirus infection with high morbidity and mortality, the vector respon- sible for both human ... Major diseases transmitted are known as Arboviral dis- eases because ...

  19. Molecular cytogenetic identification of a novel dwarf wheat line with ...

    Indian Academy of Sciences (India)

    2012-01-08

    Jan 8, 2012 ... of a pAs1 hybridization band on 2DL chromosome of 31505-1. Two SSR ... [Chen G, Zheng Q, Bao Y, Liu S, Wang H and Li X 2012 Molecular cytogenetic identification of a novel dwarf wheat line with ..... translocations (Fedak and Han 2005; Li et al. ... growth (Cambridge, UK: Cambridge University Press).

  20. Molecular study for the sex identification in Japanese quails ...

    African Journals Online (AJOL)

    In many birds' species such as Japanese quail, sex determination in young and many adult birds is very difficult. Nowadays, sex identification of animals throughout their lives is possible by molecular genetic techniques such as polymerase chain reaction (PCR). The aim of this study was to determine the sex of Japanese ...

  1. Molecular identification of Lodoicea maldivica (coco de mer seeds

    Directory of Open Access Journals (Sweden)

    Mok Chuen-shing

    2011-09-01

    Full Text Available Abstract Background The edible endosperm of Lodoicea maldivica with the common name of coco de mer is used in Chinese medicine for treating cough. Native to Seychelles, Lodoicea maldivica seeds have commanded high prices for centuries due to its scarcity. This study aims to develop a molecular identification method for the authentication of Lodoicea maldivica seeds. Methods DNA was extracted from the sample. Two polymerase chain reaction (PCR systems were developed to amplify a region of the chloroplast DNA and the nuclear phosphoribulokinase (PRK region specific to Lodoicea maldivica respectively. DNA sequence of a sample was determined and compared with that of the Lodoicea maldivica reference material. Results The PRK gene of Lodoicea maldivica was successfully amplified and sequenced for identification. Conclusion A new molecular method for the identification of Lodoicea maldivica seeds in fresh, frozen or dried forms was developed.

  2. Isolation and Metagenomic Identification of Avian Leukosis Virus Associated with Mortality in Broiler Chicken.

    Science.gov (United States)

    Bande, Faruku; Arshad, Siti Suri; Omar, Abdul Rahman

    2016-01-01

    Avian leukosis virus (ALV) belongs to the family Retroviridae and causes considerable economic losses to the poultry industry. Following an outbreak associated with high mortality in a broiler flock in northern part of Malaysia, kidney tissues from affected chickens were submitted for virus isolation and identification in chicken embryonated egg and MDCK cells. Evidence of virus growth was indicated by haemorrhage and embryo mortality in egg culture. While viral growth in cell culture was evidenced by the development of cytopathic effects. The isolated virus was purified by sucrose gradient and identified using negative staining transmission electron microscopy. Further confirmation was achieved through next-generation sequencing and nucleotide sequence homology search. Analysis of the viral sequences using the NCBI BLAST tool revealed 99-100% sequence homology with exogenous ALV viral envelope protein. Phylogenetic analysis based on partial envelope sequences showed the Malaysian isolate clustered with Taiwanese and Japanese ALV strains, which were closer to ALV subgroup J, ALV subgroup E, and recombinant A/E isolates. Based on these findings, ALV was concluded to be associated with the present outbreak. It was recommended that further studies should be conducted on the molecular epidemiology and pathogenicity of the identified virus isolate.

  3. Isolation and Metagenomic Identification of Avian Leukosis Virus Associated with Mortality in Broiler Chicken

    Directory of Open Access Journals (Sweden)

    Faruku Bande

    2016-01-01

    Full Text Available Avian leukosis virus (ALV belongs to the family Retroviridae and causes considerable economic losses to the poultry industry. Following an outbreak associated with high mortality in a broiler flock in northern part of Malaysia, kidney tissues from affected chickens were submitted for virus isolation and identification in chicken embryonated egg and MDCK cells. Evidence of virus growth was indicated by haemorrhage and embryo mortality in egg culture. While viral growth in cell culture was evidenced by the development of cytopathic effects. The isolated virus was purified by sucrose gradient and identified using negative staining transmission electron microscopy. Further confirmation was achieved through next-generation sequencing and nucleotide sequence homology search. Analysis of the viral sequences using the NCBI BLAST tool revealed 99-100% sequence homology with exogenous ALV viral envelope protein. Phylogenetic analysis based on partial envelope sequences showed the Malaysian isolate clustered with Taiwanese and Japanese ALV strains, which were closer to ALV subgroup J, ALV subgroup E, and recombinant A/E isolates. Based on these findings, ALV was concluded to be associated with the present outbreak. It was recommended that further studies should be conducted on the molecular epidemiology and pathogenicity of the identified virus isolate.

  4. Study of the Mechanical Properties and Vibration Isolation Performance of a Molecular Spring Isolator

    Directory of Open Access Journals (Sweden)

    Muchun Yu

    2016-01-01

    Full Text Available Molecular Spring Isolator (MSI is a novel passive vibration isolation technique, providing High-Static-Low-Dynamic (HSLD stiffness based on the use of molecular spring material. The molecular spring material is a solid-liquid mixture consisting of water and hydrophobic nanoporous materials. Under a certain level of external pressure, water molecules can intrude into the hydrophobic pores of nanoporous materials, developing an additional solid-liquid interface. Such interfaces are able to store, release, and transform mechanical energy, providing properties like mechanical spring. Having been only recently developed, the basic mechanic properties of a MSI have not been studied in depth. This paper focuses on the stiffness influence factors, the dynamic frequency response, and the vibration isolation performance of a MSI; these properties help engineers to design MSIs for different engineering applications. First, the working mechanism of a MSI is introduced from a three-dimensional general view of the water infiltration massive hydrophobic nanoporous pores. Next, a wide range of influence factors on the stiffness properties of MSI are studied. In addition, the frequency response functions (FRFs of the MSI vibration isolation system are studied utilizing the matching method based on equivalent piecewise linear (EPL system. Finally, the vibration isolation properties of MSI are evaluated by force transmissibility.

  5. Identification and Structural Characterization of Naturally-Occurring Broad-Spectrum Cyclic Antibiotics Isolated from Paenibacillus

    Science.gov (United States)

    Knolhoff, Ann M.; Zheng, Jie; McFarland, Melinda A.; Luo, Yan; Callahan, John H.; Brown, Eric W.; Croley, Timothy R.

    2015-08-01

    The rise of antimicrobial resistance necessitates the discovery and/or production of novel antibiotics. Isolated strains of Paenibacillus alvei were previously shown to exhibit antimicrobial activity against a number of pathogens, such as E. coli, Salmonella, and methicillin-resistant Staphylococcus aureus (MRSA). The responsible antimicrobial compounds were isolated from these Paenibacillus strains and a combination of low and high resolution mass spectrometry with multiple-stage tandem mass spectrometry was used for identification. A group of closely related cyclic lipopeptides was identified, differing primarily by fatty acid chain length and one of two possible amino acid substitutions. Variation in the fatty acid length resulted in mass differences of 14 Da and yielded groups of related MSn spectra. Despite the inherent complexity of MS/MS spectra of cyclic compounds, straightforward analysis of these spectra was accomplished by determining differences in complementary product ion series between compounds that differ in molecular weight by 14 Da. The primary peptide sequence assignment was confirmed through genome mining; the combination of these analytical tools represents a workflow that can be used for the identification of complex antibiotics. The compounds also share amino acid sequence similarity to a previously identified broad-spectrum antibiotic isolated from Paenibacillus. The presence of such a wide distribution of related compounds produced by the same organism represents a novel class of broad-spectrum antibiotic compounds.

  6. NEW PRODUCER STRAINS OF BIOBUTANOL. І. ISOLATION AND IDENTIFICATION

    Directory of Open Access Journals (Sweden)

    O. A. Tigunova

    2013-02-01

    Full Text Available Getting new, more productive strains of microorganisms that produce butanol is a topical problem. Studing of morphological and physiological characteristics of the isolated strains, improvement of their cultivation conditions, optimization of biobutanol synthesis gives the possibility to organize a cost-effective butanol production technology. The aim of this work was searching new butanol and butyric acid producer strains, their identification and studying the main steps of the selective strains biosynthesis. The objects of this study were microorganisms that had allocated from soils and sludges samples of Kiev’s lakes. Obtained cultures have been screened. Three strains were obtained as promising and identified as C. acetobutylicum, C. tyrobutylicum, C. butylicum. Selective medium have been developed and modified for the microorganisms. Producer’s features were investigated in order to maximize the accumulation of target metabolites.

  7. Isolation and identification of female DNA on postcoital penile swabs.

    Science.gov (United States)

    Cina, S J; Collins, K A; Pettenati, M J; Fitts, M

    2000-06-01

    After sexual assault, cells originating from the assailant may be recovered from the victim. Through polymerase chain reaction (PCR)-based technology, positive scientific identification of the assailant may be made from these cells. Described is a prospective study describing a method for positively identifying cells from a female sex partner obtained from postcoital swabs of the penis of the male sex partner. Swabs were taken from the penis of a man at 1- to 24-hour intervals after coitus. DNA was isolated from each swab through standard organic extraction methods. The presence of female DNA was detected using the gender-specific amelogenin marker. Extracted DNA was amplified for eight different genetic loci using the Promega PowerPlex kit (Promega) and Amplitaq Gold (Perkin Elmer). Amplified samples were electrophoresed on precast sequencing gels (Hitachi) and were analyzed fluorescently using Hitachi's FMBIO 2 fluorescent scanner and software. Each sample obtained from a penile swab or condom was compared to male and female buccal controls. Female DNA was isolated from all postcoital penile swabs as determined by exclusive amplification of the X-chromosome specific 212 base pair amelogenin marker. In all cases, scientific identification of the female DNA from the swabs was determined by coamplification of eight STR loci (PowerPlex) and was compared to female and male control profiles. Cells shed from a female victim during sexual intercourse can be retrieved from the penis of a male offender after sexual intercourse during a 1- to 24-hour postcoital interval. DNA can be extracted from these cells and can be used to scientifically identify the female sexual participant through PCR-based technology. It is suggested that penile swabs be taken from alleged perpetrators of sexual assaults to associate them with a female victim.

  8. Molecular typing of Sporothrix schenckii isolates from cats in Malaysia.

    Science.gov (United States)

    Kano, Rui; Okubo, Miki; Siew, Han Hock; Kamata, Hiroshi; Hasegawa, Atsuhiko

    2015-04-01

    Epidemiological data on the aetiologic agents of feline sporotrichosis in Malaysia have not been reported, though human sporotrichosis in Malaysia is reported to be transmitted primarily via cat scratch. To the best of our knowledge, the present report is the first study of the molecular epidemiology of Sporothrix schenckii isolates from cats with sporotrichosis in Malaysia. In the present work, we characterised 18 clinical isolates from cats in Malaysia based on molecular properties, including sequence analyses of the calmodulin gene and the rDNA ITS region and selective PCR of mating type (MAT) loci. In this study, isolates from feline sporotrichosis were identified as a S. schenckii sensu stricto by sequence analyses of the calmodulin gene and the internal transcribed spacer (ITS) region. Notably, phylogenetic analysis of the ITS confirmed assignment to clinical clade D (and not C) of S. schenckii sensu stricto. Therefore, clinical clade D of S. schenckii sensu stricto appeared to be the prevailing source of feline sporotrichosis in Malaysia. The ratio of MAT1-1-1:MAT1-2-1 in these Malaysian isolates was found to be 1 : 0. This result suggested that a clonal strain of S. schenckii is the prevailing causative agent of feline sporotrichosis in Malaysia. © 2015 Blackwell Verlag GmbH.

  9. Molecular characterization of Cryptococcus neoformans isolated from the environment in Beijing, China.

    Science.gov (United States)

    Dou, Hongtao; Wang, Huizhu; Xie, Shaowei; Chen, Xinxin; Xu, Zhipeng; Xu, Yingchun

    2017-10-01

    The molecular type of environmental Cryptococcus neoformans in Beijing was not clear. Our study aims to reveal the molecular characterization of C. neoformans complex from environment in Beijing, China. A total of 435 samples of pigeon droppings from 11 different homes in Beijing were collected from August to November in 2015. Pigeon droppings were inoculated onto caffeic acid cornmeal agar (CACA) to screen C. neoformans complex. Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed for species identification. Serotype and mating type was determined by specific primers. Restriction fragment length polymorphisms of URA5 (URA5-RFLP) were applied to genotype. Multi-locus sequence typing (MLST) was done for further identification and sequence type (ST) determination. Altogether, 81 isolates of C. neoformans AFLP1/VNI were recognized from 435 pigeon droppings in this study. The positive rate for C. neoformans AFLP1/VNI from pigeon droppings in different homes varied from 5.0% to 52.6%, the average was 20.2%. All of these cryptococcal strains were serotype A, MATα. They were genotyped as VNI by URA5-RFLP and were confirmed by MLST. No other molecular types of C. neoformans and Cryptococcus gattii isolates were isolated. Their STs were identified as ST 31 (n = 54, 66.7%), followed by ST 53 (n = 10), ST 191 (n = 8), ST 5 (n = 5), ST 57 (n = 3), and ST 38 (n = 1). We concluded that not only clinical but also environmental isolates of C. neoformans need to be investigated more deeply and more extensively. The virulence difference between ST 5 and ST 31 need to be explored in the future. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR.

    Science.gov (United States)

    Ali, Shahzad; Ali, Qurban; Melzer, Falk; Khan, Iahtasham; Akhter, Shamim; Neubauer, Heinrich; Jamal, Syed M

    2014-01-01

    Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell's serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n = 5), aborted fetuses (n = 13), and vaginal swabs (n = 12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.

  11. PREVALENCE AND IDENTIFICATION OF VIBRIO SPP. ISOLATED ON AQUACULTURED GILTHEAD SEA BREAM

    Directory of Open Access Journals (Sweden)

    C. Scarano

    2011-01-01

    Full Text Available The aim of the study was to investigate the prevalence of Vibrio spp isolated from gilthead sea bream (Sparus aurata farmed on sea cages and to identify and characterize the pathogen by molecular techniques. Eighty fish were collected from two hatcheries located on the North-Est Sardinian Mediterranean coast, and microbiological analysis were performed on different body parts such as skin, gills, muscle and intestinal tract. Subsequently 100 pure colonies with typical morphology and phenotypic characteristics were selected and submitted to the molecular identification. The analysis on the prevalence of Vibrio spp showed the effect of the hatchery rearing system (P<0.001, of the date of sampling (P<0.001, and of the body part (P<0.001. All the strains selected were confirmed to be members of the genus Vibrio spp by the molecular method/techinique/identification, whereas the rpoA gene sequence analyses allowed to identify 89 strains belonging to the species Vibrio harveyi, 6 to V. diabolicus, 2 to V. parahaemolyticus and 1 to V. mediterranei.

  12. Molecular Identification of SHV,TEM, CTX-M β lactamases Genes and Antibiotics Resistance Pattern of k.pneumoniae Isolates Collected from ICU Patients of Namazi Hospital, Shiraz, Iran

    Directory of Open Access Journals (Sweden)

    T Archin

    2014-01-01

    Full Text Available Abstract Background and aim: β-lactamase enzymes producing bacteria ESBL have spread widely throughout the world. The production of enzymes induces bacterial resistance to a wide range of antibiotics which is leading to the limitation of infection control and correct treatment. The aim of the present study was to investigate patterns of antibiotic susceptibility to antibiotics and the presence of β-lactamase genes SHV, TEM, CTX-M, in Klebsiella pneumoniae isolates from clinical specimens of intensive care. Methods: Susceptibility of isolated bacteria against 10 antibiotics was determined by agar disk diffusion method according to the CLSI guidelines. The strains (DDST were examined for the presence of the spectrum β-lactamase enzymes. Using E-test, the minimum inhibitory concentration (MIC of the antibiotic was determined to cefotaxime. Moreover, SHV, TEM, CTX-M genes were identified by, Multiplex PCR method, and some of them were sequenced. Results: The antibiotic resistance against 10 antibiotics was determined. The highest percentage of isolates was resistant to ampicillin (100% and sensitivity to imipenem was 1.66%. In this study, the majority of strains produced ESBL (60%. TEM gene in 34.38% and all three genes (TEM and SHV and CTX at 33.13% of isolates were observed. Conclusion: The present study showed that the K. pneumoniae producing ESBL in patients in ICU are common. Therefore, the use of procedures and policies for infection control in hospitals and especially ICU is necessary. Key words: Klebsiella pneumoniae, ESBL, Multiplex PCR, antibiotic sensitivity

  13. Isolation and identification of local bacteria endophyte and screening of its antimicrobial property against pathogenic bacteria and fungi

    Science.gov (United States)

    Fikri, Ahmad Syairazie Ibrahim; Rahman, Irman Abdul; Nor, Norefrina Shafinaz Md; Hamzah, Ainon

    2018-04-01

    Endophytes are organisms, often fungi and bacteria that live in living plant cells. These organisms reside in the living tissues of the host plant in a variety of relationships, ranging from symbiotic to slightly pathogenic. The endophytes may produce a plethora of substances that have potential to be used in modern medicine, agriculture and industry. The aims of this study are to isolate, identify and screening antimicrobial activity of bacterial endophytes. The endophytes were isolated using nutrient agar, incubated at 37°C for 48 hours. Identification of the isolates were done based on morphological characteristics, biochemical tests and 16S rDNA molecular analysis. Disk diffusion method was used to screen for antimicrobial activity of metabolites from endophytes against pathogenic bacteria. Screening for antifungal activity of selected endophytes was done using dual culture method againts pathogenic fungi followed by Kirby-Bauer method. Results showed endophytes designated as B2c and B7b have positive antimicrobial activity. The metabolites from isolate B2c showed antimicrobial activity against pathogenic bacteria methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus and Staphylococcus epidermis, while isolate B7b have positive activities againts MRSA, S. aureus and Pseudomonas aeruginosa. Isolates B2c displayed antifungal activity against Fusarium oxysporum, Fusarium solani, Phytophthora palmivora and Colletotrichum gloeosporioides. Identification using biochemical tests and 16S rDNA sequences identified isolate B2c as Pseudomonas resinovorans with 97% homology and isolate B7b as Bacillus subtilis with 98% homology.

  14. Isolation and biochemical and molecular characterization of Listeria monocytogenes in food

    International Nuclear Information System (INIS)

    Helel, Salma

    2008-01-01

    monocytogenes is a Gram-positive bacteria, saprophytic, non-spore. This is an extremely resistant seeds to environmental conditions outside, especially since the cold psychrotrophic. It can contaminate raw vegetables, cooked meals ready for consumption or foods to be stored in the refrigerator, such as cheese or meat. It is the bacteria responsible for listeriosis. It threatens first unborn children, infants, pregnant women, the elderly and people whose immune system is weakened. Strains of Listeria spp isolated from foods (seafood, meat, meat) were first identified at the stage of the genus by classical tests (Gram staining, catalase test, oxidase test and mobility) and stage of the test case by hemolysis, CAMP test and the gallery Api Listeria. Biochemical characterization allowed after a numerical analysis, to assign 100% of isolates to the genus Listeria. Molecular characterization was performed by PCR amplification of genes coding for protein p60 (iap), the listeriolysine O (hly), the Phosphatidylinositol Phospholipase C (PI-PLC plca) Phosphatidylcholine Phospholipase C (plcB). The result showed an amplification of the iap gene of 100% of the hly gene, plca, plcB of 31.81%. This characterization represents an identification of the collection on the genetic level and shows that 31.81% of isolates, is likely to express the genes responsible for virulence factors of L. monocytogenes, to produce listeriolysine O, phospholipase C and Lecithinase. The molecular identification was performed by microarray technique and identified isolates L. September monocytogenes (five original clinical isolates and two food-borne), fourteen L. innocua (of food) and a strain not identified by DNA chip.. (Author)

  15. Identification of lead-resistant endophytic bacteria isolated from rice

    International Nuclear Information System (INIS)

    Perez-Cordero, Alexander; Barraza-Roman, Zafiro; Martinez-Pacheco, Dalila

    2015-01-01

    Resistance of endophytic bacteria in vitro was evaluated at different lead concentrations. The tissue samples of commercial rice varieties at tillering stage were collected during the first half of 2013, in Monteria, Cordoba, Colombia. Each tissue was subjected to surface cleaning. Endophytic bacteria were isolated in agar R_2A medium. The population density (CFU/g tissue) was determined from each tissue by direct counting of R_2A medium surface. Morphotypes were classified by shape, color, size and appearance. A total of 168 morphotypes were isolated from root, tillers and leaf of different commercial varieties of rice. The lead resistance test is performed in vitro, The lead resistance test was performed in vitro, by the suspensions of endophytic bacteria in log phase and inoculation in minimal medium with five concentrations of lead as Pb (NO_3)_2. The experiment was incubated at 32 degrees celsius and agitated at 150 rpm for five days. The measure of turbidimetry at 600 nm was conduced every hour afterstarting the test. Endophytic bacteria showed the ability to grow at concentrations of 100% of Pb as Pb (NO_3) _2. The presence of Burkholderia cepacia and Pseudomonas putida, which showed resistance to differents lead concentration was confirmed as result of the identification with kit API20E. (author) [es

  16. Identification of lead- resistant endophytic bacteria isolated from rice.

    Directory of Open Access Journals (Sweden)

    Alexander Pérez-Cordero

    2015-06-01

    Full Text Available   The objective of this study was to evaluate in vitro the endophytic bacteria resistance to different lead concentrations. The sampling was undertaken in the first half of 2013, when tissue samples of commercial varieties of rice at tillering stage were collected in Montería, Cordoba, Colombia. Each tissue was subjected to surface cleaning. Endophytic bacteria in agar R2A medium were isolated. Population density (CFU/g tissue was determined from each tissue, by direct counting of R2A medium surface. morphotypes were classified by shape, color, size, and appearance. A total of 168 morphotypes were isolated from root, tillers, and leaf of different commercial varieties of rice. The lead resistance test was performed in vitro, to do that, suspensions of endophytic bacteria in log phase were prepared and inoculated in minimal medium with five concentrations of lead as Pb(NO32. The experiment was incubated at 32 °C and agitated at 150 rpm, for five days. Every hour afterstarting the test, turbidimetry measuring at 600 nm was conducted. Results showed the ability of endophytic bacteria to grow at concentrations of 100% of Pb as Pb(NO32. The results of the identification with kit API20E confirmed the presence of Burkholderia cepacia and Pseudomonas putida, which showed resistance to different lead concentrations.

  17. DNA barcode-based molecular identification system for fish species.

    Science.gov (United States)

    Kim, Sungmin; Eo, Hae-Seok; Koo, Hyeyoung; Choi, Jun-Kil; Kim, Won

    2010-12-01

    In this study, we applied DNA barcoding to identify species using short DNA sequence analysis. We examined the utility of DNA barcoding by identifying 53 Korean freshwater fish species, 233 other freshwater fish species, and 1339 saltwater fish species. We successfully developed a web-based molecular identification system for fish (MISF) using a profile hidden Markov model. MISF facilitates efficient and reliable species identification, overcoming the limitations of conventional taxonomic approaches. MISF is freely accessible at http://bioinfosys.snu.ac.kr:8080/MISF/misf.jsp .

  18. Exoenzyme activity and possibility identification of Candida dubliniensis among Candida albicans species isolated from vaginal candidiasis.

    Science.gov (United States)

    Jafari, Maryam; Salari, Samira; Pakshir, Keyvan; Zomorodian, Kamiar

    2017-09-01

    Vulvovaginal candidiasis (VVC) or vaginal candidiasis is a common fungal infection of the genitals causing inflammation, irritation, itching, and vaginal discharge. Common yeast infections are caused by the yeast species C. albicans. However, there are other species of Candida such as C. dubliniensis which are considered as the causative agents of this infection. Hydrolytic enzymes such as proteinase and coagulase are known as virulence factors. The aim of this study was the molecular confirmation and differentiation of C. dubliniensis among C. albicans strains isolated from women with vulvovaginal candidiasis by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) and the evaluation of proteinase and coagulase activities. A total of 100 C. albicans strains isolated from women with vulvovaginal candidiasis referred to Shiraz medical clinics were enrolled in the study. All the isolates were primarily identified by conventional methods. PCR-RFLP method was used for the confirmation and identification of C. albicans and C. dubliniensis. Moreover, in vitro proteinase and coagulase activities of these isolates were evaluated using bovine serum albumin media and classical rabbit plasma tube test. As a result, PCR-RFLP identified 100% of the isolates as C. albicans, and no C. dubliniensis could be identified in this study. 84% of the isolates showed proteinase activity, whereas coagulase activity was only detected in 5% of the isolates. This study reveals that C. dubliniensis plays no role in vaginal candidiasis in Iranian patients. Proteinase production could be an essential virulence factor in C. albicans pathogenicity, but coagulase activity has less potential in this matter. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Rapid molecular identification and characteristics of Lactobacillus strains.

    Science.gov (United States)

    Markiewicz, L H; Biedrzycka, E; Wasilewska, E; Bielecka, M

    2010-09-01

    Eleven type strains and 24 Lactobacillus isolates, preliminarily classified to the species due to phenotypic features, were investigated. Standard methods of identification with species-specific PCRs and typing with PFGE (with ApaI, NotI and SmaI restriction enzymes) allowed us to distinguish 16 unique strains belonging to 5 species (L. acidophilus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. salivarius). Alternative approach with 16S-23S rDNA ARDRA identification (with merely two restrictases, BsuRI and TaqI) and PCR-based typing (RAPD with two random- and rep-PCR with (GTG)(5) primers) showed to be more discriminative, i.e. 21 unique strains were classified in the same species as above. As a result, 7 out of 24 phenotypically species-assigned isolates were reclassified. The alternative procedure of rapid identification and typing of Lactobacillus isolates appeared to be equally effective and shortened from 1 week to 2-3 d (in comparison to the standard methods).

  20. Molecular characterisation of Mycobacterium caprae strains isolated in Poland.

    Science.gov (United States)

    Krajewska-Wędzina, Monika; Kozińska, Monika; Orłowska, Blanka; Weiner, Marcin; Szulowski, Krzysztof; Augustynowicz-Kopeć, Ewa; Anusz, Krzysztof; Smith, Noel H

    2018-03-10

    Bovine tuberculosis (bovine TB, bTB) is caused by bovine bacilli: Mycobacterium bovis and M caprae The studies conducted in Poland, in the National Bovine Tuberculosis Reference Laboratory in the Department of Microbiology of the National Veterinary Research Institute in Pulawy, show that animal tuberculosis in Poland is also caused by M caprae We here describe the identification and genotypic assessment of 52 isolates of M caprae obtained from Polish cattle and wild animals over the last five years. We show that strains isolated from bison have significant genotypic diversity and are distinct compared with the genotypes of strains isolated from cattle. Similarly, isolates from cattle herds can be highly genotypically variable. Formal designation of the members of the Mycobacterium tuberculosis complex is controversial in Poland; there is a gap in veterinary legislation with regard to bTB and no explicit mention of M caprae causing tuberculosis in animal. © British Veterinary Association (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  1. Molecular epidemiology of Corynebacterium pseudotuberculosis isolated from horses in California.

    Science.gov (United States)

    Haas, Dionei J; Dorneles, Elaine M S; Spier, Sharon J; Carroll, Scott P; Edman, Judy; Azevedo, Vasco A; Heinemann, Marcos B; Lage, Andrey P

    2017-04-01

    Corynebacterium pseudotuberculosis biovar Equi is an important pathogen of horses. It is increasing in frequency in the United States, and is responsible for various clinical forms of infection, including external abscesses, internal abscesses of the abdominal or thoracic cavities, and ulcerative lymphangitis. The host/pathogen factors dictating the form or severity of infection are currently unknown. Our recent investigations have shown that genotyping C. pseudotuberculosis isolates using enterobacterial repetitive intergenic consensus (ERIC)-PCR is useful for understanding the evolutionary genetics of the species as well for molecular epidemiology studies. The aims of the present study were to assess (i) the genetic diversity of C. pseudotuberculosis strains isolated from horses in California, United States and (ii) the epidemiologic relationships among isolates. One hundred and seven C. pseudotuberculosis biovar Equi isolates from ninety-five horses, and two C. pseudotuberculosis biovar Ovis strains, C. pseudotuberculosis ATCC 19410 T type strain and C. pseudotuberculosis 1002 vaccine strain, were fingerprinted using the ERIC 1+2-PCR. C. pseudotuberculosis isolated from horses showed a high genetic diversity, clustering in twenty-seven genotypes with a diversity index of 0.91. Minimal spanning tree showed four major clonal complexes with a pattern of temporal clustering. Strains isolated from the same horse showed identical ERIC 1+2-PCR genotype, with the exception of two strains isolated from the same animal that showed distinct genotypes, suggesting a co-infection. We found no strong genetic signals related to clinical form (including internal versus external infections). However, temporal clustering of genotypes was observed. Copyright © 2016. Published by Elsevier B.V.

  2. Isolation and identification of citrus psorosis virus Egyptian isolate (CPsV-EG).

    Science.gov (United States)

    Ghazal, S A; El-Dougdoug, Kh A; Mousa, A A; Fahmy, H; Sofy, A R

    2008-01-01

    Citrus psorosis ophiovirus (CPsV), is considered to be of the most serious and deter mental virus pathogen's citrus species trees in Egypt. CPsV-EG was isolated from infected citrus grapefruit (C. paradisi Macf.) at Agric. Res. Centre (ARC). The grapefruit which used for CPsV-EG isolate was found to be free from CTV, CEVd and Spiroplasma citri where as gave -ve results with DTBIA, tissue print hybridization and Diene's stain respectively. CPsV-EG was detected on the basis of biological indexing by graft inoculation which gave oak leaf pattern (OLP) on Dweet tangor and serological assay by DAS-ELISA using Mab specific CPsV. CPsV-EG was reacted with variable responses on 16 host plants belonging to 6 families. Only 8 host plants are susceptible and showed visible external symptoms which appeared as local, systemic and local followed by systemic infections. CPsV-EG isolate was transmitted from infected citrus to citrus by syringe and grafting and herbaceous plants by forefinger inoculation and syringe. The woody indicators and rootstocks were differed in response to CPsV-EG isolate which appeared as no-response, response, sensitivity and hypersensitivity. The serological characters represented as the antigenic determinants of CPsV-EG isolate related to monoclonal antibodies specific CPsV strain where as appeared precipitation reaction by DAS-ELISA and DTBIA. The partial fragment of RNA3 (coat protein gene) of CPsV-EG (-1140bp and -571bp) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from grapefruit tissues using two sets primers specific CPsV (CPV3 and CPV4) and (PS66 and PS65) respectively. The virus under study was identified as CPsV-EG isolate according to biological, serological and molecular characters.

  3. Agricultural production - Phase 2. Indonesia. Isolation of arboviruses, their identification and the identification of their culicoides vectors in Indonesia

    International Nuclear Information System (INIS)

    Mellor, P.S.

    1991-01-01

    The aims of the two-week mission were to provide assistance in studies to determine the incidence and importance of arbovirus infection in ruminants in Indonesia, specifically to help with identification of the vectors tat transmit bluetongue and related arbovirus infections, and to develop work plans for future studies under the project. The report contains detailed information on handling systems for Culicoides species, on identification of Culicoides to be used for viral isolation and on the isolation of virus from Culicoides

  4. Molecular and biochemical characterization of Pseudomonas putida isolated from bottled uncarbonated mineral drinking water

    Directory of Open Access Journals (Sweden)

    Tasić S.

    2014-01-01

    Full Text Available Pseudomonas putida belongs to a group of opportunistic pathogens that can cause disease in people with weakened or damaged immune systems. Some strains have medical significance, and for most ingestion is not the primary route of infection. If water used by predisposed subjects is contaminated by P. putida, they may become ill. The aim of this work was the biochemical and molecular characterization of strain ST3 of P. putida isolated from non-carbonated bottled drinking water from Jakov Do 4 on Mt. Vlasina. Characterization of P. putida was performed to assess the risk to human health of the indigenous strains present in the water. Biochemical characterization of strains was performed using the manual identification system ID 32 GN (BioMérieux. Identification was obtained using the database identification software ATB System (Bio-Mérieux. Molecular characterization was performed by PCR amplification and 16S rDNA “thermal cycling sequencing”. Biochemical identification of the strain ST3 was accurate (Id = 99.8%. Comparing the sequences obtained for strain ST3 with NCBI gene bank sequences for 16S rRNA, the highest similarity of our strain (96% identity with a strain of P. putida, designated as biotype A (gi|18076625|emb|AJ308311.1|.PPU308311 isolated in New Zealand, was obtained. While comparison with the NCBI collection of all deposited sequences showed that the 16S rRNA gene sequence of strain ST3 has very high homology, it is not identical, indicating indirectly that strain ST3 is an indigenous strain.

  5. An insight into the isolation, enumeration and molecular detection of Listeria monocytogenes in food

    Directory of Open Access Journals (Sweden)

    Jodi Woan-Fei Law

    2015-11-01

    Full Text Available Listeria monocytogenes, a foodborne pathogen that can cause listeriosis through the consumption of food contaminated with this pathogen. The ability of L. monocytogenes to survive in extreme conditions and cause food contaminations have become a major concern. Hence, routine microbiological food testing is necessary to prevent food contamination and outbreaks of foodborne illness. This review provides insight into the methods for cultural detection, enumeration and molecular identification of L. monocytogenes in various food samples. There are a number of enrichment and plating media that can be used for the isolation of L. monocytogenes from food samples. Enrichment media such as buffered Listeria Enrichment Broth (BLEB, Fraser broth and University of Vermont Medium (UVM Listeria enrichment broth are recommended by regulatory agencies such as FDA-BAM, USDA-FSIS and ISO. Many plating media are available for the isolation of L. monocytogenes, for instance, PALCAM, Oxford and other chromogenic media. Besides, reference methods like FDA-BAM, ISO 11290 method and USDA-FSIS method are usually applied for the cultural detection or enumeration of L. monocytogenes. MPN technique is applied for the enumeration of L. monocytogenes in the case of low level contamination. Molecular methods including polymerase chain reaction (PCR, multiplex polymerase chain reaction (mPCR, real-time/quantitative polymerase chain reaction (qPCR, nucleic acid sequence-based amplification (NASBA, loop-mediated isothermal amplification (LAMP, DNA microarray and Next Generation Sequencing (NGS technology for the detection and identification of L. monocytogenes are discussed in this review. Overall, molecular methods are rapid, sensitive, specific, time- and labour-saving. In future, there are chances for the development of new techniques for the detection and identification of foodborne with improved features.

  6. An insight into the isolation, enumeration, and molecular detection of Listeria monocytogenes in food

    Science.gov (United States)

    Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

    2015-01-01

    Listeria monocytogenes, a foodborne pathogen that can cause listeriosis through the consumption of food contaminated with this pathogen. The ability of L. monocytogenes to survive in extreme conditions and cause food contaminations have become a major concern. Hence, routine microbiological food testing is necessary to prevent food contamination and outbreaks of foodborne illness. This review provides insight into the methods for cultural detection, enumeration, and molecular identification of L. monocytogenes in various food samples. There are a number of enrichment and plating media that can be used for the isolation of L. monocytogenes from food samples. Enrichment media such as buffered Listeria enrichment broth, Fraser broth, and University of Vermont Medium (UVM) Listeria enrichment broth are recommended by regulatory agencies such as Food and Drug Administration-bacteriological and analytical method (FDA-BAM), US Department of Agriculture-Food and Safety (USDA-FSIS), and International Organization for Standardization (ISO). Many plating media are available for the isolation of L. monocytogenes, for instance, polymyxin acriflavin lithium-chloride ceftazidime aesculin mannitol, Oxford, and other chromogenic media. Besides, reference methods like FDA-BAM, ISO 11290 method, and USDA-FSIS method are usually applied for the cultural detection or enumeration of L. monocytogenes. most probable number technique is applied for the enumeration of L. monocytogenes in the case of low level contamination. Molecular methods including polymerase chain reaction, multiplex polymerase chain reaction, real-time/quantitative polymerase chain reaction, nucleic acid sequence-based amplification, loop-mediated isothermal amplification, DNA microarray, and next generation sequencing technology for the detection and identification of L. monocytogenes are discussed in this review. Overall, molecular methods are rapid, sensitive, specific, time- and labor-saving. In future, there are

  7. Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

    Science.gov (United States)

    Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai

    2014-01-01

    Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse. PMID:24945530

  8. Rapid molecular identification of human taeniid cestodes by pyrosequencing approach.

    Directory of Open Access Journals (Sweden)

    Tongjit Thanchomnang

    Full Text Available Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1 gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse.

  9. Pyrosequencing as a tool for the identification of common isolates of Mycobacterium sp.

    Science.gov (United States)

    Tuohy, Marion J; Hall, Gerri S; Sholtis, Mary; Procop, Gary W

    2005-04-01

    Pyrosequencing technology, sequencing by addition, was evaluated for categorization of mycobacterial isolates. One hundred and eighty-nine isolates, including 18 ATCC and Trudeau Mycobacterial Culture Collection (TMC) strains, were studied. There were 38 Mycobacterium tuberculosis complex, 27 M. kansasii, 27 MAI complex, 21 M. marinum, 14 M. gordonae, 20 M. chelonae-abscessus group, 10 M. fortuitum, 5 M. xenopi, 3 M. celatum, 2 M. terrae complex, 20 M. mucogenicum, and 2 M. scrofulaceum. Nucleic acid extracts were prepared from solid media or MGIT broth. Traditional PCR was performed with one of the primers biotinylated; the assay targeted a portion of the 16S rRNA gene that contains a hypervariable region, which has been previously shown to be useful for the identification of mycobacteria. The PSQ Sample Preparation Kit was used, and the biotinylated PCR product was processed to a single-stranded DNA template. The sequencing primer was hybridized to the DNA template in a PSQ96 plate. Incorporation of the complementary nucleotides resulted in light generation peaks, forming a pyrogram, which was evaluated by the instrument software. Thirty basepairs were used for isolate categorization. Manual interpretation of the sequences was performed if the quality of the 30-bp sequence was in doubt or if more than 4 bp homopolymers were recognized. Sequences with more than 5 bp of bad quality were deemed unacceptable. When blasted against GenBank, 179 of 189 sequences (94.7%) assigned isolates to the correct molecular genus or group. Ten M. gordonae isolates had more than 5 bp of bad quality sequence and were not accepted. Pyrosequencing of this hypervariable region afforded rapid and acceptable characterization of common, routinely isolated clinical Mycobacterium sp. Algorithms are recommended for further differentiation with an additional sequencing primer or additional biochemicals.

  10. Rapid Isolation and Molecular Detection of Streptomycin-Producing Streptomycetes

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    M Motovali-bashi

    2006-07-01

    Full Text Available Introduction: Streptomyces species are mycelial, aerobic gram-positive bacteria that are isolated from soil and produce a diverse range of antibiotics. Streptomyces griseus produces the antibiotic, streptomycin and forms spores even in a liquid culture. The gene cluster for the production of Streptomycin antibiotic contains strR gene that encodes StrR, a pathway-specific regulator. Then, this pathway-specific regulator induces transcription of other streptomycin production genes in the gene cluster. The overall aim of this work was rapid isolation and molecular detection of streptomycin-producing Streptomycetes, especially S. griseus, from Iranian soils in order to manipulate them for increased production of streptomycin. Methods: This research used new initiative half-specific medium for isolation of Streptomycetes from natural environments, called FZmsn. The fifty colonies of Streptomyces strains grown on the surface of FZmsn medium isolated from environmental samples were defined on the basis of their morphological characteristics and light microscope studies. A set of primers was designed to detect strR by OLIGO software. Results: In colony-PCR reactions followed by gel electrophoresis, 6 colonies from Streptomyces strains colonies were detected as S. griseus colonies. Conclusion: These native Streptomyces strains will be used for genetic manipulation of S. griseus in order to increase production levels of streptomycin.

  11. Molecular typing of Burkholderia cepacia complex isolated from patients attending an Italian Cystic Fibrosis Centre.

    Science.gov (United States)

    Teri, Antonio; Sottotetti, Samantha; Biffi, Arianna; Girelli, Daniela; D'Accico, Monica; Arghittu, Milena; Colombo, Carla; Corti, Fabiola; Pizzamiglio, Giovanna; Cariani, Lisa

    2018-04-01

    Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). Bcc infection is often extremely difficult to treat due to its intrinsic resistance to multiple antibiotics. In addition, it seems to speed up the decline of lung function and is considered a contraindication for lung transplantation in CF. This study investigates the species of the Bcc strains recovered from chronically infected CF subjects by means of: isolation, identification methods and complete recA nucleotide sequences of 151 samples. Molecular typing showed that B. cenocepacia III is the dominant strain found in the group of subjects being treated at the Milan CF Centre (Italy) and that the infection is chronically maintained by the same species. Defining species by means of molecular analysis yields important information for the clinician in order to establish the most appropriate therapy and implement correct measures for prevention of transmission among CF subjects.

  12. Isolation and Identification of L-asparaginase producing Erwinia strains which isolated from Potato Farms

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    Arastoo Badoei-Dalfard

    2016-09-01

    Full Text Available Introduction: L-Asparaginase can be effectively used for the treatment of lymphoblastic leukemia. The rapid growth of cancer cells are needed for L-asparagine abundant storage. L-asparaginase catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. The purpose of this study was to isolate and identify the L-asparaginase producing Erwinia strains from the potato farms of Jiroft. Materials and methods: Pectolytic Erwinia species isolated from crumbling potato in M9 medium. The desired L-asparaginase producing bacteria were isolated based on the color changes. Biochemical-microbial and the plant pathogenicity tests of these strains were also investigated with potato and geranium. The L-asparaginase production and molecular detection of these Erwinia strains were also investigated. Results: In this study, L-asparaginase producing Erwinia was isolated on the CVP and M9 mediums. The inoculation of Erwinia strains on the potato and geranium plants showed that Er8 and Er11 species have the ability to cause plant pathogenicity. Results showed that the maximum pathogenicity of Er8 and Er11 was observed after 48 and 15 h of inoculation in potato and geranium plants, respectively. 16S rDNA sequencing and phylogenetic analyses exhibited that Er8 and Er11 strains were similar to Erwinia chrysanthemi with 98% homology. Discussion and conclusion: Because of several applications of the Erwinia L-asparaginase in various fields, isolated Erwinia and their L-asparaginase can be suitable for applied utilization.

  13. Gender Identification in Date Palm Using Molecular Markers.

    Science.gov (United States)

    Awan, Faisal Saeed; Maryam; Jaskani, Muhammad J; Sadia, Bushra

    2017-01-01

    Breeding of date palm is complicated because of its long life cycle and heterozygous nature. Sexual propagation of date palm does not produce true-to-type plants. Sex of date palms cannot be identified until the first flowering stage. Molecular markers such as random amplified polymorphic DNA (RAPD), sequence-characterized amplified regions (SCAR), and simple sequence repeats (SSR) have successfully been used to identify the sex-linked loci in the plant genome and to isolate the corresponding genes. This chapter highlights the use of three molecular markers including RAPD, SCAR, and SSR to identify the gender of date palm seedlings.

  14. Molecular detection of an atypical, highly resistant, clonal Pseudomonas aeruginosa isolate in cystic fibrosis patients.

    LENUS (Irish Health Repository)

    Keating, Deirdre

    2013-03-01

    The identification of Pseudomonas aeruginosa (P. aeruginosa) isolates in sputum from cystic fibrosis (CF) patients can be challenging due to the multitude of phenotypic changes isolates undergo during adaptation to the microenvironment of the CF lung.

  15. Molecular characterization, fitness and mycotoxin production of benzimidazole-resistant isolates of Penicillium expansum.

    Science.gov (United States)

    Malandrakis, Anastasios A; Markoglou, Anastasios N; Konstantinou, Sotiris; Doukas, Eleftherios G; Kalampokis, John F; Karaoglanidis, George S

    2013-04-01

    Penicillium expansum field-strains resistant to benzimidazole fungicides were isolated in high frequency from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, N. Greece. In vitro fungitoxicity tests resulted in the identification of two different resistant phenotypes: highly (BEN-HR) and moderately (BEN-MR) carbendazim-resistant. Thirty seven percent of the isolated P. expansum strains belonged to the BEN-HR phenotype, carried no apparent fitness penalties and exhibited resistance levels higher than 60 based on EC50 values. Cross resistance studies with other benzimidazole fungicides showed that all BEN-HR and BEN-MR isolates were also less sensitive to benomyl and thiabendazole. Fungitoxicity tests on the response of BEN-HR isolates to fungicides belonging to other chemical classes revealed no cross-resistance relationships between benzimidazoles and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the anilinopyrimidine cyprodinil, the QoI pyraclostrobin, the imidazole imazalil and the triazole tebuconazole, indicating that a target-site modification is probably responsible for the BEN-HR phenotype observed. Contrary to the above, some BEN-MR isolates exhibited an increased sensitivity to cyprodinil compared to benzimidazole-sensitive ones. BEN-MR isolates had fitness parameters similar to the benzimidazole-sensitive isolates except for conidia production which appeared significantly decreased. Analysis of mycotoxin production (patulin and citrinin) showed that all benzimidazole-resistant isolates produced mycotoxins at concentrations significantly higher than sensitive isolates both on culture medium and on artificially inoculated apple fruit. Comparison of the β-tubulin gene DNA sequence between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in most but not all HR isolates tested. Molecular analysis of the

  16. Molecular and serological characterization of the first Leptospira santarosai strain isolated from a dog.

    Science.gov (United States)

    Miotto, Bruno Alonso; Moreno, Luisa Zanolli; Guilloux, Aline Gil Alves; Sousa, Gisele Oliveira de; Loureiro, Ana Paula; Moreno, Andrea Micke; Lilenbaum, Walter; Vasconcellos, Silvio Arruda; Heinemann, Marcos Bryan; Hagiwara, Mitika Kuribayashi

    2016-10-01

    Leptospirosis is a zoonotic disease of global importance caused by pathogenic Leptospira species. Dogs can become asymptomatically infected, acting like reservoir hosts for pathogenic Leptospira, notably Leptospira interrogans serovar Canicola. Identification of such individuals and characterization of leptospires involved in chronic infections may unravel the role of dogs in the epidemiology of particular leptospiral strains. The aim of the present work was to describe the first Leptospira santarosai strain isolated from a dog. The dog was kept in a public shelter in São Paulo city, Brazil, and presented asymptomatic urinary shedding detected by PCR. Prospective evaluation was performed to fully characterize its chronic carrier state. The dog did not present anti-Leptospira titles or clinical/laboratorial abnormalities during the evaluations; nevertheless long-term urinary shedding was confirmed by PCR and leptospires were recovered from two occasions. The isolated strain was molecularly characterized by partial 16S rRNA and secY gene sequencing and MLST analysis. Serogroup identification was performed using polyclonal antibodies. The strain was identified as Leptospira santarosai, serogroup Sejroe. This is the first evidence in the literature of the isolation of L. santarosai in dogs. Our findings show that dogs can persistently harbor leptospires other than L. interrogans. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Isolation, identification and molecular docking as cyclooxygenase (COX) inhibitors of the main constituents of Matricaria chamomilla L. extract and its synergistic interaction with diclofenac on nociception and gastric damage in rats.

    Science.gov (United States)

    Ortiz, Mario I; Fernández-Martínez, Eduardo; Soria-Jasso, Luis Enrique; Lucas-Gómez, Isaac; Villagómez-Ibarra, Roberto; González-García, Martha P; Castañeda-Hernández, Gilberto; Salinas-Caballero, Mireya

    2016-03-01

    Chamomile (Matricaria chamomilla L., Asteraceae) is a medicinal plant widely used as remedy for pain and gastric disorders. The association of non-steroidal anti-inflammatory drugs (NSAIDs) with medicinal plant extracts may increase its antinociceptive activity, permit the use of lower doses and limit side effects. The aim was to isolate and identify the main chemical constituents of Matricaria chamomilla ethanolic extract (MCE) as well as to explore their activity as cyclooxygenase (COX) inhibitors in silico; besides, to examine the interaction between MCE and diclofenac on nociception in the formalin test by isobolographic analysis, and to determine the level of gastric injury in rats. Three terpenoids, α-bisabolol, bisabolol oxide A, and guaiazulene, were isolated and identified by (1)H NMR. Docking simulation predicted COX inhibitory activity for those terpenoids. Diclofenac, MCE, or their combinations produced an antinociceptive effect. The sole administration of diclofenac and the highest combined dose diclofenac-MCE produced significant a gastric damage, but that effect was not seen with MCE alone. An isobologram was constructed and the derived theoretical ED35 for the antinociceptive effect was significantly different from the experimental ED35; hence, the interaction between diclofenac and MCE that mediates the antinociceptive effect is synergist. The MCE contains three major terpenoids with plausible COX inhibitory activity in silico, but α-bisabolol showed the highest affinity. Data suggest that the diclofenac-MCE combination can interact at the systemic level in a synergic manner and may have therapeutic advantages for the clinical treatment of inflammatory pain. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  18. Isolation and identification of Bacillus spp. from compost material, compost and mushroom casing soil active against Trichoderma spp.

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    Stanojević Olja

    2016-01-01

    Full Text Available The isolation of bacteria was carried out from samples of straw and chicken manure, compost at various stages of the composting process and casing soil used for growing button mushrooms. A preliminary screening of 108 bacterial isolates for antagonistic activity against Trichoderma aggressivum f. europaeum showed that 23 tested isolates inhibited mycelial growth of the pathogenic fungus. Further screening with four indicator isolates of fungi revealed that all 23 bacterial isolates inhibited the growth of T. aggressivum f. europaeum, T. harzianum and T. koningii, while only 13 isolates inhibited the growth of T. atroviride. T. aggressivum f. europaeum proved to be the most sensitive, with many bacterial isolates generating a high percentage of growth inhibition. Only two bacterial isolates (B-129 and B-268 were successful in inhibiting the growth of all 4 tested pathogens. All 23 bacterial isolates were characterized as Gram-positive and catalase-positive and were subjected to molecular identification based on the partial sequence, the hypervariant region of the 16S rDNA. It was shown that the obtained bacterial strains belong to Bacillus subtilis, B. amyloliquefaciens, B. licheniformis and B. pumilus species. [Projekat Ministarstva nauke Republike Srbije, br. 31043 i br. 173026

  19. Molecular epidemiology of pneumococcal isolates from children in China

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    Li-Hua Kang

    2016-04-01

    Full Text Available Objectives: To investigate the molecular epidemiology of pneumococcal isolates in Chongqing, China. Methods: In this cross-sectional study, 51 invasive Streptococcus pneumoniae (S. pneumoniae strains were from children with invasive pneumococcal disease (IPD and 32 carriage strains from healthy children from January 2010 to December 2013 at the Children’s Hospital of Chongqing Medical University, Chongqing, China. Multilocus sequence typing was used to identify the sequence types (STs. Capsular serotypes were determined by multiplex polymerase chain reaction. Drug susceptibility and resistance was determined by minimum inhibitory concentrations. Results: In this study, 11 serotypes were identified among the 83 S. pneumoniae clinical isolates tested. Prevalent serotypes were 19A (20.4%, 6A/B (20.4%, 19F (15.7%, 14 (14.5%, and 23F (10.8%. Serotype 19F was the most frequent carriage strain, and serotype 19A was the most frequent invasive strain. The ST983 was the most prevalent ST for carriage strains, and ST320 was the most prevalent ST for invasive strains. For gene analysis, psaA (99.5% and piaA (98.6% were present and much conserved in all pneumococci tested. The cps2A and pcsB genes were more frequent in invasive isolates than carriage strains. Antimicrobial resistance rates of invasive pneumococcal isolates to erythromycin, penicillin, meropenem, cefotaxime, and clindamycin were higher than the carriage isolates from children. Conclusion: Our epidemiological evidence shows that 19A, 6A/B, 19F, 14, and 23F remain the most prevalent serotypes, which can be targeted by PCV13. Genotypes and drug resistance varied between carriage and invasive strains. The PsaA and PiaA may be good protein vaccine candidates.

  20. The place of molecular methods in the identification of dermatophytes and the determination of their feasibility

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    Fatma Bıyık

    2013-03-01

    Full Text Available Background and Design: Unlike opportunistic fungi, dermatophytes cannot be isolated on the conventional culture media in a few days. Their growing periods cover approximately two weeks in a suitable media and identification are made with conventional methods as typical macroscopic and microscopic appearance. However, successful results are not always obtained with the phenotypic features, and thus, diagnostic problems and delay in diagnosis and treatment may arise. For this reason, the methods based on nucleic acid amplification have been necessary. In this study, we aimed to identify 56 dermatophytes strains, which were identified by conventional methods, by molecular methods and to investigate the correlation between the two methods and to determine the usability of molecular methods in routine laboratories. Materials and Methods: Several clinical samples of 270 patients with suspected dermatophytoses (hair+scalp, skin and nail scrapings were examined by conventional methods; Sabouraud dextrose agar, corn meal agar and potato dextrose agar were used for isolation. In case of necessity to hydrolyze urea, to be used different vitamins in Trichophyton agar media were investigated. Polymerase chain reaction (PCR and sequence analyses were done for the molecular diagnosis. Results: Using conventional methods, 37 strains (66,1% were identified as Trichophyton(T rubrum, four (7.1% - T.mentagrophytes, four (7.1% - T.tonsurans, one (1.8% - T.violaceum, eight (14.3% - Trichophyton spp., one (1.8% - Microsporum(M canis, and one (1.8% - Microsporum spp. According to the molecular and sequence analyses results (T1PCR, 25GAPCR, ITSPCR-RFLP and sequence analyses, 41 (73.8% strains were identified as T.rubrum, 10 (17.8% - T.interdigitale, one (1.8% - T. violaceum, two (3.6% - M. canis, one (1.8% - Peacilomyces lilacinus, and one (1,8% - Aspergillus fumigatus. Discussion: This study suggests that, molecular methods offer fast and reliable results in

  1. Molecular Characterisation of Salmonella enterica Serovar Typhi Isolated from Typhoidial Humans

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    Arunava Das

    2012-09-01

    Full Text Available Aims: Salmonella enterica serovar Typhi is the major causative agent for typhoidial fever around the globe among human population reported till date. Present research work was carried out for detection and molecular characterisation of Salmonella enterica serovar Typhi isolated from humans with Typhoidial fever by biochemical, phenotypical and virulence gene based polymerase chain reaction (PCR techniques. The isolated strains were also investigated for antibiotic susceptibility patterns as a control measure. Methodology and Results: A total of 16 clinical samples were collected from the same numbers of patients (7 males and 9 females from Coimbatore, Erode and Salem districts of Tamil Nadu and were processed via broth enrichment methods for isolation and identification of the causative agent S. enterica serovar Typhi. Microbiological and biochemical investigations revealed the presence of S. Typhi from 16 samples. The biotyping of the isolates showed that all the isolates belonged to biotype IV. The PCR analysis confirmed the presence of invA (Invasion gene, 244bp, tyv (Tyveloseepimerase gene, 615 bp, fliC-d (Phage-1 flagellin gene for d-antigen, 750 bp and viaB (Vi antigen gene, 439bp in all 16 clinical samples. The antibiotic susceptibility test that was carried out among the isolates against 12 antimicrobial agents, showed 100 % resistance to only ampicillin and 100 % sensitivity to carbenicillin, chloramphenicol, clindamycin, gentamycin, kanamycin and tetracycline.Conclusion, significance and impact of study: This study confirmed the association of virulent strains of S. enterica serovar Typhi from Typhoidial fever among human population and suggested that PCR based diagnostic could be very useful for the rapid detection of S. Typhi isolates. Present study emphasized the use of antibiotic like chloramphenicol or in combination with other antibiotics for the effective control of S. Typhi.

  2. Molecular epidemiology of Mycobacterium tuberculosis clinical isolates in Southwest Ireland.

    LENUS (Irish Health Repository)

    Ojo, Olabisi O

    2010-10-01

    Tuberculosis has had significant effects on Ireland over the past two centuries, causing persistently higher morbidity and mortality than in neighbouring countries until the last decade. This study describes the results of genotyping and drug susceptibility testing of 171 strains of Mycobacterium tuberculosis complex isolated between January 2004 and December 2006 in a region of Ireland centred on the city of Cork. Spoligotype comparisons were made with the SpolDB4 database and clustered 130 strains in 23 groups, forty-one strains showed unique Spoligotyping patterns. The commonest spoligotypes detected were ST0137 (X2) (16.9%), and ST0351 (15.8%) (\\'U\\' clade). The major spoligotype clades were X (26.2%), U (19.3%), T (15.2%), Beijing (5.9%), Haarlem (4.7%), LAM (4.1%), BOVIS (1.75%), with 12.9% unassigned strains. A 24-locus VNTR genotyping produced 15 clusters containing 49 isolates, with high discrimination index (HGDI>0.99). A combination of Spoligotyping and VNTR reduced the number of clustered isolates to 47 in 15 clusters (27.5%). This study identified ST351 as common among Irish nationals, and found a low rate of drug resistance with little evidence of transmission of drug resistant strains. Strain clustering was significantly associated with age under 55 years and Irish nationality. Only strains of Euro-American lineage formed clusters. Molecular typing did not completely coincide with the results of contact investigations.

  3. Rapid and Accurate Molecular Identification of the Emerging Multidrug-Resistant Pathogen Candida auris.

    Science.gov (United States)

    Kordalewska, Milena; Zhao, Yanan; Lockhart, Shawn R; Chowdhary, Anuradha; Berrio, Indira; Perlin, David S

    2017-08-01

    Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii , Candida haemulonii , and Candida lusitaniae Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species. Copyright © 2017 Kordalewska et al.

  4. Identification of Pseudallescheria and Scedosporium Species by Three Molecular Methods▿

    Science.gov (United States)

    Lu, Qiaoyun; Gerrits van den Ende, A. H. G.; Bakkers, J. M. J. E.; Sun, Jiufeng; Lackner, M.; Najafzadeh, M. J.; Melchers, W. J. G.; Li, Ruoyu; de Hoog, G. S.

    2011-01-01

    The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial β-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 103, and 5 × 102 cells/μl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species. PMID:21177887

  5. Morphological and Molecular Identification of Colletotrichum acutatum from Tomato Fruit

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    Svetlana Živković

    2010-01-01

    Full Text Available Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes, and Colletotrichum dematium are the four main species of Colletotrichum that cause tomato anthracnose. In Serbia, the occurrence of anthracnose on tomato fruit has been recorded during the last several years. Typical fruit symptoms include dark, sunken, and circular lesion with orange conidial masses. Pathogen isolates were obtained from a diseased tomato fruits, on PDA medium forming a white to gray colonies. The cultures developed black acervuli around the center of the colony. Conidia were hyaline, aseptate, and fusiform or rarely cylindrical. Appressoria were smooth, simple, clavate to ovate, and variedfrom light to dark brown. Pathogenicity tests with representative isolates were conducted on symptomless, detached tomato fruits. All tested isolates caused anthracnose lesions on tomato fruit after 7 days of incubation. Koch’s postulates were fulfilled by reisolationfrom inoculated tomato fruits. PCR analysis (using species-specific primer pair, CaInt2/ITS4 of genomic DNA from tomato isolates resulted in an amplification product of 490 bp, specific for C. acutatum, further confirming the identity of the pathogen. Based onmorphological and molecular characteristics, the isolates from tomato fruit were determined as C. acutatum.

  6. Isolation and Identification of Phenol Degrader Bacteria from Sirjan Golgohar Mine Effluent

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    Mehdi Hassanshhian

    2016-03-01

    Full Text Available Phenol and phenolic compounds are highly toxic substances that are found as monoaromatic compounds in various industrial effluents from oil refineries, petrochemical plants, (coal mines, and phenol resin plants. Their discharge into the environment, especially in water resources, causes serious toxicity. Traditionally, physicochemical methods have been used for the removal of phenol and phenolic compounds. Nowadays, bioremediation is known to be the best method for phenol removal from wastewater. The objective of the present study was twofold: isolation and identification of phenol degrading bacteria in the effluent from Golgohar Mine in Sirjan. For this purpose, samples were collected from different sections at Golgohar Mine and its effluent. Phenol degrading bacteria were isolated via enrichment of the samples in the Bushnell Hass medium with phenol used as the only source of carbon and energy. The predominant phenol degrader bacteria were selected by measuring turbidity at 600 nm. The bacteria were subsequently identified by amplification of 16S rRNA with specific primers and PCR sequencing. In this study, 17 strains of phenol degrader bacteria were isolated in soil and wastewater samples collected from different zones of the mine. Screening methods confirmed that 4 strains exhibit a better capability for phenol degradation as evidenced by their capability to degrade 0.4 g/l of phenol. Molecular identification showed that these bacteria belong to the species Pesudomonas sp, Nitrratireductor sp., and Salegentibacter sp. The results also show that the effluent from Golgohar Mine in Sirjan contains many phenol degrading bacteria. The use of these bacteria in the treatment process may lead to a significant reduction in phenol pollution in the mineral effluent.

  7. Isolation and Identification of miRNAs in Jatropha curcas

    Science.gov (United States)

    Wang, Chun Ming; Liu, Peng; Sun, Fei; Li, Lei; Liu, Peng; Ye, Jian; Yue, Gen Hua

    2012-01-01

    MicroRNAs (miRNAs) are small noncoding RNAs that play crucial regulatory roles by targeting mRNAs for silencing. To identify miRNAs in Jatropha curcas L, a bioenergy crop, cDNA clones from two small RNA libraries of leaves and seeds were sequenced and analyzed using bioinformatic tools. Fifty-two putative miRNAs were found from the two libraries, among them six were identical to known miRNAs and 46 were novel. Differential expression patterns of 15 miRNAs in root, stem, leave, fruit and seed were detected using quantitative real-time PCR. Ten miRNAs were highly expressed in fruit or seed, implying that they may be involved in seed development or fatty acids synthesis in seed. Moreover, 28 targets of the isolated miRNAs were predicted from a jatropha cDNA library database. The miRNA target genes were predicted to encode a broad range of proteins. Sixteen targets had clear BLASTX hits to the Uniprot database and were associated with genes belonging to the three major gene ontology categories of biological process, cellular component, and molecular function. Four targets were identified for JcumiR004. By silencing JcumiR004 primary miRNA, expressions of the four target genes were up-regulated and oil composition were modulated significantly, indicating diverse functions of JcumiR004. PMID:22419887

  8. Species identification of Streptococcus bovis group isolates causing bacteremia

    DEFF Research Database (Denmark)

    Agergaard, Charlotte N; Knudsen, Elisa; Dargis, Rimtas

    2017-01-01

    This study compared two MALDI-TOF MS systems (Biotyper and VITEK MS) on clinical Streptococcus bovis group isolates (n=66). The VITEK MS gave fewer misidentifications and a higher rate of correct identifications than the Biotyper. Only the identification of S. lutetiensis by the VITEK MS was reli......This study compared two MALDI-TOF MS systems (Biotyper and VITEK MS) on clinical Streptococcus bovis group isolates (n=66). The VITEK MS gave fewer misidentifications and a higher rate of correct identifications than the Biotyper. Only the identification of S. lutetiensis by the VITEK MS...

  9. Isolation and identification of indigenous lactic acid bacteria from North Sumatra river buffalo milk

    OpenAIRE

    Heni Rizqiati; Cece Sumantr; Ronny Rachman Noor; E. Damayanthi; E. I. Rianti

    2015-01-01

    Buffalo milk is a source of various lactic acid bacteria (LAB) which is potential as culture starter as well as the probiotic. This study was conducted to isolate and identify LAB from indigenous North Sumatra river buffalo milk. Lactic acid bacteria was isolated and grown in medium De Man Rogosa Sharpe Agar (MRSA). The isolation was conducted to obtain pure isolate. The identification of LAB was studied in terms of morphology, physiology, biochemistry and survival on low pH. Morphology test...

  10. Isolation and Molecular Characterization of Brucella Isolates in Cattle Milk in Uganda

    Directory of Open Access Journals (Sweden)

    Denis Rwabiita Mugizi

    2015-01-01

    Full Text Available Brucellosis is endemic in livestock and humans in Uganda and its transmission involves a multitude of risk factors like consumption of milk from infected cattle. To shed new light on the epidemiology of brucellosis in Uganda the present study used phenotypic and molecular approaches to delineate the Brucella species, biovars, and genotypes shed in cattle milk. Brucella abortus without a biovar designation was isolated from eleven out of 207 milk samples from cattle in Uganda. These isolates had a genomic monomorphism at 16 variable number tandem repeat (VNTR loci and showed in turn high levels of genetic variation when compared with other African strains or other B. abortus biovars from other parts of the world. This study further highlights the usefulness of MLVA as an epidemiological tool for investigation of Brucella infections.

  11. Identification and Characterization of Yeast Isolates from Pharmaceutical Waste Water

    Directory of Open Access Journals (Sweden)

    Marjeta Recek

    2002-01-01

    Full Text Available In order to develop an efficient an system for waste water pretreatment, the isolation of indigenous population of microorganisms from pharmaceutical waste water was done. We obtained pure cultures of 16 yeast isolates that differed slightly in colony morphology. Ten out of 16 isolates efficiently reduced COD in pharmaceutical waste water. Initial physiological characterization failed to match the 10 yeast isolates to either Pichia anomala or Pichia ciferrii. Restriction analysis of rDNA (rDNA-RFLP using three different restriction enzymes: HaeIII, MspI and CfoI, showed identical patterns of the isolates and Pichia anomala type strain. Separation of chromosomal DNAs of yeast isolates by the pulsed field gel electrophoresis revealed that the 10 isolates could be grouped into 6 karyotypes. Growth characteristics of the 6 isolates with distinct karyotypes were then studied in batch cultivation in pharmaceutical waste water for 80 hours.

  12. Molecular Characterization of Yeast Strains Isolated from Different Sources by Restriction Fragment Length Polymorphism

    International Nuclear Information System (INIS)

    Ali, M. S.; Latif, Z.

    2016-01-01

    Various molecular techniques like analysis of the amplified rDNA internal transcribed spacers (ITS), intragenic spacers and total ITS region analysis by restriction fragment length polymorphism (RFLP) has been introduced for yeast identification but there are limited databases to identify yeast species on the basis of 5.8S rDNA. In this study, twenty nine yeast strains from various sources including spoiled fruits, vegetables, foodstuffs, and concentrated juices were characterized by PCR-RFLP. PCR-RFLP has been used to characterize yeasts present in different spoiled food samples after isolation of the yeasts. By using this technique, the isolated yeast strains were characterized by direct 5.8S-ITS rDNA region amplification. RFLP analysis was applied to each of the amplification products (varied from 400bp to 800bp) detected, and the corresponding yeast identifications were made according to each specific restriction patterns obtained after treatment with two endonucleases TaqI and HaeIII which yielded a specific banding pattern for each species. For further confirmation amplified products of eleven selected isolates were sequenced and blast on NCBI. Both RFLP and sequence analyses of the strains with accession nos. KF472163, KF472164, KF472165, KF472166, KF472167, KF472168, KF472169, KF472170, KF472171, KF472172, KF472173 gave significantly similar results. The isolates were found to belong five different yeast species including; Candida spp., Pichia spp., Kluyveromyces spp., Clavispora spp. and Hanseniaspora spp. This method provides a fast, easy, reliable and authentic way for determining yeast population present in different type of samples, as compared to traditional characterization technique. (author)

  13. Isolation, identification and application in lignin degradation of an ...

    African Journals Online (AJOL)

    This study was undertaken to isolate an ascomycete producing ligninolytic enzyme and characterize its lignin degradation capability. Among 20 isolates, GHJ-4 was isolated from decayed wood of Salix matsudana Koidz in Mount Tai, China, by different indicator compounds assay. The taxonomy of the fungi was ...

  14. Identification of Turnip mosaic virus isolated from canola in northeast ...

    African Journals Online (AJOL)

    Phylogenetic analyses based on ClustalW multiple alignments with previously reported 33 isolates indicated 88 to 98% similarity in nucleotide and 94 to 99% in amino acid levels among isolates. TuMV-IRN GSK represented the highest identity to another Iranian isolate (IRN TRa6). Phylogenetic tree clustered all sequences ...

  15. Isolating strong-field dynamics in molecular systems

    Science.gov (United States)

    Orenstein, Gal; Pedatzur, Oren; Uzan, Ayelet J.; Bruner, Barry D.; Mairesse, Yann; Dudovich, Nirit

    2017-05-01

    Strong-field ionization followed by recollision provides a unique pump-probe measurement which reveals a range of electronic processes, combining sub-Angstrom spatial and attosecond temporal resolution. A major limitation of this approach is imposed by the coupling between the spatial and temporal degrees of freedom. In this paper we focus on the study of high harmonic generation and demonstrate the ability to isolate the internal dynamics—decoupling the temporal information from the spatial one. By applying an in situ approach we reveal the universality of the intrinsic pump-probe measurement and establish its validity in molecular systems. When several orbitals are involved we identify the fingerprint of the transition from the single-channel case into the multiple-channel dynamics, where complex multielectron phenomena are expected to be observed.

  16. COMPARATIVE ANALYSIS OF METHODS FOR IDENTIFICATION OF NONTUBERCULOUS MYCOBACTERIA ISOLATED FROM CLINICAL MATERIAL

    Directory of Open Access Journals (Sweden)

    A. V. Lyamin

    2017-01-01

    Full Text Available Recently there has been a significant increase in the incidence of mycobacteriosis, which is due to an increase in the proportion of immunosuppressed patients, the presence of these various comorbid conditions, as well as the improvement of diagnostic methods. Selecting the most accurate method of identification is extremely important in determining treatment strategy of patients. The aim of the study was to conduct a comparative analysis of modern methods of identification NTMB isolated from clinical specimens in 2015 in the Samara region. The work was carried out identification of 78 strains of microorganisms. Laboratory diagnosis was carried out using the DNA hybridization method and MALDI-ToF mass spectrometry. When microbial identification using MALDI-ToF mass spectrometry was isolated 16 strains (20.5% M. kansasii; 11 strains (14.1% M. avium and M. fortuitum; 9 strains (11.5% M. gordonae; strain 3 (3.8% M. peregrinum, M. szulgai, M. chimera intracellulare group, strain 2 (2.6% M. abscessus, M. septicum, M. paragordonae, M. senegalence, 1 strain (1.3% M. chelonae, M. frederiksbergense, M. monacense, M. lentiflavum. By using mass spectrometry, it was identified 15 types NTMB compared with 9 types — by DNA hybridization. Full match identification results was observed only in 45 (57.7% strains of divergent strains were found in 16 (20.5%. Most often when using the DNA hybridization method, discrepancy was detected in slow-growing cultures (9 strains with a predominance of microorganisms identified as M. gordonae. Among the representatives of fast-growing NTMB, seven investigations were identified in the identification, more often among representatives of the M. fortuitum and M. peregrinum groups. Particular attention should be paid to the identification of the M. kansasii strain by a molecular genetic method, which mass spectrometry was defined as M. bovis. Both cultures of M. tuberculosis complex, which were identified by MALDI

  17. Isolation and identification of a bovine viral diarrhea virus from sika deer in china

    OpenAIRE

    Gao, Yugang; Wang, Shijie; Du, Rui; Wang, Quankai; Sun, Changjiang; Wang, Nan; Zhang, Pengju; Zhang, Lianxue

    2011-01-01

    Abstract Background Bovine viral diarrhea virus (BVDV) infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical...

  18. SEROLOGICAL DETECTION AND MOLECULAR CHARACTERIZATION OF A BEGOMOVIRUS ISOLATE OBTAINED FROM Macroptilium lathyroides

    Directory of Open Access Journals (Sweden)

    JOSÉ ALBERSIO DE ARAUJO LIMA

    2012-01-01

    Full Text Available The viruses from the genus Begomovirus, family Geminiviridae are considered emergent pathogens, mainly because of the population explosion of their insect vectors. For this reason, more attention needs to be directed to the correct virus species identification inside the genus. The present paper had the objectives of serologically detecting a begomovirus in Macroptilium lathyroides plants in the State of Ceará, and developing biological, serological and molecular studies with a virus isolate obtained from M. lathyroides. Indirect ELISA with antiserum for Macroptilium golden mosaic virus (MaGMV demonstrated that the samples collected from M. lathyroides showing golden mosaic in the field were infected with a begomovirus. The virus isolate obtained was transmitted by grafting to eight species of the family Leguminosae, four species of Solonaceae, and one species in the family Amaranthaceae. The virus also was transmitted from M. lathyroides to M. lathyroides by the whitefly Bemisia tabaci biotype B. A DNA fragment of 1.2 kb was obtained by PCR with the primers PAL1v 1978 and PAR1c 496 for component A, and a DNA fragment of 0.5 kb was obtained with the primers PBL1v 2040 and PCR cl for component B, confirming the presence of a begomovirus infecting M. lathyroides. Molecular studies indicated that the begomovirus isolate showed 77% genomic similarity with Bean golden mosaic virus and 75% with Cowpea golden mosaic virus for their cp and rep genes, indicating the possibility that the isolate is a distinct virus species of the Begomovirus genus.

  19. Molecular characterization of Prunus necrotic ringspot virus isolated from rose in Brazil.

    OpenAIRE

    FAJARDO, T. V. M.; NASCIMENTO, M. B.; EIRAS, M.; NICKEL, O.; PIO-RIBEIRO, G.

    2016-01-01

    ABSTRACT: There is no molecular characterization of Brazilian isolates of Prunus necrotic ringspot virus (PNRSV), except for those infecting peach. In this research, the causal agent of rose mosaic was determined and the movement (MP) and coat (CP) protein genes of a PNRSV isolate from rose were molecularly characterized for the first time in Brazil. The nucleotide and deduced amino acid sequences of MP and CP complete genes were aligned and compared with other isolates. Molecular analysis of...

  20. Molecular identification and phylogenetic study of Demodex caprae.

    Science.gov (United States)

    Zhao, Ya-E; Cheng, Juan; Hu, Li; Ma, Jun-Xian

    2014-10-01

    The DNA barcode has been widely used in species identification and phylogenetic analysis since 2003, but there have been no reports in Demodex. In this study, to obtain an appropriate DNA barcode for Demodex, molecular identification of Demodex caprae based on mitochondrial cox1 was conducted. Firstly, individual adults and eggs of D. caprae were obtained for genomic DNA (gDNA) extraction; Secondly, mitochondrial cox1 fragment was amplified, cloned, and sequenced; Thirdly, cox1 fragments of D. caprae were aligned with those of other Demodex retrieved from GenBank; Finally, the intra- and inter-specific divergences were computed and the phylogenetic trees were reconstructed to analyze phylogenetic relationship in Demodex. Results obtained from seven 429-bp fragments of D. caprae showed that sequence identities were above 99.1% among three adults and four eggs. The intraspecific divergences in D. caprae, Demodex folliculorum, Demodex brevis, and Demodex canis were 0.0-0.9, 0.5-0.9, 0.0-0.2, and 0.0-0.5%, respectively, while the interspecific divergences between D. caprae and D. folliculorum, D. canis, and D. brevis were 20.3-20.9, 21.8-23.0, and 25.0-25.3, respectively. The interspecific divergences were 10 times higher than intraspecific ones, indicating considerable barcoding gap. Furthermore, the phylogenetic trees showed that four Demodex species gathered separately, representing independent species; and Demodex folliculorum gathered with canine Demodex, D. caprae, and D. brevis in sequence. In conclusion, the selected 429-bp mitochondrial cox1 gene is an appropriate DNA barcode for molecular classification, identification, and phylogenetic analysis of Demodex. D. caprae is an independent species and D. folliculorum is closer to D. canis than to D. caprae or D. brevis.

  1. Isolation and identification of methicillin-resistant Staphylococcus ...

    African Journals Online (AJOL)

    Moreover, this study compared the efficacy of the different identification tests with gold standard, polymerase chain reaction (PCR) assay. The tests used were tube coagulase, DNase agar test, antibiogram, several routine biochemical identification tests and PCR assays. PCR assay used specific primers for resistance or ID ...

  2. Isolation and molecular identification of a Serratia strain from ...

    African Journals Online (AJOL)

    ONOS

    2010-04-05

    Apr 5, 2010 ... family Tupaiidae, mainly found in Southern China, India and Southeast Asia. The 17 known species of tree shrews ... for pathogenic coccus cultivation and nutrient agar culture dish and. MacConkey agar for pathogenic rod ...

  3. Molecular identification of Giardia lamblia isolates from adult human ...

    African Journals Online (AJOL)

    ARL

    2013-02-27

    Feb 27, 2013 ... Giardia lamblia is a flagellated protozoa that infects the intestinal tract of a wide range of mammalian hosts, including both wild and domestic animals as well as humans. Two genotypes A and B are commonly reported among humans. The purpose of this study was to investigate the genotypes of G.

  4. Isolation and molecular identification of Vibrio spp. by sequencing of ...

    African Journals Online (AJOL)

    Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6% of samples of meat and meat products showed colonies on TCBS.

  5. Isolation and identification mould micoflora inhabiting plant leaf litter from Mount Lawu, Surakarta, Central Java

    Directory of Open Access Journals (Sweden)

    MUHAMMAD ILYAS

    2007-04-01

    Full Text Available A study on isolation and identification mould inhabiting plant leaf litter had been conducted. The objective of the study was to isolate and identify mould inhabiting plant leaf litter from Mount Lawu, Surakarta, Central Java. The mould isolation was based on washing and filtering with membrane isolation method. The result showed that 39 moulds generas with 55 species varians, one group identified in class level, and three groups of unidentified mould isolates had been isolated. Taxas distributions showed that there were endophyte and phytopatogen mould isolates had been isolated such as Fusarium, Pestalotiopsis, Phoma, and Coelomycetes. However, typical soil taxa and common saprobic fungi such as Aspergillus, Cunninghamella, Mucor, Paecilomyces, Penicillium, Rhizopus, and Trichoderma remain dominated the resulted isolates.

  6. Molecular identification of Malaysian Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart) using life stage specific mitochondrial DNA.

    Science.gov (United States)

    Kavitha, R; Tan, T C; Lee, H L; Nazni, W A; Sofian, A M

    2013-06-01

    DNA identification of blow fly species can be a very useful tool in forensic entomology. One of the potential benefits that mitochondrial DNA (mtDNA) has offered in the field of forensic entomology is species determination. Conventional identification methods have limitations for sibling and closely related species of blow fly and stage and quality of the specimen used. This could be overcome by DNA-based identification methods using mitochondrial DNA which does not demand intact or undamaged specimens. Mitochondrial DNA is usually isolated from whole blow fly and legs. Alternate sources for mitochondrial DNA isolation namely, egg, larva, puparium and empty puparium were explored in this study. The sequence of DNA obtained for each sample for every life cycle stage was 100% identical for a particular species, indicating that the egg, 1st instar, 2nd instar, 3rd instar, pupa, empty puparium and adult from the same species and obtained from same generation will exhibit similar DNA sequences. The present study also highlighted the usefulness of collecting all life cycle stages of blow fly during crime scene investigation with proper preservation and subsequent molecular analysis. Molecular identification provides a strong basis for species identification and will prove an invaluable contribution to forensic entomology as an investigative tool in Malaysia.

  7. Isolation and identification of fungi responsible for leaf spots disease ...

    African Journals Online (AJOL)

    The diseased plant leaves were taken to the laboratory for culture, isolation, and ... These included; Alternaria longipes, Aspergillus fumigatus, Aspergillus niger, ... Phomopsis mangiferae, Pseudofusicoccum spp. and Rhizopus oryzae.

  8. GC-MS Quantitation and Identification of Bisphenol-A Isolated from Water

    Science.gov (United States)

    Mead, Ralph N.; Seaton, Pamela J.

    2011-01-01

    Isolation and identification of organic compounds is a necessary skill chemistry students must be able to do with proficiency. In this upper-level undergraduate laboratory, students isolate bisphenol-A (BPA; 4-4'-isopropylidenediphenol) from water using solid-phase extraction (SPE) followed by derivatization with analysis by GC-MS. The students…

  9. Identification of Bifidobacterium Strains Isolated from Kashk-e Zard: A Traditional Iranian Fermented Cereal-Dairy Based Food

    Directory of Open Access Journals (Sweden)

    Mashak

    2016-09-01

    Full Text Available Objectives The genus Bifidobactrium enjoys considerable significance among the probiotic bacteria for having appropriately adapted to the human gastrointestinal tract. As the properties of Bifidobacteria are strain-oriented and niche-dependent, there is growing interest in studying the different sources of these probiotics. Kashk-e Zard, a traditional fermented food produced from wheat and yogurt through a two-week, two-step fermentation process, is rich in probiotics and is worthy of study in this regard. The present study aimed to identify Bifidobacterium spp. in Kashk-e Zard. Methods Twenty-three samples of Kashk-e Zard were collected and subjected to Bifidobacterium identification experiments. Polymerase chain reaction (PCR and sequencing methods were applied for bacterial identification. Results Twelve of the isolates obtained were G +, rod-shaped, and catalase-, whereas only three of them identified positive for fructose 6-phosphate phosphoketolase (F6PPK a Bifidobacterium specific test and mupirocin resistance. These three isolates were then considered for further identification using the 16SrDNA sequencing technique. Conclusions Although carbohydrate fermentation patterns specified these three isolates as B. infantis, B. bifidum, and B. longum, the molecular results did not confirm B. longum, which is still also controversial in the literature. Overall, our results demonstrated that Kashk-e Zard is a rich potential source of probiotic bacteria and further investigations should be undertaken.

  10. Isolation and identification of fungal flora associated with groundnut ...

    African Journals Online (AJOL)

    A total of 25 colonies were isolated from all the samples from which 6 fungal species were identified, namely Mucor, Aspergillus, Rhizophus, Curvularia, Pencillium and Fusarium spp. Of these, Mucor and Rhizopus were most prevalent having been isolated from the three storage facilities studied. Curvularia and Penicillium ...

  11. Isolation and Identification of Spoilage Fungi Associated With Rice ...

    African Journals Online (AJOL)

    The spoilage fungi isolated were Aspergillus species, Rhizopus, Penicilluim, Fusarium, Eurotium, Mucor, Geotrichum, Alternaria, Cladosporium and Actinomyces species. The predominant spoilage fungi in the grains were Aspergillus species. The populations of some spoilage fungi isolated from the grains were not high ...

  12. Screening and identification of lactic acid bacteria isolated from ...

    African Journals Online (AJOL)

    The lactic acid bacteria (LAB) isolated from sorghum (Sorghum bicolor. L.) silage were identified during different periods of evolution of sorghum silage in west Algeria. Morphological, physiological, biochemical and technological techniques were used to characterize lactic acid bacteria isolates. A total number of 27 ...

  13. Isolation, Identification, and Characterization of Cadmium Resistant Pseudomonas sp. M3 from Industrial Wastewater

    OpenAIRE

    Syed Zaghum Abbas; Mohd Rafatullah; Norli Ismail; Japareng Lalung

    2014-01-01

    The present study deals with the isolation, identification, and characterization of the cadmium resistant bacteria from wastewater collected from industrial area of Penang, Malaysia. The isolate was selected based on high level of the cadmium and antibiotic resistances. On the basis of morphological, biochemical characteristics, 16S rDNA gene sequencing and phylogeny analysis revealed that the strain RZCd1 was authentically identified as Pseudomonas sp. M3. The industrial isolate showed more ...

  14. [Molecular characterization of Echinococcus granulosus isolates obtained from different hosts].

    Science.gov (United States)

    Erdoğan, Emrah; Özkan, Bora; Mutlu, Fatih; Karaca, Serkan; Şahin, İzzet

    2017-01-01

    Echinococcus granulosus is a parasite that can be seen throughout the world. So far, five species of genus Echinococcus have been identified as parasite in people: E.granulosus, E.multilocularis, E.vogeli, E.oligarthrus, E.shiquicus. Larval (metacestod) form of parasite settles in internal organs of hoofed animals (cattle, goats, pigs, horses, sheep, etc.) and human; the adult form is found in small intestine of final host, canine. Disease caused by parasite called as "Cystic echinococcosis" (CE) is an important health problem and causes economic losses in many countries including our country that livestock is common. Infective eggs cause infections in intermediate hosts by taking oral way and rarely inhalation. Received egg opens in the stomach and intestines of intermediate host and oncosphere is released. Oncosphere quickly reaches the lamina propria of the villus epithelium by its histolytic enzymes and hooks. It usually transported from here to the liver and lungs, less frequently, muscle, brain, spleen, kidney and to other organs through the veins. By molecular studies, five species have been validated taxonomically and 10 different variants or strains of E.granulosus have been identified. Host and developmental differences between strains may negatively affect control studies and fight against the parasite. This study aimed to determinate E.granulosus strains obtained from cyst material of different intermediate hosts from different regions of Turkey by molecular methods. In the study, 25 human, 8 cattle, 6 sheep and 2 goat cysts material has been collected. Total genomic DNA was isolated from protoscoleces in cyst fluid and analyzed by PCR with COX-1 (L) and COX-1 (S) genes specific primers. DNA sequence analysis for each PCR product has been made. DNA sequence analysis results evaluated phylogenetically by MEGA analyze and BLAST software. As a result of this study, all isolates were identified as E.granulosus sensu stricto (G1) by DNA sequence analysis. CE

  15. Isolation and identification of Mycobacterium bovis in milk from cows in northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Joelson Marcolino Ramos

    Full Text Available ABSTRACT: Milk samples from 16 cows that tested positive on the tuberculin test in the state of Paraíba, northeastern Brazil, were used for mycobacteria isolation and identification. Mycobacteria were isolated from five (31.25% of the 16 milk samples; three samples were classified as M. bovis, and two as belonging to the Mycobacterium genus. This is probably the first study of isolation and identification of M. bovis in milk from cows in Northeastern Brazil, which suggests that humans are at risk of contamination by ingestion.

  16. Morphological and molecular identification of Fusarium tricinctum and Fusarium acuminatum as causal agents of garlic bulbs rot in Serbia

    Directory of Open Access Journals (Sweden)

    Ignjatov Maja V.

    2017-01-01

    Full Text Available Garlic (Allium sativum L. is considered to be one of the oldest crops in the world. During 2016, infected garlic bulbs occurred in storages on several localities of the Province of Vojvodina. Symptomatic cloves showed typical rot symptoms such as softened and spongy areas covered with white fungal growth with deep lesions formed on the cloves which became dry over time. A total of 36 isolates of Fusarium species were obtained from diseased cloves of garlic. Colony morphology and microscopic properties of isolated Fusarium species were recorded from the cultures grown on PDA and CLA, respectively. Identification of two chosen isolates was performed by sequencing the EF-1α gene. The TEF sequence of isolate JBL12 showed 100% similarity with several F. tricinctum sequences and sequence of JBL539 showed 99% identity with several F. acuminatum sequences and they were deposited in the NCBI GenBank. Based on the results of the morphological and molecular identification, isolates JBL12 and JBL539 were identified as F. tricinctum and F. acuminatum, respectively, as new causal agents of garlic bulbs rot in Serbia. Specific primers were designed for the PCR identification of the F. tricinctum. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR31030

  17. Rapid identification of ascomycetous yeasts from clinical specimens by a molecular method based on flow cytometry and comparison with identifications from phenotypic assays.

    Science.gov (United States)

    Page, Brent T; Shields, Christine E; Merz, William G; Kurtzman, Cletus P

    2006-09-01

    This study was designed to compare the identification of ascomycetous yeasts recovered from clinical specimens by using phenotypic assays (PA) and a molecular flow cytometric (FC) method. Large-subunit rRNA domains 1 and 2 (D1/D2) gene sequence analysis was also performed and served as the reference for correct strain identification. A panel of 88 clinical isolates was tested that included representatives of nine commonly encountered species and six infrequently encountered species. The PA included germ tube production, fermentation of seven carbohydrates, morphology on corn meal agar, urease and phenoloxidase activities, and carbohydrate assimilation tests when needed. The FC method (Luminex) employed species-specific oligonucleotides attached to polystyrene beads, which were hybridized with D1/D2 amplicons from the unidentified isolates. The PA identified 81 of 88 strains correctly but misidentified 4 of Candida dubliniensis, 1 of C. bovina, 1 of C. palmioleophila, and 1 of C. bracarensis. The FC method correctly identified 79 of 88 strains and did not misidentify any isolate but did not identify nine isolates because oligonucleotide probes were not available in the current library. The FC assay takes approximately 5 h, whereas the PA takes from 2 h to 5 days for identification. In conclusion, PA did well with the commonly encountered species, was not accurate for uncommon species, and takes significantly longer than the FC method. These data strongly support the potential of FC technology for rapid and accurate identification of medically important yeasts. With the introduction of new antifungals, rapid, accurate identification of pathogenic yeasts is more important than ever for guiding antifungal chemotherapy.

  18. Identification of new IS711 insertion sites in Brucella abortus field isolates.

    Science.gov (United States)

    Mancilla, Marcos; Ulloa, Marcos; López-Goñi, Ignacio; Moriyón, Ignacio; María Zárraga, Ana

    2011-08-03

    Brucellosis is a zoonosis caused by Brucella spp., a group of highly homogeneous bacteria. The insertion sequence IS711 is characteristic of these bacteria, and occurs in variable numbers and positions, but always constant within a given species. This species-associated polymorphism is used in molecular typing and identification. Field isolates of B. abortus, the most common species infecting cattle, typically carry seven IS711 copies (one truncated). Thus far, IS711 transposition has only been shown in vitro and only for B. ovis and B. pinnipedialis, two species carrying a high number of IS711 copies, but never in other Brucella species, neither in vitro nor in field strains. We found several B. abortus strains isolated from milk and aborted fetuses that carried additional IS711 copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marR transcriptional regulator gene. Interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. Sequence analyses revealed that the new copies probably resulted from the transposition of a single IS711 copy common to all Brucella species sequenced so far. Our results show that the replicative transposition of IS711 can occur under field conditions. Therefore, it represents an active mechanism for the emergence of genetic diversity in B. abortus thus contributing to intra-species genetic polymorphism.

  19. Identification of new IS711 insertion sites in Brucella abortus field isolates

    Directory of Open Access Journals (Sweden)

    Moriyón Ignacio

    2011-08-01

    Full Text Available Abstract Background Brucellosis is a zoonosis caused by Brucella spp., a group of highly homogeneous bacteria. The insertion sequence IS711 is characteristic of these bacteria, and occurs in variable numbers and positions, but always constant within a given species. This species-associated polymorphism is used in molecular typing and identification. Field isolates of B. abortus, the most common species infecting cattle, typically carry seven IS711 copies (one truncated. Thus far, IS711 transposition has only been shown in vitro and only for B. ovis and B. pinnipedialis, two species carrying a high number of IS711 copies, but never in other Brucella species, neither in vitro nor in field strains. Results We found several B. abortus strains isolated from milk and aborted fetuses that carried additional IS711 copies in two hitherto undescribed insertion sites: one in an intergenic region near to the 3' end of a putative lactate permease gene and the other interrupting the sequence of a marR transcriptional regulator gene. Interestingly, the second type of insertion was identified in isolates obtained repeatedly from the same herd after successive brucellosis outbreaks, an observation that proves the stability and virulence of the new genotype under natural conditions. Sequence analyses revealed that the new copies probably resulted from the transposition of a single IS711 copy common to all Brucella species sequenced so far. Conclusions Our results show that the replicative transposition of IS711 can occur under field conditions. Therefore, it represents an active mechanism for the emergence of genetic diversity in B. abortus thus contributing to intra-species genetic polymorphism.

  20. [Molecular techniques applied in species identification of Toxocara].

    Science.gov (United States)

    Fogt, Renata

    2006-01-01

    Toxocarosis is still an important and actual problem in human medicine. It can manifest as visceral (VLM), ocular (OLM) or covert (CT) larva migrans syndroms. Complicated life cycle of Toxocara, lack of easy and practical methods of species differentiation of the adult nematode and embarrassing in recognition of the infection in definitive hosts create difficulties in fighting with the infection. Although studies on human toxocarosis have been continued for over 50 years there is no conclusive answer, which of species--T. canis or T. cati constitutes a greater risk of transmission of the nematode to man. Neither blood serological examinations nor microscopic observations of the morphological features of the nematode give the satisfied answer on the question. Since the 90-ths molecular methods were developed for species identification and became useful tools being widely applied in parasitological diagnosis. This paper cover the survey of methods of DNA analyses used for identification of Toxocara species. The review may be helpful for researchers focused on Toxocara and toxocarosis as well as on detection of new species. The following techniques are described: PCR (Polymerase Chain Reaction), RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA) and SSCP (Single Strand Conformation Polymorphism).

  1. Isolation, Characterization and Identification of Microalgae from the Red Sea

    KAUST Repository

    Luque Alaní s, Patricio

    2013-01-01

    Eukaryotic microalgae from the Red Sea were isolated, characterized and identified with the purpose of building a culture collection that will serve future research activities in the area of industrial microbiology. Seven sampling locations were

  2. Isolation and identification of Metarhizium anisopliae from Chilo ...

    African Journals Online (AJOL)

    agar culture media: potato dextrose agar medium (PDA), potato dextrose with 1% (w/v) peptone agar medium (PPDA), and oatmeal agar medium (OMA). 16 different isolates were identified as Metarhizium anisopliae (Metschnikoff) based on ...

  3. Isolation and identification of antimicrobial-producing lactic acid ...

    African Journals Online (AJOL)

    , Proteus species, Staphylococcus aureus, Salmonella species, Pseudomonas flourescence, P. aeruginosa, Serratia species and Pediococcus acidilactici. Of the 42 antimicrobial producing isolates characterized, 16, 12, 6 and 8 were identified ...

  4. Isolation, identification and screening of potential cellulase-free ...

    African Journals Online (AJOL)

    compaq

    2012-09-05

    Sep 5, 2012 ... Eight fungal species were selected for further study based on clearing zones ... lignolytic enzymes, instead of chlorine, are used to break the xylan, cellulose, lignin ... purchased from Sigma chemicals Co., USA. Isolation and ...

  5. Isolation, Characterization and Identification of Microalgae from the Red Sea

    KAUST Repository

    Luque Alanís, Patricio

    2013-05-01

    Eukaryotic microalgae from the Red Sea were isolated, characterized and identified with the purpose of building a culture collection that will serve future research activities in the area of industrial microbiology. Seven sampling locations were surveyed using an in-house designed isolation protocol. Microalgae enrichment was carried out in vitro using the streak plate method and fluorescence activated cell sorting approaches. Colonial and cellular microscopy, growth media preference assays, as well as temperature, pH and salinity tolerance tests were carried out to describe the isolates. DNA extraction, PCR amplification, template sequencing and in silico analyses were carried out to identify the isolates and arrange them in a proper phylogenetic description. In total, 129 isolates were obtained. From these, only 39 were selected for characterization given their increased ability of accumulating large amounts of biomass in solid and liquid media in relatively short periods of time. All of these have a green color, are unicellular, non-motile, photosynthetic organisms and have a cell size ranging from 5 to 8 µm. More than half of them showed growth preference in Walne media, followed by F/2, MN and BG-11 SW. Maximum temperature tolerance of all organisms was around 38 ºC, while optimum growth was observed close to 25 ºC. pH preference was diverse and three groups were identified: acidic (6), intermediate (8 - 9) and alkaline (> 10) growing isolates. Salinity tests showed an overall growth preference at 25 PSU, approximately 10 units lower than that found at the sampling stations. Most isolates showed diminished growth at high salinity and high pH, except for OS3S1b which grew well in both cases, and could be an interesting strain to study further. Twenty four isolates were related to Ulvophyceae sp. MBIC10591 by BLAST approaches with a maximum identity of 96 - 97%. A maximum likelihood phylogenetic tree was created for these isolates, relative to the BLAST hits

  6. Isolation and identification of an ester from a crude oil

    Science.gov (United States)

    Phillips, H.F.; Breger, I.A.

    1958-01-01

    A dioctylphthalate has been isolated from a crude oil by means of adsorption column chromatography. The ester was identified by means of elemental analysis, refractive index, and its infra-red absorption spectrum. Saponification of the isolate and examination of the resultant alcohol by means of infrared absorption spectra led to the conclusion that the ester is a branched chain dioctylphthalate. This is the first reported occurrence of an ester in crude petroleum. ?? 1958.

  7. Molecular Characterization of Vibrio cholerae Isolated From Clinical Samples in Kurdistan Province, Iran.

    Science.gov (United States)

    Ramazanzadeh, Rashid; Rouhi, Samaneh; Shakib, Pegah; Shahbazi, Babak; Bidarpour, Farzam; Karimi, Mohammad

    2015-05-01

    Vibrio cholerae causes diarrhoeal disease that afflicts thousands of people annually. V. cholerae is classified on the basis of somatic antigens into serovars or serogroups and there are at least 200 known serogroup. Two serogroups, O1 and O139 have been associated with epidemic diseases. Virulence genes of these bacteria are OmpW, ctxA and tcpA. Due to the importance of V. cholerae infection and developing molecular diagnostics of this organism in medical and microbiology sciences, this study aimed to describe molecular characterization of V. cholerae isolated from clinical samples using a molecular method. In this study, 48 samples were provided during summer 2013 (late August and early September) by reference laboratory. Samples were assessed using biochemical tests initially. The primer of OmpW, ctxA and tcpA genes was used in Polymerase Chain Reaction (PCR) protocols. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR and Repetitive Extragenic Palindromic (REP)-PCR methods were used to subtype V. cholerae. In this study, from a total of 48 clinical stool samples 39 (81.2 %) were positive for V. cholerae in biochemical tests and bacteria culture tests. The PCR results showed that of 39 positive isolates 35 (89.7%), 34 (87.1%) and 37 (94.8%) were positive for ctxA, tcpA and OmpW gene, respectively. Also, in the REP-PCR method with ERIC primer strains were divided into 10 groups. In the REP-PCR method with REP primer, strains were divided into 13 groups. Polymerase chain reaction has specificity and accuracy for identification of the organism and is able to differentiate biotypes. Enterobacterial repetitive intergenic consensus sequence is one of the informative and discriminative methods for the analysis of V. cholerae diversity. The REP-PCR is a less informative and discriminative method compared to other methods for the analysis of V. cholerae diversity.

  8. Strategy for identification & characterization of Bartonella henselae with conventional & molecular methods

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    Kavita Diddi

    2013-01-01

    Full Text Available Background & objectives: Bartonella henselae is a fastidious gram-negative bacterium usually causing self limiting infections in immunocompetent individuals but often causes potentially life threatening infection, such as bacillary angiomatosis in immunocompromised patients. Both diagnosis of infections and research into molecular mechanisms of pathogenesis have been hindered by lack of appropriate and reliable diagnostic techniques. We undertook this study to standardize methods to characterize B. henselae in clinical samples to diagnose Bartonella infection correctly. Methods: B. henselae ATCC 49882 strain was procured from American type culture collection, USA. This strain was revived and maintained in the laboratory, and identification and characterization of this strain was done by conventional and molecular techniques, which included culture on various media, staining by different methods including electron microscopy, biochemical analysis by conventional methods and API, polymerase chain reaction (PCR for amplification of citrate synthase gene followed by restriction fragment length polymorphism (RFLP. Results: This organism was biochemically inert due to slow growth and generated unique identification code with API. The amplification of the citrate-synthase gene with primers yielded a 381 bp product followed by specific RFLP profile for B. henselae. Interpretation & conclusions: Bartonella is fastidious and fragile organism and should be handled carefully. Extra effort and careful observation are required to isolate and characterize this organism.

  9. Molecular variability among isolates of Fusarium oxysporum associated with root rot disease of Agave tequilana.

    Science.gov (United States)

    Vega-Ramos, Karla L; Uvalle-Bueno, J Xavier; Gómez-Leyva, Juan F

    2013-04-01

    In this study, 115 isolates of Fusarium oxysporum from roots of Agave tequilana Weber cv azul plants and soil in commercial plantations in western Mexico were characterized using morphological and molecular methods. Genetic analyses of monosporic isolates included restriction enzyme analysis of rDNA (ARDRA) using HaeIII and HinfI, and genetic diversity was determined using Box-PCR molecular markers. Box-PCR analysis generated 14 groups. The groups correlated highly with the geographic location of the isolate and sample type. These results demonstrate the usefulness of ARDRA and Box-PCR techniques in the molecular characterization of the Fusarium genus for the discrimination of pathogenic isolates.

  10. Identification and characterization of filamentous fungi isolated from fermentation starters for Hong Qu glutinous rice wine brewing.

    Science.gov (United States)

    Lv, Xu-Cong; Huang, Zhi-Qing; Zhang, Wen; Rao, Ping-Fan; Ni, Li

    2012-01-01

    Hong Qu glutinous rice wine is one of the most popular traditional rice wines in China. Traditionally, this wine is brewed from glutinous rice with the addition of wine fermentation starters (Hong Qu (also called red yeast rice) and White Qu). The objective of this study was to investigate the variability of filamentous fungi associated with traditional fermentation starters through a traditional culture-dependent method and a molecular identification approach. In this study, forty-three filamentous fungi were separated by traditional culture-dependent means (macro- and microscopic characteristics) from 10 fermentation starters and classified into 16 different species based on morphological examination and the internal transcribed spacer (ITS) sequences analysis. It was observed that the genus Aspergillus had the highest number (14 isolates) of isolates followed by Rhizopus (11 isolates), Monascus (5 isolates) and Penicillium (4 isolates). The species R. oryzae, A. niger, A. flavus and M. purpureus were frequently found in wine starter samples, among which R. oryzae was the most frequent species. The enzyme-producing properties (glucoamylase, α-amylase and protease) of all fungal isolates from different starters were also evaluated. A. flavus, R. oryzae and M. purpureus were found to be better glucoamylase producers. A. flavus, R. oryzae and A.oryzae exhibited higher activity of α-amylase. A. flavus and A. oryzae had higher protease activity. However, some fungal isolates of the same species exhibited a significant variability in the production levels for all determined enzyme activity. This study is the first to identify filamentous fungi associated with the starter of Hong Qu glutinous rice wine using both traditional and molecular methods. The results enrich our knowledge of liquor-related micro-organisms, and can be used to promote the development of the traditional fermentation technology.

  11. Molecular assay to fraud identification of meat products.

    Science.gov (United States)

    Doosti, Abbas; Ghasemi Dehkordi, Payam; Rahimi, Ebrahim

    2014-01-01

    Detection of species fraud in meat products is important for consumer protection and food industries. A molecular technique such as PCR method for detection of beef, sheep, pork, chicken, donkey, and horse meats in food products was established. The purpose of this study was to identification of fraud and adulteration in industrial meat products by PCR-RFLP assay in Iran. In present study, 224 meat products include 68 sausages, 48 frankfurters, 55 hamburgers, 33 hams and 20 cold cut meats were collected from different companies and food markets in Iran. Genomic DNA was extracted and PCR was performed for gene amplification of meat species using specific oligonucleotid primers. Raw meat samples are served as the positive control. For differentiation between donkey's and horse's meat, the mitochondrial DNA segment (cytochrome-b gene) was amplified and products were digested with AluI restriction enzyme. Results showed that 6 of 68 fermented sausages (8.82%), 4 of 48 frankfurters (8.33%), 4 of 55 hamburgers (7.27%), 2 of 33 hams (6.6%), and 1 of 20 cold cut meat (5%) were found to contain Haram (unlawful or prohibited) meat. These results indicate that 7.58% of the total samples were not containing Halal (lawful or permitted) meat and have another meat. These findings showed that molecular methods such as PCR and PCR-RFLP are potentially reliable techniques for detection of meat type in meat products for Halal authentication.

  12. An investigation on non-invasive fungal sinusitis; Molecular identification of etiologic agents

    Directory of Open Access Journals (Sweden)

    Abdolrasoul Mohammadi

    2017-01-01

    Full Text Available Background: Fungal sinusitis is increasing worldwide in the past two decades. It is divided into two types including invasive and noninvasive. Noninvasive types contain allergic fungal sinusitis (AFS and fungus ball. AFS is a hypersensitivity reaction to fungal allergens in the mucosa of the sinonasal tract in atopic individuals. The fungus ball is a different type of noninvasive fungal rhinosinusitis which is delineated as an accumulation of debris and fungal elements inside a paranasal sinus. Fungal sinusitis caused by various fungi such as Aspergillus species, Penicillium, Mucor, Rhizopus, and phaeohyphomycetes. The aim of the present study is to identify fungal species isolated from noninvasive fungal sinusitis by molecular methods. Materials and Methods: During 2015–2016, a total of 100 suspected patients were examined for fungal sinusitis. Functional endoscopic sinus surgery was performed using the Messerklinger technique. Clinical samples were identified by phenotypic and molecular methods. Polymerase chain reaction (PCR sequencing of ITS1-5.8S-ITS2 region and PCR-restriction fragment length polymorphism with Msp I restriction enzyme was performed for molecular identification of molds and yeasts, respectively. Results: Twenty-seven out of 100 suspected cases (27% had fungal sinusitis. Nasal congestion (59% and headache (19% were the most common clinical signs among patients. Fifteen patients (55.5% were male and 12 patients (44.5% were female. Aspergillus flavus was the most prevalent fungal species (26%, followed by Penicillium chrysogenum (18.5% and Candida glabrata species complex (15%. Conclusion: Since clinical manifestations, computed tomography scan, endoscopy, and histopathological findings are very nonspecific in AFS and fungus ball; therefore, molecular investigations are compulsory for precise identification of etiologic agents and appropriate management of these fungal infections.

  13. MALDI-TOF MS enables the rapid identification of the major molecular types within the Cryptococcus neoformans/C. gattii species complex.

    Directory of Open Access Journals (Sweden)

    Carolina Firacative

    Full Text Available BACKGROUND: The Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level. METHODOLOGY: Protein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied. RESULTS: The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established. CONCLUSIONS: MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this

  14. Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis.

    Science.gov (United States)

    Turk, Nenad; Milas, Zoran; Mojcec, Vesna; Ruzic-Sabljic, Eva; Staresina, Vilim; Stritof, Zrinka; Habus, Josipa; Postic, Daniele

    2009-11-01

    A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

  15. Isolation and identification of indigenous lactic acid bacteria from North Sumatra river buffalo milk

    Directory of Open Access Journals (Sweden)

    Heni Rizqiati

    2015-06-01

    Full Text Available Buffalo milk is a source of various lactic acid bacteria (LAB which is potential as culture starter as well as the probiotic. This study was conducted to isolate and identify LAB from indigenous North Sumatra river buffalo milk. Lactic acid bacteria was isolated and grown in medium De Man Rogosa Sharpe Agar (MRSA. The isolation was conducted to obtain pure isolate. The identification of LAB was studied in terms of morphology, physiology, biochemistry and survival on low pH. Morphology tests were conducted by Gram staining and cell forming; physiology tests were conducted for growing viability at pH 4.5 and temperature at 45oC; whereas biochemistry tests were conducted for CO2, dextran and NH3 productions. Determination of LAB species was conducted using Analytical Profile Index (API test CHL 50. Results of identification showed that 41 isolates were identified as LAB with Gram-positive, catalase-negative, rod and round shaped characteristics. Resistance test done to low pH (pH 2 for the lactic acid bacteria showed decrease of bacteria viability up to1.24±0.68 log cfu/ml. The resistant isolates at low pH were L12, L16, L17, L19, L20, M10, P8, S3, S19 and S20. Identification with API test CHL 50 for 10 isolates showed that four isolates were identified as Lactobacillus plantarum, L. brevis, L. pentosus and Lactococuslactis.

  16. Morphological, molecular, and mycotoxigenic identification of dominant filamentous fungi from moldy civil cheese.

    Science.gov (United States)

    Cakmakci, Songul; Cetin, Bulent; Gurses, Mustafa; Dagdemir, Elif; Hayaloglu, Ali Adnan

    2012-11-01

    Moldy Civil is a mold-ripened variety of cheese produced mainly in eastern Turkey. This cheese is produced with Civil cheese and whey curd cheese (Lor). Civil cheese has had a geographical presence since 2009 and is manufactured with skim milk. In the production of Moldy Civil cheese, Civil cheese or a mixture of Civil and Lor cheese is pressed into goat skins or plastic bags and ripened for 3 months or longer. During the ripening period, natural contaminating molds grow on the surface of and inside the cheese. In this study, 186 mold strains were isolated from 41 samples of Moldy Civil cheese, and 165 of these strains were identified as Penicillium roqueforti. Identification and mycotoxicologic analyses were conducted using morphotypic and molecular methods. PCR amplicons of the ITS1-5.8S-ITS4 region were subjected to sequence analysis. This research is the first using molecular methods on Moldy Civil cheese. Mycotoxicologic analyses were conducted using thin-layer chromatography, and random amplified polymorphic DNA genotypes were determined using the ari1 primer. Of 165 isolates, only 28 produced no penicillic acid, P. roqueforti toxin, or roquefortine.

  17. Identification and isolation of gene differentially expressed on scrotal ...

    African Journals Online (AJOL)

    Yomi

    2012-01-05

    Jan 5, 2012 ... 1Laboratory of Molecular Biology and Bovine Breeding, Institute ... there were eight sequences highly identified to be Bos taurus mRNA for proline-rich protein P-B and ..... orchestrate microtubule dynamics and determine the.

  18. Pigments Characterization and Molecular Identification of Bacterial Symbionts of Brown Algae Padinasp. Collected from Karimunjawa Island

    Directory of Open Access Journals (Sweden)

    Damar Bayu Murti

    2016-06-01

    Full Text Available The search for carotenoids in nature has been extensively studied because of their applications in foods. One treasure of the biopigment source is symbiotic-microorganisms with marine biota. The advantages of symbiont bacteria are easy to culture and sensitize pigments. The use of symbiont bacteria helps to conserve fish, coral reefs, seagrass, and seaweed. Therefore, the bacteria keeps their existence in their ecosystems. In this study, bacterial symbionts were successfully isolated from brown algae Padina sp. The bacterial symbionts had yellow pigment associated with carotenoids. The pigments were characterized using High Performance Liquid Chromatography (HPLC with a Photo Diode Array (PDA detector. The carotenoid pigments in the bacterial symbionts were identified as dinoxanthin, lutein and neoxanthin. Molecular identification by using a 16S rRNA gene sequence method, reveals that the bacterial symbionts were closely related to Bacillus marisflavi with a homology of 99%. Keywords :carotenoid pigments, brown algae, Padina, bacterial symbionts, 16S rRNA

  19. Extraction, isolation and identification of flavonoid from Euphorbia neriifolia leaves

    Directory of Open Access Journals (Sweden)

    Veena Sharma

    2017-05-01

    Full Text Available The flavonoids contained in Euphorbia neriifolia leaves were extracted, identified and characterized. Direct and sequential soxhlet extraction and its concentrated fractions were subjected to thin layer chromatography and high performance thin layer chromatography. The results showed that maximum yield of the flavonoid (6.53 g was obtained from ethanolic extract. The Rf value of isolated flavonoid and phytochemical screening has been compared with standard Quercetin. Characterization of isolated flavonoid was done by IR, 1H NMR, and MS. On the basis of chemical and spectral analysis structure was elucidated as 2-(3,4-dihydroxy-5-methoxy-phenyl-3,5-dihydroxy-6,7-dimethoxychromen-4-one, a flavonoid. This compound was isolated for the first time from this plant.

  20. A grass molecular identification system for forensic botany: a critical evaluation of the strengths and limitations.

    Science.gov (United States)

    Ward, Jodie; Gilmore, Simon R; Robertson, James; Peakall, Rod

    2009-11-01

    Plant material is frequently encountered in criminal investigations but often overlooked as potential evidence. We designed a DNA-based molecular identification system for 100 Australian grasses that consisted of a series of polymerase chain reaction assays that enabled the progressive identification of grasses to different taxonomic levels. The identification system was based on DNA sequence variation at four chloroplast and two mitochondrial loci. Seventeen informative indels and 68 single-nucleotide polymorphisms were utilized as molecular markers for subfamily to species-level identification. To identify an unknown sample to subfamily level required a minimum of four markers or nine markers for species identification. The accuracy of the system was confirmed by blind tests. We have demonstrated "proof of concept" of a molecular identification system for trace botanical samples. Our evaluation suggests that the adoption of a system that combines this approach with DNA sequencing could assist the morphological identification of grasses found as forensic evidence.

  1. IDENTIFICATION OF THE ISOLATED COMPOUNDS FROM Zingiber amaricans BL. RHIZOME

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    Sugeng Riyanto

    2010-06-01

    Full Text Available Five extracts were obtained from extraction of rhizomes of Zingiber amaricans. Hexane, dichloromethane and methanol extracts were obtained by maceration, while dichloromethane and acetone extracts the resulted of soxhlet extraction. By column chromatography technique 2,6,9-humulantrien-9-one (zerumbone was isolated as the major constituent of the hexane, dichloromethane and methanol extracts. The minor constituents were phytosterol mixtures isolated from hexane and dichloromethane extracts. The mixtures consisted cholesterol, campesterol, stigmasterol and b-sitosterol. The structure elucidations of zerumbone was confirmed by spectroscopic method, whereas the phytosterol mixtures was identified by gas chromatography-mass.   Keywords: zerumbone, phytosterol, zingiber amaricans, spectroscopy

  2. Molecular identification of nematode larvae different from those of the Trichinella genus detected by muscle digestion.

    Science.gov (United States)

    Marucci, Gianluca; Interisano, Maria; La Rosa, Giuseppe; Pozio, Edoardo

    2013-05-20

    Although larvae of the genus Trichinella are the most common parasite species detected in vertebrate muscles using artificial digestion, nematode larvae belonging to other genera are sometimes detected and incorrectly identified as Trichinella. However, it is often very difficult to identify these larvae at the species, genus or family level using microscopy because of the absence of specific morphological characters or cuticle damage, and the only means of identification is PCR and sequencing of specific molecular markers (12S mtDNA; COI; 18S rDNA; and ITS1). From 2008 to 2011, 18 nematode isolates not belonging to the genus Trichinella were collected from different host species. Eleven of these isolates were successfully identified at the species, genus or superfamily level: larvae from two common kestrels, three hooded crows, a hen harrier and a domestic pig were identified as Toxocara cati; larvae from a badger were identified as Toxocara canis; larvae from a domestic pig were identified as a free-living nematode of the genus Panagrolaimus; larvae from a wild boar were identified as belonging to the Metastrongylus genus; and larvae from a rough-legged buzzard were identified as belonging to the superfamily Filarioidea. The recovery of nematodes belonging to genera other than Trichinella during routine meat inspection suggests that the persons performing the analyses need to be informed of the possibility of false positives and that a molecular-based identification system that allows for a rapid and reliable response must be adopted (i.e., a DNA barcoding-like system). Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Haqqani Arsalan S

    2013-01-01

    Full Text Available Abstract Background In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes and ectosomes (originating from direct budding/shedding of plasma membranes. Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit. Methods To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed. Results A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially

  4. Molecular identification of the chitinase genes in Plasmodium relictum.

    Science.gov (United States)

    Garcia-Longoria, Luz; Hellgren, Olof; Bensch, Staffan

    2014-06-18

    Malaria parasites need to synthesize chitinase in order to go through the peritrophic membrane, which is created around the mosquito midgut, to complete its life cycle. In mammalian malaria species, the chitinase gene comprises either a large or a short copy. In the avian malaria parasites Plasmodium gallinaceum both copies are present, suggesting that a gene duplication in the ancestor to these extant species preceded the loss of either the long or the short copy in Plasmodium parasites of mammals. Plasmodium gallinaceum is not the most widespread and harmful parasite of birds. This study is the first to search for and identify the chitinase gene in one of the most prevalent avian malaria parasites, Plasmodium relictum. Both copies of P. gallinaceum chitinase were used as reference sequences for primer design. Different sequences of Plasmodium spp. were used to build the phylogenetic tree of chitinase gene. The gene encoding for chitinase was identified in isolates of two mitochondrial lineages of P. relictum (SGS1 and GRW4). The chitinase found in these two lineages consists both of the long (PrCHT1) and the short (PrCHT2) copy. The genetic differences found in the long copy of the chitinase gene between SGS1 and GRW4 were higher than the difference observed for the cytochrome b gene. The identification of both copies in P. relictum sheds light on the phylogenetic relationship of the chitinase gene in the genus Plasmodium. Due to its high variability, the chitinase gene could be used to study the genetic population structure in isolates from different host species and geographic regions.

  5. Molecular identification and characterization of Fusarium spp. associated with sorghum seeds.

    Science.gov (United States)

    Divakara, Shetty Thimmappa; Santosh, Parthasarathy; Aiyaz, Mohammed; Ramana, Mudili Venkata; Hariprasad, Puttaswamy; Nayaka, Siddaih Chandra; Niranjana, Siddapura Ramachandrappa

    2014-04-01

    Fusarium spp. are not only pathogenic to plants but are also known as toxin producers that negatively affect animal and human health. The identification of Fusarium spp. remains one of the most critical issues in fungal taxonomy. In this study, different strains of Fusarium spp. were isolated from sorghum seed samples and identified at the molecular level by tef-1α gene amplification. A multiplex polymerase chain reaction (mPCR) assay was developed to differentiate toxigenic and non-toxigenic Fusarium spp. by designing a primer for the Fum21 gene along with the Fum1 and Fum8 genes. A competitive direct enzyme-linked immunosorbent assay (CD-ELISA) was employed to assess the fumonisin-producing ability of Fusarium spp. Phylogenetic analyses were performed using partial sequences of tef-1α and inter-simple sequence repeat (ISSR) markers of different Fusarium spp. All 27 isolates of Fusarium spp. were positive for the tef-1α gene and revealed the presence of F. verticillioides, F. thapsina and F. cf. incarnatum-equiseti complex. The standardized mPCR assay distinguished toxigenic and non-toxigenic F. verticillioides. Further, mPCR fumonisin-positive F. verticillioides isolates were also positive by CD-ELISA. The tef-1α gene sequence was found to be useful in revealing intraspecific polymorphism to some extent. ISSR markers revealed a high level of polymorphism among different isolates of Fusarium spp., and the dendrogram of ISSR analyses grouped the 27 isolates into two major clusters. The present method provided rapid and reliable detection of fumonisin-producing Fusarium spp. The mPCR assay could be an alternative strategy to current conventional mycotoxin analytical techniques and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities. © 2013 Society of Chemical Industry.

  6. Identification of six potato virus Y isolates from Saudi Arabia

    African Journals Online (AJOL)

    Sherif

    2012-05-22

    May 22, 2012 ... PAGE) indicated the 34 kDa viral coat protein and agarose gel of the immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) products indicated the 800 bp cp gene. The sequences were aligned together, narrowed to six (one PVY-N and five PVY-O isolates) and then aligned.

  7. Identification of potential local isolated for biosurfactant production

    Science.gov (United States)

    Shafiei, Zahra; Yusoff, Wan Mohtar Wan; Hamid, Aidil Abdul; Moazami, Nasrin; Hamzah, Ainon; Fooladi, Taybeh

    2013-11-01

    Biosurfactant are amphiphilic molecule that have received increasing attention in recent years because of their role in the growth of microorganisms on water-insoluble hydrophobic materials such as hydrocarbons as well as their commercial potential in the cosmetics, food, oil recovery and agricultural industries. In this study a potential biosurfactant producing strain was isolated from several soil samples of Terengganu oil refinery, Malaysia and selected during preliminary screening using hemolytic activity, oil spreading and drop collapsed technique. Isolates with at least more than one positive response to these three methods were subjected to complementary screening by measuring surface tension reduction as well as emulsification capacity. The biosurfactant produced by isolated 5M was able to reduced surface tension of culture medium from 60 mN/m to30mN/m. The biochemical and morphological characterization, 16SrRNA gene sequencing showed that the isolated 5M belongs to bacillus groups. The maximum production of biosurfactant by Bacillus 5M was observed after 48 h of incubation.

  8. Isolation and identification of phenol degrading bacteria from Lake ...

    African Journals Online (AJOL)

    Phenol and its components are extremely toxic and can easily be isolated from different industrial sewage such as oil refinery, petrochemical industry and mines, especially collier and chemical factories. Hence the presence of these compounds in the environment could cause environmental pollution, especially in water ...

  9. Isolation and Identification of Fungi Associated with the Spoilage of ...

    African Journals Online (AJOL)

    This study was carried out in Sokoto Metropolis to isolate and identify fungi associated with the deterioration of sweet orange fruits. A total of one hundred samples of fresh sweet Oranges (Citrus sinensis L) were used. First, a total of seventy samples were obtained from the three selected marketing centres in Sokoto ...

  10. Isolation, Identification and Determination of Six Nucleosides and ...

    African Journals Online (AJOL)

    Methods: The compounds in bamboo shoots were isolated and identified by ultraviolet (UV) spectroscopy, mass spectrometry (MS), and nuclear magnetic resonance (NMR). Quantitative analysis was performed by reversed-phase high performance liquid chromatography (RP-HPLC) using a C18 column and a mixture ...

  11. Isolation and identification of fungal species from dried date palm ...

    African Journals Online (AJOL)

    A total of 360 dried date palm (Phoenix dactylifera) fruits were collected from hawkers, shops and market places within Maiduguri metropolis for the detection of the presence of fungal species. Investigation was based on cultural, microscopically and biochemical tests. Of the 327 (90.83%) fungal isolates recovered on ...

  12. Isolation, identification and antimicrobial suscep- tibility profiles of ...

    African Journals Online (AJOL)

    determine the in vitro antimicrobial resistance profiles of the isolates. A to- tal of 266 samples ... meats), cross contamination through direct contact of foods to contaminated surfaces such as stainless steel, hanging ... of contaminated milk and meat products (Endrias Zewdu and Cornelius 2009), through mutation, acquisition ...

  13. Isolation and identification of the microorganisms most prevalent in ...

    African Journals Online (AJOL)

    Infections of the external eye account for a significant percentage of ocular inflammations, some of which lead to visual losses as result of corneal involvement. This study purely isolated and identified the microorganisms most prevalent in external eye infections in Owerri urban (as seen Mercy Eye clinic). With the aid of ...

  14. Isolation and Identification of a Brevibacterium linens strain ...

    African Journals Online (AJOL)

    nitrophenol (PNP). Subsequent subcultures in agar, nutrient agar plates and agar slants by streaking led to isolation of pure colonies. The pure culture could degrade up to 300 mg/L PNP in presence of yeast extract. It was Gram positive rods, ...

  15. Identification of Cryptococcus neoformans isolates using Staib agar without creatinine

    OpenAIRE

    Nardelli, Vanessa; Pérez, Celina; Mata-Essayag, Sofía; Colella, María Teresa; Roselló, Arantza; Hartung de Capriles, Claudia; Landaeta, María Eugenia; Olaizola, Carolina; Magaldi, Sylvia

    2005-01-01

    In mycology, culture is the best way to demonstrate and identify pathogenic fungi. Many kinds of media have been developed and modified, using fungal biochemical properties to recognize them. The most common media are Sabouraud, Malt Agar, Cornmeal agar, Potato-dextrose agar, and Staib agar. Staib agar has been widely used for identification of yeasts from the genus Cryptococcus and other fungi. The aim of this study was to demonstrate the usefulness of Staib agar, and of a modified Staib med...

  16. Isolation and identification of cellulolytic bacteria from termites gut (Cryptotermes sp.)

    Science.gov (United States)

    Peristiwati; Natamihardja, Y. S.; Herlini, H.

    2018-05-01

    The energy and environmental crises developed due to a huge amount of cellulosic materials are disposed of as “waste.” Cellulose is the most abundant biopolymer on Earth. The hydrolysis of cellulose to glucose and soluble sugars has thus become a subject of intense research. Termites are one of the most important soil insects that efficiently decompose lignocelluloses with the aid of their associated microbial symbionts to a simpler form of sugars. The steps of this study consisted of cellulose isolation, cellulolytic bacteria isolation and identification. Cellulose degrading bacteria from termite (Cryptotermes sp.) gut flora were isolated, screened and their identification was studied which showed halo zones due to CMC agar. Among 12 isolates of bacteria, six isolates were cellulolytic. MLC-A isolate had shown a maximum in a cellulolytic index (1.32). Each isolate was identified based on standard physical and biochemical tests. Three isolates were identified in the genus of Clostridium, one isolate be placed in the group of Mycobacteriaceae, Lactobacillaceae or Coryneform and the last one in the genus Proteus.

  17. The Use of Fta Card on Dna Sample Preparation for Molecular of Plant Disease Identification

    OpenAIRE

    Sulistyawati, Purnamila; Rimbawanto, Anto

    2007-01-01

    Accurate and guick identification of pathogen is key to control the spread of plant disesases. Morphological identification is often ineffective because it requires fruit body which often are not presence, rely on characters which may be highly variable within and among species and can be slow and time consuming. Molecular identification of plant disease can overcome most of the shortcomings of morphological identification. Application of FTA Cardn for sample collection is crucial for the su...

  18. Isolation and identification of differentially expressed genes between ...

    African Journals Online (AJOL)

    Plants have evolved sophisticated molecular defense mechanisms in order to survive disease conditions. So far, a number of pathogen resistance (R) genes have been reported in plants. These R genes are thought to be involved in activating the signals that lead to disease resistance. The structural specificity of R genes ...

  19. Isolation and identification of a new intracellular antimicrobial peptide produced by Paenibacillus alvei AN5.

    Science.gov (United States)

    Alkotaini, Bassam; Anuar, Nurina; Kadhum, Abdul Amir Hassan; Sani, Asmahani Azira Abdu

    2014-04-01

    A wild-type, Gram-positive, rod-shaped, endospore-forming and motile bacteria has been isolated from palm oil mill sludge in Malaysia. Molecular identification using 16S rRNA gene sequence analysis indicated that the bacteria belonged to genus Paenibacillus. With 97 % similarity to P. alvei (AUG6), the isolate was designated as P. alvei AN5. An antimicrobial compound was extracted from P. alvei AN5-pelleted cells using 95 % methanol and was then lyophilized. Precipitates were re-suspended in phosphate buffered saline (PBS), producing an antimicrobial crude extract (ACE). The ACE showed antimicrobial activity against Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 29522, Bacillus cereus ATCC 14579 and Lactobacillus plantarum ATCC 8014. By using SP-Sepharose cation exchange chromatography, Sephadex G-25 gel filtration and Tricine SDS-PAGE, the ACE was purified, which produced a ~2-kDa active band. SDS-PAGE and infrared (IR) spectroscopy indicated the proteinaceous nature of the antimicrobial compound in the ACE, and liquid chromatography electrospray ionization mass spectroscopy and de novo sequencing using an automatic, Q-TOF premier system detected a peptide with the amino acid sequence F-C-K-S-L-P-L-P-L-S-V-K (1,330.7789 Da). This novel peptide was designated as AN5-2. The antimicrobial peptide exhibited stability from pH 3 to 12 and maintained its activity after being heated to 90 °C. It also remained active after incubation with denaturants (urea, SDS and EDTA).

  20. Isolation and Identification of Burkholderia glumae from Symptomless Rice Seeds

    Directory of Open Access Journals (Sweden)

    Bo Zhu

    2008-06-01

    Full Text Available A survey on isolation and detection of the casual organism of bacterial grain rot of rice was conducted during 1997–2006. In 2006, six pathogenic bacterial strains were isolated from two symptomless seed samples of rice (Oryza sativa L. originally produced in Hainan Province and then planted in Zhejiang Province, China. They were identified as Burkholderia glumae which is the causal organism of bacterial grain rot of rice by physiological characteristics, colony morphology, pathogenicity test, Biolog, fatty acid methyl ester (FAME analysis and RAPD-PCR compared with the four standard reference strains. It is confirmed that there is the infection of B. glumae in so-called ‘health looking seeds’.

  1. Procedure of identification of fullerenes isolated from iron-carbon alloys

    International Nuclear Information System (INIS)

    Zakirnichnaya, M.M.

    2001-01-01

    A method of fullerenes isolation from the structure of iron-carbon alloys and their identification using physical methods which provide determination of the different parameters of nanoobjects is developed. Qualitative (mass-spectrometry of positive and negative ions, small angle X-ray scattering) and quantitative (IR-spectrometry, liquid chromatography) evaluation of fullerenes in the samples obtained from iron-carbon alloys and their visual observation using scanning tunnel microscopy are performed. It is found that the method provides isolation and identification of fullerenes present in the structure of steels and irons [ru

  2. Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus

    DEFF Research Database (Denmark)

    Dalsgaard, A.; Dalsgaard, Inger; Høi, L.

    1996-01-01

    Methods for the identification and isolation of environmental isolates of Vibrio vulnificus were evaluated. Alkaline peptone water supplemented with polymyxin B and colistin-polymyxin B- cellobiose agar were employed for the isolation of suspected V. vulnificus from water, sediment and shellfish ...... hybridizing with the probe. The results indicated that, compared with colony hybridization, the API 20E assay was not adequate for the identification of environmental isolates of V. vulnificus....

  3. Identification and diversity of Fusarium species isolated from tomato fruits

    Directory of Open Access Journals (Sweden)

    Murad Nur Baiti Abd

    2016-07-01

    Full Text Available Fruit rot of tomato is a serious disease caused by Fusarium species. Sampling was conducted throughout Selangor, Malaysia and fungal species identification was conducted based on morphological and gene encoding translation elongation factor 1-α (tef1-α sequence analysis. Five species of Fusarium were discovered namely F. oxysporum (including F. oxysporum f. sp. lycopersici, F. solani, F. equiseti, F. proliferatum and F. verticillioides. Our results provide additional information regarding the diversity of Fusarium species associated with fruit rot disease of tomato.

  4. Isolation and identification of bacterial pathogen from mastitis milk in Central Java Indonesia

    Science.gov (United States)

    Harjanti, D. W.; Ciptaningtyas, R.; Wahyono, F.; Setiatin, ET

    2018-01-01

    Mastitis is a multi-etiologic disease of the mammary gland characterized mainly by reduction in milk production and milk quality due to intramammary infection by pathogenic bacteria. Nearly 83% of lactating dairy cows in Indonesia are infected with mastitis in various inflammation degrees. This study was conducted to isolate and identify the pathogen in milk collected from mastitis-infected dairy cows. The study was carried out in ten smallholder dairy farms in Central Java Indonesia based on animal examination, California mastitis test, isolation bacterial pathogens, Gram staining, Catalase and Coagulase test, and identification of bacteria species using Vitek. Bacteriological examination of milk samples revealed 15 isolates where Streptococcus was predominant species (73.3%) and the coagulase negative Staphylococcus species was identified at the least bacteria (26.7%). The Streptococcus bacteria found were Streptococcus uberis (2 isolates), Streptococcus sanguinis(6 isolates), Streptococcus dysgalactiaessp dysgalactiae(1 isolate) , Streptococcus mitis (1 isolate) and Streptococcus agalactiae (1 isolate). The Staphylococcus isolates comprising of Staphylococcus simulans (1 isolate) and Staphylococcus chromogens (3 isolates). Contamination of raw milkwith pathogenic bacteria can cause outbreaks of human disease (milk borne disease). Thus, proper milk processing method that couldinhibit the growth or kill these pathogenic bacteria is important to ensure the safety of milk and milk products.

  5. Characterization and identification of newly isolated Acinetobacter baumannii strain serdang 1 for phenol removal

    Science.gov (United States)

    Yadzir, Z. H. M.; Shukor, M. Y.; Nazir, M. S.; Abdullah, M. A.

    2012-09-01

    A new indigenous bacterial strain from Malaysian soil contaminated with petroleum waste had been successfully isolated, characterized and identified for phenol removal. The gram negative bacteria showed 98% identity with Acinetobacter baumannii based on Biolog{trade mark, serif} Identification System and the determination of a partial 16S ribosomal RNA (rRNA) sequence. The isolate clustered with species belonging to Acinetobacter clade in a 16S rDNA-based neighbour-joining phylogenetic tree.

  6. Isolation and identification of lactic acid bacteria from koumiss in Eastern Inner Mongolia of China

    Science.gov (United States)

    Bai, Lijuan; Ji, Shujuan

    2017-01-01

    Koumiss is a traditional fermented dairy product and known as its unique physiological actions. Isolation and identification of LAB in it will yield valuable knowledge. In total, 55 LAB strains were isolated and identified of 12 koumiss samples collected in limited regions of Eastern Inner Mongolia. 16S rRNA sequence analysis results showed that were Lactobacillus helveticus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus delbrueckii, Enterococcus. durans, Leuconostoc lactis and Leuconostoc mesenteroides. It is benefit to further research on koumiss.

  7. Commercialized non-Camellia tea: traditional function and molecular identification

    Directory of Open Access Journals (Sweden)

    Ping Long

    2014-06-01

    Full Text Available Non-Camellia tea is a part of the colorful Chinese tea culture, and is also widely used as beverage and medicine in folk for disease prevention and treatment. In this study, 37 samples were collected, including 33 kinds of non-Camellia teas and 4 kinds of teas (Camellia. Traditional functions of non-Camellia teas were investigated. Furthermore, non-Camellia teas of original plants were characterized and identified by molecular methods. Four candidate regions (rbcL, matK, ITS2, psbA-trnH were amplified by polymerase chain reaction. In addition, DNA barcodes were used for the first time to discriminate the commercial non-Camellia tea and their adulterants, and to evaluate their safety. This study showed that BLASTN and the relevant phylogenetic tree are efficient tools for identification of the commercial non-Camellia tea and their adulterants. However, some sequences from original plants have not been found and there is a limitation of sequence number of original plants in GenBank. Submitting more original plant sequences to the GenBank will be helpful for evaluating the safety of non-Camellia teas.

  8. Molecular characterization of Histoplasma capsulatum isolated from an outbreak in treasure hunters Histoplasma capsulatum in treasure hunters

    Directory of Open Access Journals (Sweden)

    Muñoz Bertha

    2010-09-01

    Full Text Available Abstract Background In Mexico, primary pulmonary histoplasmosis is the most relevant clinical form of the disease. The geographical distribution of specific strains of Histoplasma capsulatum circulating in Mexico has not been fully established. Outbreaks must be reported in order to have current, updated information on this disease, identifying new endemic areas, manner of exposure to the fungi, and molecular characterization of the causative agents. We report a recent outbreak of histoplasmosis in treasure hunters and the molecular characterization of two isolates obtained from these patients. Methods Six patients admitted to the National Institute of Respiratory Diseases (INER in Mexico City presented severe respiratory symptoms suggestive of histoplasmosis. They acquired the infection in the Veracruz (VZ endemic zone. Diagnosis was made by X-ray and Computed tomography (CT, liver function, immunological techniques, and culture. Identification of H. capsulatum isolates was confirmed by using Polymerase chain reaction (PCR was conducted with a probe from the M antigen, and the isolates were characterized by means of Random amplification of polymorphic DNA (RAPD-PCR employed the 1253 oligonucleotide and a mixture of oligonucleotides 1281 and 1283. These were compared to eight reference strain isolates from neighboring areas. Results X-ray and CT revealed disseminated micronodular images throughout lung parenchyma, as well as bilateral retrocaval, prevascular, subcarinal, and hilar adenopathies, hepatosplenomegaly, and altered liver function tests. Five of the six patients developed disseminated histoplasmosis. Two H. capsulatum strains were isolated. The same band profile was detected in both strains, indicating that both isolates corresponded to the sole H. capsulatum strain. Molecular characterization of the isolates was similar in 100% with the EH-53 Hidalgo human (HG strain (reference strain integrated into the LAm A clade described for

  9. Molecular identification of Tribolium castaneum and T. confusum ...

    Indian Academy of Sciences (India)

    ful alternative method to identify the two sibling flour beetle species. ∗ ... represents a valuable addition or alternative to traditional phenotypic identification methods. Also, DNA ..... Buczkowski G. and Bennett G. 2009 Survey and identification.

  10. Molecular docking of viscosine as a new lipoxygenase inhibitor isolated from Dodonaea viscosa

    Directory of Open Access Journals (Sweden)

    Amir Zada Khan

    2013-03-01

    Full Text Available Viscosine was isolated from Dodonaea viscosa, which showed significant lipoxygenase inhibitory activity (IC50: value 39 ± 0.17. Molecular docking simulations were conducted to explore molecular binding mode, and to help elucidate molecular mechanism behind its significant inhibitory activity. Molecular interactions of viscosine with catalytic triad (His523, His518, Ile875 inside active site of lipoxygenase via hydrogen bonding, seems to be the major factor involved in its significant lipoxygenase inhibitory activity.

  11. Molecular characterization of two isolates of sweet potato leaf curl ...

    African Journals Online (AJOL)

    Comparison analysis showed that DNA-A sequence of JS1 isolate was closely related to that of sweet potato leaf curl virus (SPLCV) from United States with nucleotide sequence identity of 97.0% and DNA-A of Y338 showed highest sequence identity at 97.8% with an isolate of SPLCV from China. Phylogenetic analysis ...

  12. Molecular characterization of Azotobacter spp. nifH gene Isolated ...

    African Journals Online (AJOL)

    The Azotobacter species was isolated from marine source in two different seasons. They were cultivated under laboratory conditions using Nitrogen free Azotobacter specific medium. We observed that they were present in both seasons. The phylogenetic tree revealed that our isolated Azotobacter spp. was distantly related ...

  13. Molecular characterization of bacteria isolated from the Kingdom of ...

    African Journals Online (AJOL)

    ola

    2012-11-06

    Nov 6, 2012 ... Esherichia coli strains isolated from different habitats in relation to their antimicrobial activity against pathogenic fungi causing dermatological diseases. MATERIALS AND METHODS. Microbial strains, media and culture conditions. Bacterial isolates from soil samples were collected from different localities ...

  14. Isolation and identification of a novel radio-resistant strain

    International Nuclear Information System (INIS)

    Zhang Zhidong; Mao Jun; Wang Wei; Tang Qiyong; Shi Yuhu

    2008-01-01

    A novel radio-resistant strain named RL2 was studied polyphasically, which was isolated from the soils in the Gurban-Tunggut Desert, Xinjiang. The strain is Gam-positive, sphere-shaped and pink pigmented; The DNA (G+C) contents of RL2 is 71.62mo1%; The 16S rDNA genes of RL2 and D. radiodurans type strain DSM20539 shows a high level of similarity (97.2%). According to phenotypic characteristics and phylogenetic analysis, it can be suggested that the strain RL2 has been identified as Deinococcus. sp and it may be a novel species. (authors)

  15. Isolation and identification of an allelopathic substance from Hibiscus sabdariffa.

    Science.gov (United States)

    Suwitchayanon, Prapaipit; Pukclai, Piyatida; Ohno, Osamu; Suenaga, Kiyotake; Kato-Noguchi, Hisashi

    2015-05-01

    In this study, an allelopathic substance was isolated from an aqueous methanol extract of Hibiscus sabdariffa L. by column chromatography and reverse phase HPLC. The chemical structure of the substance was determined by 1H NMR spectroscopy and mass spectrometry as trimethyl allo-hydroxycitrate. Trimethyl allo-hydroxycitrate inhibited the growth of cress hypocotyls and roots at concentrations greater than 10 mM. The concentrations required for 50% growth inhibition of the hypocotyls and roots of cress were 20.3 and 14.4 mM, respectively. The inhibitory activity of trimethyl allo-hydroxycitrate suggests that the substance may act as an allelopathic substance of H. sabdariffa.

  16. Environmental issue identification for the Basalt Waste Isolation Project

    International Nuclear Information System (INIS)

    Carrell, D.J.; Jones, K.A.

    1980-04-01

    A preliminary evaluation of environmental issues is provided in this report. It contains summary of the thought process that was used during the area characterization studies for a geological repository for high-level radioactive wastes. Environmental issues are discussed separately for construction, operation, and long term isolation aspects of a repository in basalt. During construction the primary environmental concerns are public perception and water resources; intermediate concerns are air quality, ecosystems, physical resources, and cultural and social resources. During operation, the primary environmental issues concern the transport of radioactive materials and physical resources. Long term environmental issues envolve water resources and borehole plugging

  17. Molecular identification and histopathological study of natural Streptococcus agalactiae infection in hybrid tilapia (Oreochromis niloticus).

    Science.gov (United States)

    Laith, A A; Ambak, Mohd Azmi; Hassan, Marina; Sheriff, Shahreza Md; Nadirah, Musa; Draman, Ahmad Shuhaimi; Wahab, Wahidah; Ibrahim, Wan Nurhafizah Wan; Aznan, Alia Syafiqah; Jabar, Amina; Najiah, Musa

    2017-01-01

    The main objective of this study was to emphasize on histopathological examinations and molecular identification of Streptococcus agalactiae isolated from natural infections in hybrid tilapia ( Oreochromis niloticus ) in Temerloh Pahang, Malaysia, as well as to determine the susceptibility of the pathogen strains to various currently available antimicrobial agents. The diseased fishes were observed for variable clinical signs including fin hemorrhages, alterations in behavior associated with erratic swimming, exophthalmia, and mortality. Tissue samples from the eyes, brain, kidney, liver, and spleen were taken for bacterial isolation. Identification of S. agalactiae was screened by biochemical methods and confirmed by VITEK 2 and 16S rRNA gene sequencing. The antibiogram profiling of the isolate was tested against 18 standard antibiotics included nitrofurantoin, flumequine, florfenicol, amoxylin, doxycycline, oleandomycin, tetracycline, ampicillin, lincomycin, colistin sulfate, oxolinic acid, novobiocin, spiramycin, erythromycin, fosfomycin, neomycin, gentamycin, and polymyxin B. The histopathological analysis of eyes, brain, liver, kidney, and spleen was observed for abnormalities related to S. agalactiae infection. The suspected colonies of S. agalactiae identified by biochemical methods was observed as Gram-positive chained cocci, β-hemolytic, and non-motile. The isolate was confirmed as S. agalactiae by VITEK 2 (99% similarity), reconfirmed by 16S rRNA gene sequencing (99% similarity) and deposited in GenBank with accession no. KT869025. The isolate was observed to be resistance to neomycin and gentamicin. The most consistent gross findings were marked hemorrhages, erosions of caudal fin, and exophthalmos. Microscopic examination confirmed the presence of marked congestion and infiltration of inflammatory cell in the eye, brain, kidney, liver, and spleen. Eye samples showed damage of the lens capsule, hyperemic and hemorrhagic choroid tissue, and retina

  18. Molecular identification and histopathological study of natural Streptococcus agalactiae infection in hybrid tilapia (Oreochromis niloticus

    Directory of Open Access Journals (Sweden)

    Laith Abdul Razzak

    2017-01-01

    Full Text Available Aim: The main objective of this study was to emphasize on histopathological examinations and molecular identification of Streptococcus agalactiae isolated from natural infections in hybrid tilapia (Oreochromis niloticus in Temerloh Pahang, Malaysia, as well as to determine the susceptibility of the pathogen strains to various currently available antimicrobial agents. Materials and Methods: The diseased fishes were observed for variable clinical signs including fin hemorrhages, alterations in behavior associated with erratic swimming, exophthalmia, and mortality. Tissue samples from the eyes, brain, kidney, liver, and spleen were taken for bacterial isolation. Identification of S. agalactiae was screened by biochemical methods and confirmed by VITEK 2 and 16S rRNA gene sequencing. The antibiogram profiling of the isolate was tested against 18 standard antibiotics included nitrofurantoin, flumequine, florfenicol, amoxylin, doxycycline, oleandomycin, tetracycline, ampicillin, lincomycin, colistin sulfate, oxolinic acid, novobiocin, spiramycin, erythromycin, fosfomycin, neomycin, gentamycin, and polymyxin B. The histopathological analysis of eyes, brain, liver, kidney, and spleen was observed for abnormalities related to S. agalactiae infection. Results: The suspected colonies of S. agalactiae identified by biochemical methods was observed as Gram-positive chained cocci, β-hemolytic, and non-motile. The isolate was confirmed as S. agalactiae by VITEK 2 (99% similarity, reconfirmed by 16S rRNA gene sequencing (99% similarity and deposited in GenBank with accession no. KT869025. The isolate was observed to be resistance to neomycin and gentamicin. The most consistent gross findings were marked hemorrhages, erosions of caudal fin, and exophthalmos. Microscopic examination confirmed the presence of marked congestion and infiltration of inflammatory cell in the eye, brain, kidney, liver, and spleen. Eye samples showed damage of the lens capsule

  19. Isolation and identification of a bovine viral diarrhea virus from sika deer in china.

    Science.gov (United States)

    Gao, Yugang; Wang, Shijie; Du, Rui; Wang, Quankai; Sun, Changjiang; Wang, Nan; Zhang, Pengju; Zhang, Lianxue

    2011-02-25

    Bovine viral diarrhea virus (BVDV) infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical sign of BVDV to isolation and identification of BVDV from sika deer. we isolated a suspected BVDV strain from livers of an aborted fetus from sika deer in Changchun (China) using MDBK cell lines, named as CCSYD strain, and identified it by cytopathic effect (CPE), indirect immunoperoxidase test (IPX) and electron microscopy(EM). The results indicated that this virus was BVDV by a series of identification. The structural proteins E0 gene was cloned and sequenced. The obtained E0 gene sequence has been submitted to GenBank with the accession number: FJ555203. Alignment with other 9 strains of BVDV, 7 strains of classical swine fever virus (CSFV) and 3 strains of border disease virus(BDV) in the world, showed that the homology were 98.6%-84.8%, 76.0%-74.7%, 76.6%-77.0% for nucleotide sequence, respectively. The phylogenetic analysis indicated that new isolation and identification CCSYD strain belonged to BVDV1b. To the best of our knowledge, this is the first report that BVDV was isolated and identified in sika deer. This current research contributes development new BVDV vaccine to prevent and control of BVD in sika deer.

  20. Isolation and identification of a bovine viral diarrhea virus from sika deer in china

    Directory of Open Access Journals (Sweden)

    Wang Nan

    2011-02-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV infections continue to cause significantly losses in the deer population. Better isolation and identification of BVDV from sika deer may contribute significantly to the development of prophylactic therapeutic, and diagnostic reagents as well as help in prevention and control of BVDV. However, isolation and identification of BVDV from sika deer is seldom reported in literature. In this study, we collected some samples according to clinical sign of BVDV to isolation and identification of BVDV from sika deer. Results we isolated a suspected BVDV strain from livers of an aborted fetus from sika deer in Changchun (China using MDBK cell lines, named as CCSYD strain, and identified it by cytopathic effect (CPE, indirect immunoperoxidase test (IPX and electron microscopy(EM. The results indicated that this virus was BVDV by a series of identification. The structural proteins E0 gene was cloned and sequenced. The obtained E0 gene sequence has been submitted to GenBank with the accession number: FJ555203. Alignment with other 9 strains of BVDV, 7 strains of classical swine fever virus (CSFV and 3 strains of border disease virus(BDV in the world, showed that the homology were 98.6%-84.8%, 76.0%-74.7%, 76.6%-77.0% for nucleotide sequence, respectively. The phylogenetic analysis indicated that new isolation and identification CCSYD strain belonged to BVDV1b. Conclusion To the best of our knowledge, this is the first report that BVDV was isolated and identified in sika deer. This current research contributes development new BVDV vaccine to prevent and control of BVD in sika deer.

  1. Chemicals from Agave sisalana Biomass: Isolation and Identification

    Science.gov (United States)

    Santos, Jener David Gonçalves; Vieira, Ivo Jose Curcino; Braz-Filho, Raimundo; Branco, Alexsandro

    2015-01-01

    Agave sisalana (sisal) is known worldwide as a source of hard fibers, and Brazil is the largest producer of sisal. Nonetheless, the process of removing the fibers of the sisal leaf generates 95% waste. In this study, we applied chemical sequential steps (hydrothermal extraction, precipitation, liquid-liquid extraction, crystallization, SiO2 and Sephadex LH 20 column chromatography) to obtain pectin, mannitol, succinic acid, kaempferol and a mixture of saponins as raw chemicals from sisal biomass. The structural identification of these compounds was performed though spectrometric methods, such as Infrared (IR), Ultraviolet (UV), Mass spectrometry (MS) and Nuclear magnetic resonance (NMR). All the sisal chemicals found in this work are used by both the chemical and pharmaceutical industries as excipients or active principles in products. PMID:25903149

  2. Chemicals from Agave sisalana Biomass: Isolation and Identification

    Directory of Open Access Journals (Sweden)

    Jener David Gonçalves Santos

    2015-04-01

    Full Text Available Agave sisalana (sisal is known worldwide as a source of hard fibers, and Brazil is the largest producer of sisal. Nonetheless, the process of removing the fibers of the sisal leaf generates 95% waste. In this study, we applied chemical sequential steps (hydrothermal extraction, precipitation, liquid-liquid extraction, crystallization, SiO2 and Sephadex LH 20 column chromatography to obtain pectin, mannitol, succinic acid, kaempferol and a mixture of saponins as raw chemicals from sisal biomass. The structural identification of these compounds was performed though spectrometric methods, such as Infrared (IR, Ultraviolet (UV, Mass spectrometry (MS and Nuclear magnetic resonance (NMR. All the sisal chemicals found in this work are used by both the chemical and pharmaceutical industries as excipients or active principles in products.

  3. Identification of a molecular pH sensor in coral.

    Science.gov (United States)

    Barott, Katie L; Barron, Megan E; Tresguerres, Martin

    2017-11-15

    Maintaining stable intracellular pH (pHi) is essential for homeostasis, and requires the ability to both sense pH changes that may result from internal and external sources, and to regulate downstream compensatory pH pathways. Here we identified the cAMP-producing enzyme soluble adenylyl cyclase (sAC) as the first molecular pH sensor in corals. sAC protein was detected throughout coral tissues, including those involved in symbiosis and calcification. Application of a sAC-specific inhibitor caused significant and reversible pHi acidosis in isolated coral cells under both dark and light conditions, indicating sAC is essential for sensing and regulating pHi perturbations caused by respiration and photosynthesis. Furthermore, pHi regulation during external acidification was also dependent on sAC activity. Thus, sAC is a sensor and regulator of pH disturbances from both metabolic and external origin in corals. Since sAC is present in all coral cell types, and the cAMP pathway can regulate virtually every aspect of cell physiology through post-translational modifications of proteins, sAC is likely to trigger multiple homeostatic mechanisms in response to pH disturbances. This is also the first evidence that sAC modulates pHi in any non-mammalian animal. Since corals are basal metazoans, our results indicate this function is evolutionarily conserved across animals. © 2017 The Author(s).

  4. Molecular identification of Lactobacillus spp. associated with puba, a Brazilian fermented cassava food

    Directory of Open Access Journals (Sweden)

    S.M. Crispim

    2013-01-01

    Full Text Available Puba or carimã is a Brazilian staple food obtained by spontaneous submerged fermentation of cassava roots. A total of 116 lactobacilli and three cocci isolates from 20 commercial puba samples were recovered on de Man, Rogosa and Sharpe agar (MRS; they were characterized for their antagonistic activity against foodborne pathogens and identified taxonomically by classical and molecular methods. In all samples, lactic acid bacteria were recovered as the dominant microbiota (7.86 ± 0.41 log10 CFU/g. 16S-23S rRNA ARDRA pattern assigned 116 isolates to the Lactobacillus genus, represented by the species Lactobacillus fermentum (59 isolates, Lactobacillus delbrueckii (18 isolates, Lactobacillus casei (9 isolates, Lactobacillus reuteri (6 isolates, Lactobacillus brevis (3 isolates, Lactobacillus gasseri (2 isolates, Lactobacillus nagelii (1 isolate, and Lactobacillus plantarum group (18 isolates. recA gene-multiplex PCR analysis revealed that L. plantarum group isolates belonged to Lactobacillus plantarum (15 isolates and Lactobacillus paraplantarum (3 isolates. Genomic diversity was investigated by molecular typing with rep (repetitive sequence-based PCR using the primer ERIC2 (enterobacterial repetitive intergenic consensus. The Lactobacillus isolates exhibited genetic heterogeneity and species-specific fingerprint patterns. All the isolates showed antagonistic activity against the foodborne pathogenic bacteria tested. This antibacterial effect was attributed to acid production, except in the cases of three isolates that apparently produced bacteriocin-like inhibitory substances. This study provides the first insight into the genetic diversity of Lactobacillus spp. of puba.

  5. Microbiological and molecular characterization of Staphylococcus hominis isolates from blood.

    Directory of Open Access Journals (Sweden)

    Soraya Mendoza-Olazarán

    Full Text Available BACKGROUND: Among Coagulase-Negative Staphylococci (CoNS, Staphylococcus hominis represents the third most common organism recoverable from the blood of immunocompromised patients. The aim of this study was to characterize biofilm formation, antibiotic resistance, define the SCCmec (Staphylococcal Chromosomal Cassette mec type, and genetic relatedness of clinical S. hominis isolates. METHODOLOGY: S. hominis blood isolates (n = 21 were screened for biofilm formation using crystal violet staining. Methicillin resistance was evaluated using the cefoxitin disk test and the mecA gene was detected by PCR. Antibiotic resistance was determined by the broth microdilution method. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE and SCCmec typed by multiplex PCR using two different methodologies described for Staphylococcus aureus. RESULTS: Of the S. hominis isolates screened, 47.6% (10/21 were categorized as strong biofilm producers and 23.8% (5/21 as weak producers. Furthermore, 81% (17/21 of the isolates were methicillin resistant and mecA gene carriers. Resistance to ampicillin, erythromycin, and trimethoprim was observed in >70% of isolates screened. Each isolate showed a different PFGE macrorestriction pattern with similarity ranging between 0-95%. Among mecA-positive isolates, 14 (82% harbored a non-typeable SCCmec type: eight isolates were not positive for any ccr complex; four contained the mec complex A ccrAB1 and ccrC, one isolate contained mec complex A, ccrAB4 and ccrC, and one isolate contained the mec complex A, ccrAB1, ccrAB4, and ccrC. Two isolates harbored the association: mec complex A and ccrAB1. Only one strain was typeable as SCCmec III. CONCLUSIONS: The S. hominis isolates analyzed were variable biofilm producers had a high prevalence of methicillin resistance and resistance to other antibiotics, and high genetic diversity. The results of this study strongly suggested that S. hominis isolates harbor

  6. Fusarium proliferatum isolated from garlic in Spain: identification, toxigenic potential and pathogenicity on related Allium species

    Directory of Open Access Journals (Sweden)

    Daniel PALMERO

    2012-05-01

    Full Text Available Fusarium proliferatum has been reported on garlic in the Northwest USA, Spain and Serbia, causing water-soaked tan-colored lesions on cloves. In this work, Fusarium proliferatum was isolated from 300 symptomatic garlic bulbs. Morphological identification of Fusarium was confirmed using species-specific PCR assays and EF-1α sequencing. Confirmation of pathogenicity was conducted with eighteen isolates. Six randomly selected F. proliferatum isolates from garlic were tested for specific pathogenicity and screened for fusaric acid production. Additionally, pathogenicity of each F. proliferatum isolate was tested on healthy seedlings of onion (Allium cepa, leek (A. porrum, scallions (A. fistulosum, chives (A. schoenoprasum and garlic (A. sativum. A disease severity index (DSI was calculated as the mean severity on three plants of each species with four test replicates. Symptoms on onion and garlic plants were observed three weeks after inoculation. All isolates tested produced symptoms on all varieties inoculated. Inoculation of F. proliferatum isolates from diseased garlic onto other Allium species provided new information on host range and pathogenicity. The results demonstrated differences in susceptibility with respect to host species and cultivar. The F. proliferatum isolates tested all produced fusaric acid (FA; correlations between FA production and isolate pathogenicity are discussed. Additionally, all isolates showed the presence of the FUM1 gene suggesting the ability of Spanish isolates to produce fumonisins.

  7. Molecular identification of bacteria associated with canine periodontal disease.

    Science.gov (United States)

    Riggio, Marcello P; Lennon, Alan; Taylor, David J; Bennett, David

    2011-06-02

    Periodontal disease is one of the most common diseases of adult dogs, with up to 80% of animals affected. The aetiology of the disease is poorly studied, although bacteria are known to play a major role. The purpose of this study was to identify the bacteria associated with canine gingivitis and periodontitis and to compare this with the normal oral flora. Swabs were obtained from the gingival margin of three dogs with gingivitis and three orally healthy controls, and subgingival plaque was collected from three dogs with periodontitis. Samples were subjected to routine bacterial culture. The prevalent species identified in the normal, gingivitis and periodontitis groups were uncultured bacterium (12.5% of isolates), Bacteroides heparinolyticus/Pasteurella dagmatis (10.0%) and Actinomyces canis (19.4%), respectively. Bacteria were also identified using culture-independent methods (16S rRNA gene sequencing) and the predominant species identified were Pseudomonas sp. (30.9% of clones analysed), Porphyromonas cangingivalis (16.1%) and Desulfomicrobium orale (12.0%) in the normal, gingivitis and periodontitis groups, respectively. Uncultured species accounted for 13.2%, 2.0% and 10.5%, and potentially novel species for 38.2%, 38.3% and 35.3%, of clones in the normal, gingivitis and periodontitis groups, respectively. This is the first study to use utilise culture-independent methods for the identification of bacteria associated with this disease. It is concluded that the canine oral flora in health and disease is highly diverse and also contains a high proportion of uncultured and, in particular, potentially novel species. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Isolation and Identification of Active Compound Cause Light Emmitting of Bacterial Photobacterium phosphoreum Isolated from the Indonesia Jepara Marine Squid

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    Idam Arif

    2005-04-01

    Full Text Available This research carried out to study the bioluminescence process of bacterial Photobacterium phosphoreum isolated from Indonesia marine squid. The method used in the present study involved isolation, purification, electrophoresis, and the absorbance and light intensity measurement. This result show that the luciferace enzyme of bacterial Photobacterium phosphoreum or called LBPP catalyzes the emission of visible light from the reaction of reduced flavin mononucleotide (FMNH2, molecular oxygen (O2, and an aldehyde (RCOH. The electrophoresis data show that LBPP comprised of two different subunits α and βwith 41kD and 38 kD molecular weights. The absorb pattern showed that the bioluminescence process centered around 516 nm and are consistent with the fluorescence data. This result concluded that the excitation state formed after LBPP bind subtracts and the ground state formed after LBPP releases product and visible light.

  9. Isolation and identification of the human homolog of a new p53-binding protein, Mdmx

    NARCIS (Netherlands)

    Shvarts, A.; Bazuine, M.; Dekker, P.; Ramos, Y. F.; Steegenga, W. T.; Merckx, G.; van Ham, R. C.; van der Houven van Oordt, W.; van der Eb, A. J.; Jochemsen, A. G.

    1997-01-01

    We recently reported the identification of a mouse cDNA encoding a new p53-associating protein that we called Mdmx because of its structural similarity to Mdm2, a well-known p53-binding protein. Here we report the isolation of a cDNA encoding the human homolog of Mdmx. The ORF of the cDNA encodes a

  10. Evaluation of the Vitek 2 ANC card for identification of clinical isolates of anaerobic bacteria.

    Science.gov (United States)

    Lee, E H L; Degener, J E; Welling, G W; Veloo, A C M

    2011-05-01

    An evaluation of the Vitek 2 ANC card (bioMérieux, Marcy l'Etoile, France) was performed with 301 anaerobic isolates. Each strain was identified by 16S rRNA gene sequencing, which is considered to be the reference method. The Vitek 2 ANC card correctly identified 239 (79.4%) of the 301 clinical isolates to the genus level, including 100 species that were not represented in the database. Correct species identification was obtained for 60.1% (181/301) of the clinical isolates. For the isolates not identified to the species level, a correct genus identification was obtained for 47.0% of them (47/100), and 16 were accurately designated not identified. Although the Vitek 2 ANC card allows the rapid and acceptable identification of the most common clinically important anaerobic bacteria within 6 h, improvement is required for the identification of members of the genera Fusobacterium, Prevotella, and Actinomyces and certain Gram-positive anaerobic cocci (GPAC).

  11. Identification of unusual Campylobacter-like isolates from poultry products as Helicobacter pullorum

    DEFF Research Database (Denmark)

    Atabay, H.I.; Corry, J.E.L.; On, Stephen L.W.

    1998-01-01

    %) suggests that routine isolation and identification methods should be amended to enable a thorough evaluation of its role in human gastroenteritis and avian hepatitis. Some phenotypic characters useful in identifying poultry campylobacteria are presented which could be utilized, along with other technique...

  12. Molecular characterization of Prunus necrotic ringspot virus isolated from rose in Brazil

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    Thor Vinícius Martins Fajardo

    2015-12-01

    Full Text Available ABSTRACT: There is no molecular characterization of Brazilian isolates of Prunus necrotic ringspot virus (PNRSV, except for those infecting peach. In this research, the causal agent of rose mosaic was determined and the movement (MP and coat (CP protein genes of a PNRSV isolate from rose were molecularly characterized for the first time in Brazil. The nucleotide and deduced amino acid sequences of MP and CP complete genes were aligned and compared with other isolates. Molecular analysis of the MP and CP nucleotide sequences of a Brazilian PNRSV isolate from rose and others from this same host showed highest identities of 96.7% and 98.6%, respectively, and Rose-Br isolate was classified in PV32 group.

  13. Proteomic identification of gender molecular markers in Bothrops jararaca venom.

    Science.gov (United States)

    Zelanis, André; Menezes, Milene C; Kitano, Eduardo S; Liberato, Tarcísio; Tashima, Alexandre K; Pinto, Antonio F M; Sherman, Nicholas E; Ho, Paulo L; Fox, Jay W; Serrano, Solange M T

    2016-04-29

    Variation in the snake venom proteome is a well-documented phenomenon; however, sex-based variation in the venom proteome/peptidome is poorly understood. Bothrops jararaca shows significant sexual size dimorphism and here we report a comparative proteomic/peptidomic analysis of venoms from male and female specimens and correlate it with the evaluation of important venom features. We demonstrate that adult male and female venoms have distinct profiles of proteolytic activity upon fibrinogen and gelatin. These differences were clearly reflected in their different profiles of SDS-PAGE, two-dimensional electrophoresis and glycosylated proteins. Identification of differential protein bands and spots between male or female venoms revealed gender-specific molecular markers. However, the proteome comparison by in-solution trypsin digestion and label-free quantification analysis showed that the overall profiles of male and female venoms are similar at the polypeptide chain level but show striking variation regarding their attached carbohydrate moieties. The analysis of the peptidomes of male and female venoms revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles. Furthermore we confirmed the ubiquitous presence of four BPPs that lack the C-terminal Q-I-P-P sequence only in the female venom as gender molecular markers. As a result of these studies we demonstrate that the sexual size dimorphism is associated with differences in the venom proteome/peptidome in B. jararaca species. Moreover, gender-based variations contributed by different glycosylation levels in toxins impact venom complexity. Bothrops jararaca is primarily a nocturnal and generalist snake species, however, it exhibits a notable ontogenetic shift in diet and in venom proteome upon neonate to adult transition. As is common in the Bothrops genus, B. jararaca shows significant sexual dimorphism in snout-vent length and weight, with females being

  14. Isolation, screening and identification of novel isolates of Actinomycetes from India for antimicrobial applications

    Directory of Open Access Journals (Sweden)

    Vineeta Singh

    2016-12-01

    Full Text Available The search for novel bioactive compounds from the natural environment has been rapidly increased with the increase in multi-drug resistant (MDR pathogens. In the present study, the antimicrobial potential of novel actinomycetes has been evaluated by initial screening of six soil samples. Primary and secondary screening was performed against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans, Candida tropicalis, Trichophyton rubrum, and other MDR bacterial and fungal test strains, and at the end thirteen active isolates were selected for further study. Microbial strains were identified on the basis of growth conditions and other biochemical characters. Five most active microbial strains were identified using 16S rRNA sequence homology and designated as Streptomyces xanthophaeus MTCC 11938, Streptomyces variabilis MTCC 12266, Streptomyces xanthochromogenes MTCC 11937, Streptomyces levis EU 124569 and Streptomyces sp. NCIM 5500. Four antibacterial and three antifungal compounds isolated from the above five isolates were purified and partially characterized using UV absorption and IR spectra. Two antibacterial metabolites, belong to chromone and peptide antibiotic, respectively. The antifungal compounds were found to be of non-polyene nature. In conclusion, we study the isolation of novel bacterial strains of actinomycetes for producing novel compounds having antibacterial and antifungal activities from the unexplored agro-ecological niches of India. Also, this study paves the way for further characterization of these isolates of Streptomyces sp. for their optimum utilization for antimicrobial purposes.

  15. Comparison of Enzymatic Method Rapid Yeast Plus System with RFLP-PCR for Identification of Isolated Yeast from Vulvovaginal Candidiasis.

    Science.gov (United States)

    Hossein, Moallaei; Mirhendi, Seied Hossein; Brandão, João; Mirdashti, Reza; Rosado, Laura

    2011-09-01

    To compare two identification methods, i.e., restriction fragment length polymorphism (RFLP)-PCR analysis and enzymatic method Rapid TM Yeast Plus System to identify different species causing vulvovaginal candidiasis (VVC). Vaginal discharges of women who had attended the gynecology outpatient clinic of Mobini Hospital in Sabzevar, Iran were collected using cotton swabs and were cultured on Sabouraud dextrose agar. Isolated yeasts were identified by germ-tube testing and Rapid TM Yeast Plus System (Remel USA). For molecular identification, the isolated DNA was amplified with ITS1 and ITS4 universal primers and PCR products digested with the enzyme HpaІІ followed by agarose gel electrophoresis. Epidemiological and clinical features of women with respect to identified species were also evaluated. Out of 231 subjects enrolled, 62 VVC cases were detected. The isolated species were identified as follows: Candida albicans, 24 (38.7%), C. glabrata, 15 (24.2%), C. kefyr, 13 (21.0%) C. krusei, 9 (14.5%), and Saccharomyces cerevisiae, 1 (1.6%) by RFLP-PCR method; whereas findings by Rapid TM Yeast Plus System were C. albicans, 24 (38.7%), C. glabrata, 5 (8%), C. kefyr, 11 (17.7%) C. krusei, 2 (3.2%), S. cerevisiae, 9 (14.5%), and C. tropicalis, 6 (9.6%) as well as other nonpathogenic yeasts, 4 (6.9%). Statistical comparison showed that there is no significant difference in identification of C. albicans by the two methods; although, in this study, it was not true about other species of yeasts. A correlation between clinical and laboratory findings is important as it enables us to administer an appropriate treatment on time.

  16. 9 CFR 130.15 - User fees for veterinary diagnostic isolation and identification tests performed at NVSL...

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false User fees for veterinary diagnostic isolation and identification tests performed at NVSL (excluding FADDL) or other authorized site. 130.15... AGRICULTURE USER FEES USER FEES § 130.15 User fees for veterinary diagnostic isolation and identification...

  17. An optimised protocol for molecular identification of Eimeria from chickens☆

    Science.gov (United States)

    Kumar, Saroj; Garg, Rajat; Moftah, Abdalgader; Clark, Emily L.; Macdonald, Sarah E.; Chaudhry, Abdul S.; Sparagano, Olivier; Banerjee, Partha S.; Kundu, Krishnendu; Tomley, Fiona M.; Blake, Damer P.

    2014-01-01

    Molecular approaches supporting identification of Eimeria parasites infecting chickens have been available for more than 20 years, although they have largely failed to replace traditional measures such as microscopy and pathology. Limitations of microscopy-led diagnostics, including a requirement for specialist parasitological expertise and low sample throughput, are yet to be outweighed by the difficulties associated with accessing genomic DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the production of a standardised protocol that includes sample collection and DNA template preparation, as well as primer selection from the numerous PCR assays now published. Such a protocol will facilitate development of valuable epidemiological datasets which may be easily compared between studies and laboratories. The outcome of an optimisation process undertaken in laboratories in India and the UK is described here, identifying four steps. First, samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally, nested PCR was carried out using previously published primers targeting the internal transcribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in the presence of faecal material, DNA extraction using a traditional phenol/chloroform protocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using the morphometric tool COCCIMORPH for the first time with field samples. PMID:24138724

  18. Isolation and identification of acetogenic bacteria obtained from deer rumen and their potential for methanogenesis inhibitor

    Directory of Open Access Journals (Sweden)

    Amlius Thalib

    2008-10-01

    Full Text Available Methanogenesis can be inhibited by various chemicals through different mechanism reaktion. The use of acetogenic bacteria as H2 sink is assumed to be a promising approach. Isolation and identification of acetogenic bacteria obtained from deer rumen had been conducted. Two types of media used for isolation were hydrogen-carbondioxide utilizing acetogens and carbonmonoxide utilizing acetogens. Identification of species of acetogens isolates was based on descriptions of morphology, Gram type, motility, bioreaction results, and oksygen requirement. The compositions of methane and volatile fatty acids (VFA were determined on minimal media or added with sheep rumen liquid innoculated with pure isolates. The identification results showed that the isolate cultured on media of hydrogen-carbondioxide utilizing acetogens was Acetoanaerobium noterae and the ones cultured on media of carbonmonoxide utilizing acetogens was Acetobacterium woodii. Inoculumn of A. noterae and A. woodii could decreased the composition of methane resulted from substrate fermented by fresh rumen liquid of sheep (CRDF, that is culture of A. noterae added FPM and defaunator decreased methane production by 28.8% (P CH3COOH + 2H2O by which reduction of CO2 with H2 producing CH4 can be inhibited or decreased. Their function as methanogenesis inhibitor would be more significant when they are combined with microbial growth factors and defaunator.

  19. [Isolation and identification of cow-origin Cryptosporidium isolates in Hefei].

    Science.gov (United States)

    Sun, Tao; Liu, Wei; Wang, Ju-Hua; Xue, Xiu-Heng; Zhao, Chang-Cheng; Li, Pei-Ying

    2011-12-01

    To isolate cow-origin Cryptosporidium in Hefei, and identify its species. 285 dairy cattle fecal samples collected from a farm in Hefei were examined by using floating saturated solution of sucrose and modified acid-fast staining. Cryptosporidium oocysts were isolated and purified from positive fecal samples. Genetic DNA was extracted to be the template. According to the sequence of 18S rRNA gene and HSP70 gene from Cryptosporidium sp., the primers were designed and synthesized. The PCR products were amplified by PCR and nested-PCR. The nested PCR products were cloned and sequenced. Homology searches and phylogenic tree construction were done by DNAStar software. Five fecal samples were positive by morphological methods with an infection rate of 1.8% (5/285). Oocysts from the 5 positive fecal samples were elliptical or ovoid detected by using floating saturated solution of sucrose and modified acid-fast staining with the size of 7.37 microm x 6.13 microm and 7.58 microm x 6.20 microm, and a shape index of 1.20 and 1.22, respectively. Nested-PCR resulted in a 18S rRNA and HSP70 gene fragments with approximately 250 bp and 325 bp, respectively. The five isolates showed a high level of nucleic acid identity with sequence data of the 18S rRNA gene of Cryptosporidium andersoni (DQ989573), and they were clustered in the same clade. The highest HSP70 gene sequence identity was found among the five isolates and other reported C. andersoni isolates (AY954892 and DQ989576), and they were placed into the same clade. The cow-origin Cryptosporidium isolates derived from Hefei is Cryptosporidium andersoni.

  20. Identification and antimicrobial susceptibility patterns of Staphylococcus spp. isolated from canine chronic otitis externa

    Directory of Open Access Journals (Sweden)

    Silva N.

    2001-01-01

    Full Text Available Swab samples obtained from 96 dogs with chronic otitis externa were cultured for the isolation of Staphylococcus species. Of 57 staphylococcal strains, 41 (72% were coagulase-negative (CNS. The identification of staphylococci strains was made by standard procedures for the routine identification of staphylococci in clinical practice. S. sciuri was the most frequent species isolated (22.8% from chronic otitis externa in dogs followed by S. intermedius (12.3%, S. auricularis (10.5% and S. aureus (8.8%. Three (5.2% CNS strains could not be identified. Bacterial isolates were susceptible to enrofloxacin, gentamicin, cephalothin, chloramphenicol and neomycin. Resistance was most common to penicillin G, oxacillin and ampicillin.

  1. Identification of clinically relevant Corynebacterium strains by Api Coryne, MALDI-TOF-mass spectrometry and molecular approaches.

    Science.gov (United States)

    Alibi, S; Ferjani, A; Gaillot, O; Marzouk, M; Courcol, R; Boukadida, J

    2015-09-01

    We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 97 Corynebacterium clinical in comparison to identification strains by Api Coryne and MALDI-TOF-MS using 16S rRNA gene and hypervariable region of rpoB genes sequencing as a reference method. C. striatum was the predominant species isolated followed by C. amycolatum. There was an agreement between Api Coryne strips and MALDI-TOF-MS identification in 88.65% of cases. MALDI-TOF-MS was unable to differentiate C. aurimucosum from C. minutissimum and C. minutissimum from C. singulare but reliably identify 92 of 97 (94.84%) strains. Two strains remained incompletely identified to the species level by MALDI-TOF-MS and molecular approaches. They belonged to Cellulomonas and Pseudoclavibacter genus. In conclusion, MALDI-TOF-MS is a rapid and reliable method for the identification of Corynebacterium species. However, some limits have been noted and have to be resolved by the application of molecular methods. Copyright © 2015. Published by Elsevier SAS.

  2. Short communication: Molecular characteristics, antimicrobial susceptibility, and pathogenicity of clinical Nocardia cyriacigeorgica isolates from an outbreak of bovine mastitis.

    Science.gov (United States)

    Chen, Wei; Liu, Yongxia; Barkema, Herman W; Gao, Jian; De Buck, Jeroen; Kastelic, John P; Liu, Gang; Ali, Tariq; Shahid, Muhammad; Han, Bo

    2017-10-01

    The occurrence of nocardial mastitis, mostly in the context of outbreaks, has been reported in many countries. However, there is a paucity of reports regarding detailed characterization of Nocardia cyriacigeorgica from bovine mastitis. Thus, herein we report characteristics, antimicrobial susceptibility patterns, molecular identification, and pathogenicity of N. cyriacigeorgica isolated from an outbreak of clinical mastitis in a dairy herd in northern China. A total of 182 (80.2%) lactating cows had clinical mastitis with severe inflammation and firmness of the udder, reduced milk production, and anorexia, with no apparent clinical response to common antibiotics. Out of 22 mastitic milk samples submitted to our laboratory, 12 N. cyriacigeorgica were isolated and characterized using standard microbiological analysis, 16S rRNA gene sequencing, random amplified polymorphic DNA PCR analysis, biochemical assays, and antibiotic susceptibility testing. Additionally, in vivo experiments were done to determine pathogenicity of these clinical mastitis isolates. All isolates were resistant to ampicillin, amoxicillin-clavulanic acid, ciprofloxacin, minocycline, rifampicin, and aminoglycosides (type VI pattern). Additionally, intramammary inoculation of mice with N. cyriacigeorgica caused chronic inflammatory changes, including hyperemia, edema, and infiltration of lymphocytes and neutrophils, as well as hyperplasia of lymph nodules in mammary glands. Therefore, we concluded that N. cyriacigeorgica was involved in the current outbreak of mastitis. To our best knowledge, this is the first report to characterize N. cyriacigeorgica isolated from cases of bovine mastitis in China. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  3. Growth medium for the rapid isolation and identification of anthrax

    Science.gov (United States)

    Kiel, Johnathan L.; Parker, Jill E.; Grubbs, Teri R.; Alls, John L.

    2000-07-01

    Anthrax has been recognized as a highly likely biological warfare or terrorist agent. The purpose of this work was to design a culture technique to rapidly isolate and identify `live' anthrax. In liquid or solid media form, 3AT medium (3-amino-L-tyrosine, the main ingredient) accelerated germination and growth of anthrax spores in 5 to 6 hours to a point expected at 18 to 24 hours with ordinary medium. During accelerated growth, standard definitive diagnostic tests such as sensitivity to lysis by penicillin or bacteriophage can be run. During this time, the bacteria synthesized a fluorescent and thermochemiluminescent polymer. Bacteria captured by specific antibody are, therefore, already labeled. Because living bacteria are required to generate the polymer, the test converts immunoassays for anthrax into viability assays. Furthermore, the polymer formation leads to the death of the vegetative form and non-viability of the spores produced in the medium. By altering the formulation of the medium, other microbes and even animal and human cells can be grown in it and labeled (including viruses grown in the animal or human cells).

  4. Genetic Identification of Orientobilharzia turkestanicum from Sheep Isolates in Iran.

    Directory of Open Access Journals (Sweden)

    Reza Tabaripour

    2015-03-01

    Full Text Available Adult worms of Orientobilharzia turkestanicum live in the portal veins, or intestinal veins of cattle, sheep, goat and many other mammals causing orientobilharziasis. Orientobilharziasis causes significant economic losses to livestock industry of Iran. However, there is limited information about genotypes of O. turkestanicum in Iran.In this study, 30 isolates of O. turkestanicum obtained from sheep were characterized by sequencing mitochondrial cytochrome c oxidase subunit 1 (cox1 and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1 gene. The mitochondrial cox1 and nad1 DNA were amplified by polymerase chain reaction (PCR and then sequenced and compared with O. turkestanicum and that of other members of the Schistosomatidae available in Gen-Bank(™.Phylogenetic relationships between them were re-constructed using the maximum parsimony method. Phylogenetic analyses done in present study placed O. turkestanicum within the Schistosoma genus, and indicates that O. turkestanicum was phylogenetically closer to the African schistosome group than to the Asian schistosome group.Comparison of nad1 and cox1 sequences of O. turkestanicum obtained in this study with corresponding sequences available in Genbank(™ revealed some sequence variations and provided evidence for presence of microvarients in Iran.

  5. Molecular, ultrastructural, and biological characterization of Pennsylvania isolates of Plum pox virus.

    Science.gov (United States)

    Schneider, William L; Damsteegt, Vernon D; Gildow, Fred E; Stone, Andrew L; Sherman, Diana J; Levy, Laurene E; Mavrodieva, Vessela; Richwine, Nancy; Welliver, Ruth; Luster, Douglas G

    2011-05-01

    Plum pox virus (PPV) was identified in Pennsylvania in 1999. The outbreak was limited to a four-county region in southern Pennsylvania. Initial serological and molecular characterization indicated that the isolates in Pennsylvania belong to the D strain of PPV. The Pennsylvania isolates were characterized by sequence analysis, electron microscopy, host range, and vector transmission to determine how these isolates related to their previously studied European counterparts. Genetically, Pennsylvania (PPV-Penn) isolates were more closely related to each other than to any other PPV-D strains, and isolates from the United States, Canada, and Chile were more closely related to each other than to European isolates. The PPV-Penn isolates exist as two clades, suggesting the possibility of multiple introductions. Electron microscopy analysis of PPV-Penn isolates, including cytopathological studies, indicated that the virions were similar to other Potyvirus spp. PPV-Penn isolates had a herbaceous host range similar to that of European D isolates. There were distinct differences in the transmission efficiencies of the two PPV-Penn isolates using Myzus persicae and Aphis spiraecola as vectors; however, both PPV-Penn isolates were transmitted by M. persicae more efficiently than a European D isolate but less efficiently than a European M isolate.

  6. Isolation and identification of main mastitis pathogens in Mexico

    Directory of Open Access Journals (Sweden)

    H. Castañeda Vázquez

    2013-04-01

    Full Text Available The present work is a large epidemiological study aiming to detect the prevalence of subclinical mastitis and to investigate the major udder pathogens in Jalisco State, western Mexico. For this purpose, 2205 dairy cows, representing 33 Mexican dairy herds, were involved. Of 2205 cows, 752 mastitic animals were diagnosed and only 2,979 milk samples could be obtained for further investigation. All 2979 milk samples were subjected to California Mastitis Test (CMT to differentiate clinical cases from subclinical ones where 1996 samples (67 % reacted positively. Of these, 1087 samples (54.5% came from cows suffering from clinical cases of mastitis. Bacteriological identification of the causative agents revealed the presence of a major group of pathogens including the Coagulase negative staphylococci (CNS, S.aureus, S.agalactiae, Corynebacterium spp. and Coliform bacteria which were detected in 464 (15.6%, 175 (5.9%, 200 (6.8%, 417 (14% and 123 (4.1% of the 2927 investigated quarters, 295 (15.4%, 118 (15.7%, 111 (14.8%, 227 (30.2% and 109 (14.5% of the 752 examined cows and in 33 (100%, 22 (66.7%, 19 (57.6%, 30 (90.1% and 27 (81.8% of the 33 herds involved, respectively. Other pathogens could be detected in the investigated milk samples such as S. dysgalactiae (0.4%, S.uberis (0.37%, Bacillus spp. (1%, Nocardia spp. (0.6% und Candida spp. (0.1%. Meanwhile, others were present in a negligible ratio; including the Aerococcus viridans, and Enterococcus spp., Lactococcus lactis, S. bovis.

  7. Molecular characterization of Azotobacter spp. nifH gene Isolated ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... Available online at http://www.academicjournals.org/AJB. ISSN 1684–5315 ... Molecular characterization of Azotobacter spp. nifH .... MATERIALS AND METHODS ..... rapidly expanding and is currently composed of over.

  8. Molecular identification of Nocardia species using the sodA gene

    Directory of Open Access Journals (Sweden)

    K. Sánchez-Herrera

    2017-09-01

    Full Text Available Currently for bacterial identification and classification the rrs gene encoding 16S rRNA is used as a reference method for the analysis of strains of the genus Nocardia. However, it does not have enough polymorphism to differentiate them at the species level. This fact makes it necessary to search for molecular targets that can provide better identification. The sodA gene (encoding the enzyme superoxide dismutase has had good results in identifying species of other Actinomycetes. In this study the sodA gene is proposed for the identification and differentiation at the species level of the genus Nocardia. We used 41 type species of various collections; a 386 bp fragment of the sodA gene was amplified and sequenced, and a phylogenetic analysis was performed comparing the genes rrs (1171 bp, hsp65 (401 bp, secA1 (494 bp, gyrB (1195 bp and rpoB (401 bp. The sequences were aligned using the Clustal X program. Evolutionary trees according to the neighbour-joining method were created with the programs Phylo_win and MEGA 6. The specific variability of the sodA genus of the genus Nocardia was analysed. A high phylogenetic resolution, significant genetic variability, and specificity and reliability were observed for the differentiation of the isolates at the species level. The polymorphism observed in the sodA gene sequence contains variable regions that allow the discrimination of closely related Nocardia species. The clear specificity, despite its small size, proves to be of great advantage for use in taxonomic studies and clinical diagnosis of the genus Nocardia.

  9. Relative Stabilities and Reactivities of Isolated Versus Conjugated Alkenes: Reconciliation Via a Molecular Orbital Approach

    Science.gov (United States)

    Sotiriou-Leventis, Chariklia; Hanna, Samir B.; Leventis, Nicholas

    1996-04-01

    The well-accepted practice of generating a pair of molecular orbitals, one of lower energy and another of higher energy than the original pair of overlapping atomic orbitals, and the concept of a particle in a one-dimensional box are implemented in a simplified, nonmathematical method that explains the relative stabilities and reactivities of alkenes with conjugated versus isolated double bonds. In this method, Huckel-type MO's of higher polyenes are constructed by energy rules of linear combination of atomic orbitals. One additional rule is obeyed: bonding molecular orbitals overlap only with bonding molecular orbitals, and antibonding molecular orbitals overlap only with antibonding molecular orbitals.

  10. Molecular analysis of clinical isolates previously diagnosed as Mycobacterium intracellulare reveals incidental findings of "Mycobacterium indicus pranii" genotypes in human lung infection.

    Science.gov (United States)

    Kim, Su-Young; Park, Hye Yun; Jeong, Byeong-Ho; Jeon, Kyeongman; Huh, Hee Jae; Ki, Chang-Seok; Lee, Nam Yong; Han, Seung-Jung; Shin, Sung Jae; Koh, Won-Jung

    2015-09-30

    Mycobacterium intracellulare is a major cause of Mycobacterium avium complex lung disease in many countries. Molecular studies have revealed several new Mycobacteria species that are closely related to M. intracellulare. The aim of this study was to re-identify and characterize clinical isolates from patients previously diagnosed with M. intracellulare lung disease at the molecular level. Mycobacterial isolates from 77 patients, initially diagnosed with M. intracellulare lung disease were re-analyzed by multi-locus sequencing and pattern of insertion sequences. Among the 77 isolates, 74 (96 %) isolates were designated as M. intracellulare based on multigene sequence-based analysis. Interestingly, the three remaining strains (4 %) were re-identified as "Mycobacterium indicus pranii" according to distinct molecular phylogenetic positions in rpoB and hsp65 sequence-based typing. In hsp65 sequevar analysis, code 13 was found in the majority of cases and three unreported codes were identified. In 16S-23S rRNA internal transcribed spacer (ITS) sequevar analysis, all isolates of both species were classified within the Min-A ITS sequevar. Interestingly, four of the M. intracellulare isolates harbored IS1311, a M. avium-specific element. Two of three patients infected with "M. indicus pranii" had persistent positive sputum cultures after antibiotic therapy, indicating the clinical relevance of this study. This analysis highlights the importance of precise identification of clinical isolates genetically close to Mycobacterium species, and suggests that greater attention should be paid to nontuberculous mycobacteria lung disease caused by "M. indicus pranii".

  11. Molecular typing of mycobacterium tuberculosis isolates circulating in Jiangsu Province, China

    Directory of Open Access Journals (Sweden)

    Dong Haiyan

    2011-10-01

    Full Text Available Abstract Background Globally, China is the second place with high burden of tuberculosis (TB. To explore the characteristics of the pathogens of Mycobacterium tuberculosis (MTB circulating in this area is helpful for understanding and controlling the spread of the strains. Recent developments in molecular biology have allowed prompt identification and tracking specific strains of MTB spreading through the population. Methods Spacer-oligonucleotide typing (spoligotyping and mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR were performed in combination to yield specific genetic profiles of 260 MTB strains isolated from 30 counties of Jiangsu province in China between June and July 2010. The spoligotyping results were in comparison to the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4. Drug susceptibility test (DST was performed on all strains by proportion method on Lowenstein-Jensen (LJ culture media. Results Based on the spoligotyping method, 246 strains displayed known patterns and 14 were absent in the database. Predominant spoligotypes belonged to the Beijing family (80.4%. By using the 24-loci VNTR typing scheme, 224 different patterns were identified, including 20 clusters and 204 unique patterns. The largest clade comprised 195 strains belonging to the Beijing family. The combination of spoligotyping and 24-loci MIRU-VNTR demonstrated maximal discriminatory power. Furthermore, we observed a significant association between Beijing family strains and drug-resistant phenotypes. The Beijing family strains presented increased risks for developing multi-drug resistant TB, with the OR (95% CI of 11.07(1.45-84.50. Conclusions The present study demonstrated that Beijing family isolates were the most prevalent strains circulating in Jiangsu province of China. The utility of spoligotyping in combination with 24-loci MIRU-VNTR might be a useful tool for epidemiological analysis of MTB

  12. Molecular Typing of Vibrio parahaemolyticus Strains Isolated from Mollusks in the North Adriatic Sea.

    Science.gov (United States)

    Rahman, Mohammad Shamsur; Carraro, Roberta; Cardazzo, Barbara; Carraro, Lisa; Meneguolo, Davide Boscolo; Martino, Maria Elena; Andreani, Nadia Andrea; Bordin, Paola; Mioni, Renzo; Barco, Lisa; Novelli, Enrico; Balzan, Stefania; Fasolato, Luca

    2017-08-01

    Vibrio parahaemolyticus is an emerging foodborne pathogen in the Mediterranean, usually associated with shellfish consumption. The increase in the number of outbreaks in Europe is primarily associated with the global warming of the ocean that has a great impact on the spread and genetic selection of waterborne pathogens. The primary role of Italy in Europe's mollusk production, together with the fact that cases of infections with V. parahaemolyticus are not always notified to the European community, highlighted the necessity of acquiring new information about the epidemiological involvement of shellfish products. The aim of the study was to provide useful insights into the first steps of the Risk Assessment associated with V. parahaemolyticus through the molecular characterization of isolates from commercialized mollusks. A total of 102 strains identified as V. parahaemolyticus were investigated as part of a larger sampling (1-year survey) from several shellfish species collected from the Venice lagoon and the North Adriatic sea. All strains were characterized by multilocus sequence typing and tested for the presence of virulence genes (trh and tdh). The study of sampling/environmental factors and epidemiological analyses was performed to describe the behaviors of the different genetic populations. The population structure analysis highlighted three genetic clusters that could be subject to temperature selection during cold (≤15°C) and warm (>16°C) seasons. Moreover, other factors, such as molluscan species (clams/mussels), probably played a role in the distribution of genetic clusters. Although few strains carried the virulence factors (n = 6 trh + ), epidemiological links with clinical isolates and a local dissemination of some sequence types were underlined. This work provides a useful background on the genotype spread as a first step in the Hazard Identification in light of future climate changes.

  13. Caracterização morfofisiológica e identificação molecular de isolados de Guignardia citricarpa, agente patogênico da mancha preta dos citros = Morphophysiological characterization and molecular identification of isolates of Guignardia citricarpa, a pathogenic agent of the citrus black spot

    Directory of Open Access Journals (Sweden)

    Marilda Pereira Caixeta

    2008-12-01

    Full Text Available O presente trabalho teve como objetivo identificar 11 isolados de Guignardia citricarpa, agente causal da mancha preta dos citros (MPC, obtidos de frutas cítricas sintomáticas de diferentes regiões geográficas, por meio da PCR e caracterização morfofisiológica das estruturas propagativas, esporulação e crescimento micelial emdiferentes meios de cultura, temperaturas e regimes de luz, nas condições de laboratório. Pelo teste de PCR, todos os isolados foram identificados como o patógeno G. citricarpa. Os isolados caracterizados foram submetidos às temperaturas de 20, 25 e 30ºC, em regime de luz contínua, escuro contínuo e fotoperíodos de 12 horas, durante 24 dias. Utilizaram-se os meios de cultura aveia-ágar (AA, batata-dextrose-ágar (BDA e cenoura-dextrose-ágar (CDA. Os resultados mostraram que ocorreu interação entre os diferentes meios de cultura, temperaturas e fotoperíodos. O meio de cultura que melhor estimulou o crescimento micelial foi o CDA a 25ºC sob o fotoperíodo de 12h. A maior produção de esporos (conídios foi verificada no meio BDA a 20ºC, no fotoperíodo de 12 horas. No meio CDA, não ocorreu esporulação de nenhum isolado. Sob a temperatura de 30ºC, foiverificada apenas a produção de hifas e picnídios para a maioria dos isolados, em todos os meios de cultura e fotoperíodo testados.Thepresent work aims to identify 11 isolates of Guignardia citricarpa, the causal agent of the citrus black spot (CBS, obtained from affected fruit in different geographical regions, through PCR and morphophysiological characterization of propagative structures, sporing and mycelial growth in different means of culture, temperatures and photoperiods, under laboratory conditions. Through the PCR test, all isolates were identified as being the G.citricarpa pathogen. The characterized isolates were subjected to evaluations at temperatures of 20, 25 and 30ºC, in continuous light, continuous darkness, and alternating 12

  14. Rapid isolation of high molecular weight DNA from single dried ...

    African Journals Online (AJOL)

    ANAND

    For studying genetic diversity in populations of predatory coccinellid, Cryptolaemus montrouzieri. Mulsant (Coccinellidae: Coleoptera), our attempts to isolate high quality DNA from individual adult beetle using several previously reported protocols and even modifications were quite unsuccessful as the insect size was small ...

  15. Molecular detection and isolation of avian metapneumovirus in Mexico.

    Science.gov (United States)

    Rivera-Benitez, José Francisco; Martínez-Bautista, Rebeca; Ríos-Cambre, Francisco; Ramírez-Mendoza, Humberto

    2014-01-01

    We conducted a longitudinal study to detect and isolate avian metapneumovirus (aMPV) in two highly productive poultry areas in Mexico. A total of 968 breeder hens and pullets from 2 to 73 weeks of age were analysed. Serology was performed to detect aMPV antibodies and 105 samples of tracheal tissue were collected, pooled by age, and used for attempted virus isolation and aMPV nested reverse transcriptase-polymerase chain reaction (nRT-PCR). The serological analysis indicated that 100% of the sampled chickens showed aMPV antibodies by 12 weeks of age. Five pools of pullet samples collected at 3 to 8 weeks of age were positive by nRT-PCR and the sequences obtained indicated 98 to 99% similarity with the reported sequences for aMPV subtype A. Virus isolation of nRT-PCR-positive samples was successfully attempted using chicken embryo lung and trachea mixed cultures with subsequent adaptation to Vero cells. This is the first report of detection and isolation of aMPV in Mexico.

  16. Isolation, Identification, and Characterization of a New Highly Pathogenic Field Isolate of Mycobacterium avium spp. avium

    Directory of Open Access Journals (Sweden)

    Liangquan Zhu

    2018-01-01

    Full Text Available Avian tuberculosis is a chronic, contagious zoonotic disease affecting birds, mammals, and humans. The disease is most often caused by Mycobacterium avium spp. avium (MAA. Strain resources are important for research on avian tuberculosis and vaccine development. However, there has been little reported about the newly identified MAA strain in recent years in China. In this study, a new strain was isolated from a fowl with symptoms of avian tuberculosis by bacterial culture. The isolated strain was identified to be MAA by culture, staining, and biochemical and genetic analysis, except for different colony morphology. The isolated strain was Ziehl-Zeelsen staining positive, resistant to p-nitrobenzoic acid, and negative for niacin production, Tween-80 hydrolysis, heat stable catalase and nitrate production. The strain had the DnaJ gene, IS1245, and IS901, as well. Serum agglutination indicated that the MAA strain was of serotype 1. The MAA strain showed strong virulence via mortality in rabbits and chickens. The prepared tuberculin of the MAA strain had similar potency compared to the MAA reference strain and standard tuberculin via a tuberculin skin test. Our studies suggested that this MAA strain tends to be a novel subtype, which might enrich the strain resource of avian tuberculosis.

  17. Identification, Typing, Antifungal Resistance Profile, and Biofilm Formation of Candida albicans Isolates from Lebanese Hospital Patients

    Directory of Open Access Journals (Sweden)

    Ibrahim Bitar

    2014-01-01

    Full Text Available As leading opportunistic fungal pathogens identification and subtyping of Candida species are crucial in recognizing outbreaks of infection, recognizing particularly virulent strains, and detecting the emergence of drug resistant strains. In this study our objective was to compare identification of Candida albicans by the hospitals through the use of conventional versus identification based on the ITS (Internal Transcribed Spacer and to assess biofilm forming capabilities, drug resistance patterns and correlate these with MLST typing. ITS typing revealed a 21.2% hospital misidentification rate. Multidrug resistance to three drugs out of four tested was detected within 25% of the isolates raising concerns about the followed treatment regimens. Drug resistant strains as well as biofilm formers were phylogenetically related, with some isolates with significant biofilm forming capabilities being correlated to those that were multidrug resistant. Such isolates were grouped closely together in a neighbor-joining tree generated by MLST typing indicating phylogenetic relatedness, microevolution, or recurrent infection. In conclusion, this pilot study gives much needed insight concerning C. albicans isolates circulating in Lebanese hospitals and is the first study of its kind correlating biofilm formation, antifungal resistance, and evolutionary relatedness.

  18. First isolation and molecular characterization of Ehrlichia canis in Costa Rica, Central America.

    Science.gov (United States)

    Romero, L E; Meneses, A I; Salazar, L; Jiménez, M; Romero, J J; Aguiar, D M; Labruna, M B; Dolz, G

    2011-08-01

    The present study investigated Ehrlichia species in blood samples from dogs suspected of clinical ehrlichiosis, using molecular and isolation techniques in cell culture. From a total of 310 canine blood samples analyzed by 16S rRNA nested PCR, 148 (47.7%) were positive for Ehrlichia canis. DNA from Ehrlichia chaffeensis or Ehrlichia ewingii was not detected in any sample using species-specific primers in separated reactions. Leukocytes from five PCR-positive dogs were inoculated into DH82 cells; successful isolation of E. canis was obtained in four samples. Partial sequence of the dsb gene of eight canine blood samples (including the five samples for in vitro isolation) was obtained by PCR and their analyses through BLAST showed 100% of identity with the corresponding sequence of E. canis in GenBank. This study represents the first molecular diagnosis, isolation, and molecular characterization of E. canis in dogs from Costa Rica. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Molecular Characterization of Clostridium difficile Isolates in China From 2010 to 2015

    Directory of Open Access Journals (Sweden)

    Xiao-shu Liu

    2018-04-01

    Full Text Available Clostridium difficile infection (CDI has become a worldwide public health problem causing high mortality and a large disease burden. Molecular typing and analysis is important for surveillance and infection control of CDI. However, molecular characterization of C. difficile across China is extremely rare. Here, we report on the toxin profiles, molecular subtyping with multilocus sequence typing (MLST and PCR ribotyping, and epidemiological characteristics of 199 C. difficile isolates collected between 2010 through 2015 from 13 participating centers across China. We identified 35 STs and 27 ribotypes (RTs among the 199 C. difficile isolates: ST35 (15.58%, ST3 (15.08%, ST37 (12.06%, and RT017 (14.07%, RT001 (12.06%, RT012 (11.56% are the most prevalent. One isolate with ST1 and 8 isolates with ST 11 were identified. We identified a new ST in this study, denoted ST332. The toxin profile tcdA+tcdB+tcdC+tcdR+tcdE+CDT- (65.83% was the predominant profile. Furthermore, 11 isolates with positive binary toxin genes were discovered. According to the PCR ribotyping, one isolate with RT 027, and 6 isolates with RT 078 were confirmed. The epidemiological characteristics of C. difficile in China shows geographical differences, and both the toxin profile and molecular types exhibit great diversity across the different areas.

  20. Spa typing and identification of pvl genes of meticillin-resistant Staphylococcus aureus isolated from a Libyan hospital in Tripoli.

    Science.gov (United States)

    Ahmed, Mohamed O; Baptiste, Keith E; Daw, Mohamed A; Elramalli, Asma K; Abouzeed, Yousef M; Petersen, Andreas

    2017-09-01

    The purpose of the study was to investigate the molecular characteristics of meticillin-resistant Staphylococcus aureus (MRSA) isolated from clinical sources in Tripoli, Libya. A total of 95 MRSA strains collected at the Tripoli medical Centre were investigated by spa typing and identification of the Panton-Valentine Leukocidin (pvl) genes. A total of 26 spa types were characterized and distributed among nine clonal complexes; CC5 (n=32), CC80 (n=18), CC8 (n=17) and CC22 (n=12) were the most prevalent clonal complexes. In total, 34% of the isolates were positive for PVL. This study demonstrated the presence of CA-MRSA and pvl positive strains in hospital settings and underlines the importance of using molecular typing to investigate the epidemiology of MRSA. Preventative measures and surveillance systems are needed to control and minimize the spread of MRSA in the Libyan health care system. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

  1. The isolation and identification of endophytic bacteria from mangrove (Sonneratia alba) that produces gelatinase

    Science.gov (United States)

    Nursyam, H.; Prihanto, A. A.; Warasari, N. I.; Saadah, M.; Masrifa, R. E.; Nabila, N. A.; Istiqfarin, N.; Siddiq, I. J.

    2018-04-01

    Gelatinase is an enzyme that hydrolyze gelatin into gelatin hydrolyzate. The purpose of this study was to isolate and to identify endophytic bacteria from Sonneratia alba mangrove which able to produce gelatinase enzyme. Sonneratia alba mangroves was obtained from Bajul Mati Beach, Malang Regency. The samples in this study were, stems, and leaves. Pure cultured bacteria were investigated for its capability for producing gelatinase enzyme by using gelatin media. Best producer would further be analyzed its species using microbact system. Screening process resulted in 3 positive isolates, namely code isolate of R, B, and L. R which was isolate from root of S. alba was the best producer for gelatinase. Identification process with morphology and microbact system revelaed that A. SBM is a Gram-negative bacterium that has a basil cell shape, with a diameter colony of 2.19 mm. Based on the microbact system test carried out, the bacteria is Pseudomonas aeruginosa.

  2. A PCR-based strategy for simple and rapid identification of rough presumptive Salmonella isolates

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Baggesen, Dorte Lau; Porting, P.H.

    1999-01-01

    The purpose of the present study was to investigate the application of ready-to-go Salmonella PCR tests, based on dry chemistry, for final identification of rough presumptive Salmonella isolates. The results were compared with two different biotyping methods performed at two different laboratories......, which did not result in any DNA band. A total of 32 out of the 36 rough presumptive isolates were positive in the PCR. All but one isolate were also identified as Salmonella by the two biochemical methods. All 80 Salmonella strains were also tested in the two multiplex serogroup tests based on PCR beads....... The sensitivity of the BAX Salmonella PCR test was assessed by testing a total of 80 Salmonella isolates, covering most serogroups, which correctly identified all the Salmonella strains by resulting in one 800-bp band in the sample tubes. The specificity of the PCR was assessed using 20 non-Salmonella strains...

  3. Molecular characterization of multidrug-resistant Klebsiella pneumoniae isolates

    Directory of Open Access Journals (Sweden)

    Xiang-hua Hou

    2015-09-01

    Full Text Available Klebsiella pneumoniae is an important cause of healthcare-associated infections worldwide. Selective pressure, the extensive use of antibiotics, and the conjugational transmission of antibiotic resistance genes across bacterial species and genera facilitate the emergence of multidrug-resistant (MDR K. pneumoniae. Here, we examined the occurrence, phenotypes and genetic features of MDR K. pneumoniae isolated from patients in intensive care units (ICUs at the First Affiliated Hospital of Xiamen University in Xiamen, China, from January to December 2011. Thirty-eight MDR K. pneumoniae strains were collected. These MDR K. pneumoniae isolates possessed at least seven antibiotic resistance determinants, which contribute to the high-level resistance of these bacteria to aminoglycosides, macrolides, quinolones and β-lactams. Among these isolates, 24 strains were extended-spectrum β-lactamase (ESBL producers, 2 strains were AmpC producers, and 12 strains were both ESBL and AmpC producers. The 38 MDR isolates also contained class I (28/38 and class II integrons (10/38. All 28 class I-positive isolates contained aacC1, aacC4, orfX, orfX’ and aadA1 genes. β-lactam resistance was conferred through blaSHV (22/38, blaTEM (10/38, and blaCTX-M (7/38. The highly conserved blaKPC-2 (37/38 and blaOXA-23(1/38 alleles were responsible for carbapenem resistance, and a gyrAsite mutation (27/38 and the plasmid-mediated qnrB gene (13/38 were responsible for quinolone resistance. Repetitive-sequence-based PCR (REP-PCR fingerprinting of these MDR strains revealed the presence of five groups and sixteen patterns. The MDR strains from unrelated groups showed different drug resistance patterns; however, some homologous strains also showed different drug resistance profiles. Therefore, REP-PCR-based analyses can provide information to evaluate the epidemic status of nosocomial infection caused by MDR K. pneumoniae; however, this test lacks the power to discriminate some

  4. Isolation and molecular genetic characterization of a yeast strain ...

    African Journals Online (AJOL)

    The yeast was identified by molecular genetics technique based on sequence analysis of the variable D1/D2 domain of the large subunit (26S) ribosomal DNA. Subsequent 26S rRNA gene sequencing showed 100% base sequence homology and it was identified as Candida viswanathii. The degradation of PAHs

  5. Isolation and Molecular Structure of Hexacyanoruthenate(III)

    DEFF Research Database (Denmark)

    Bendix, J; Steenberg, P; Søtofte, Inger

    2003-01-01

    The [Ru(CN)(6)](3-) ion is synthesized in aqueous solution and isolated as [Ph(4)AS](3)[Ru(CN)(6)].2H(2)O (1). Compound 1 crystallizes as orange needles in the monoclinic space group P2(1)/n with cell parameters a = 11.346(2) Angstrom, b = 23.107(5) Angstrom, c = 25.015(5) Angstrom, beta = 99...

  6. Improving time in molecular identification of Mycosphaerella fijiensis ...

    African Journals Online (AJOL)

    cantocanche

    2012-04-24

    Apr 24, 2012 ... List of Mycosphaerella isolates used in this study. Isolate. Fungi. Geographical ... taxonomy studies of pathogenic fungi given their simplicity ... simple in-house method for DC-PCR. This direct ..... proposal for a standardized protocol. J. Ind. Microbiol. .... This study. *qPCR, Quantitative PCR or real time PCR.

  7. Isolation and molecular characterization of Newcastle disease viruses from raptors.

    Science.gov (United States)

    Jindal, Naresh; Chander, Yogesh; Primus, Alexander; Redig, Patrick T; Goyal, Sagar M

    2010-12-01

    The present study was undertaken to detect and characterize Newcastle disease virus (NDV) in raptors. Cloacal and oropharyngeal swab samples were collected from 60 casualty raptors during January to March 2009 in Minnesota. Inoculation of all these samples (n=120) in 9-day-old embryonated hens' eggs resulted in isolation of haemagglutinating viruses in three samples from two bald eagles and one great horned owl. These three haemagglutinating viruses were confirmed as NDV by reverse transcription-polymerase chain reaction (RT-PCR) using fusion gene-specific primers, and were negative for avian influenza virus by RT-PCR. Further characterization revealed that all three possessed (112)GKQGRL(117) at the fusion gene cleavage site, indicating that they were lentogenic strains. Phylogenetic analysis revealed that all three isolates clustered with published class II genotype II NDVs. The nucleotide sequence homology of the three NDV isolates among themselves was 98.4 to 99.6% and the sequence homology with lentogenic strains from wild birds used for comparison varied between 94.5 and 100%. Detection of NDV strains from raptors merits further epidemiological studies to determine the prevalence of different NDV strains in raptors and their impact in relation to transmission to domestic poultry.

  8. Molecular identification of tsetse fly (Diptera: Glossinidae) species ...

    African Journals Online (AJOL)

    Christopher

    2015-05-13

    May 13, 2015 ... plates (Bauer and Wetzel, 1976). The blood is usually collected ... number of males and females, although sex was not considerd in the identification. .... Basic local alignment search tool (BLAST) of the DNA sequences of COII ...

  9. Comparison of Three Commercial Systems for Identification of Yeasts Commonly Isolated in the Clinical Microbiology Laboratory

    Science.gov (United States)

    Wadlin, Jill K.; Hanko, Gayle; Stewart, Rebecca; Pape, John; Nachamkin, Irving

    1999-01-01

    We evaluated three commercial systems (RapID Yeast Plus System; Innovative Diagnostic Systems, Norcross, Ga.; API 20C Aux; bioMerieux-Vitek, Hazelwood, Mo.; and Vitek Yeast Biochemical Card, bioMerieux-Vitek) against an auxinographic and microscopic morphologic reference method for the ability to identify yeasts commonly isolated in our clinical microbiology laboratory. Two-hundred one yeast isolates were compared in the study. The RapID Yeast Plus System was significantly better than either API 20C Aux (193 versus 167 correct identifications; P clinically relevant yeasts. PMID:10325356

  10. Isolation and Identification of Lactic Acid Bacteria Isolated from a Traditional Jeotgal Product in Korea

    Science.gov (United States)

    Cho, Gyu Sung; Do, Hyung Ki

    2006-06-01

    Seventeen lactic acid bacterial strains (LAB) were isolated using MRS agar medium from Jeotgal, a Korean fermented food, purchased at the Jukdo market of Pohang. To identify the strains isolated, they were tested by examining their cell morphologies, gram-staining, catalase activity, arginine hydrolase activity, D-L lactate form and carbohydrate fermentation. According to the phenotypic characteristics, three strains were tent atively identified as Lactobacillus spp., ten were Enterococcus spp. (or Streptococcus spp., or Pediococcus spp.) and the rest were Leuconostoc spp. (or Weissella spp.). Five strains among 17 were chosen by preliminary bacteriocin activity test. Four bacterial strains which inhibited both indicator microorganisms were identified by 16S rRNA sequencing. The results are as follows; Leuconostoc mesenteroides (HK 4), Leuconostoc mesenteroides (HK 5), Leuconostoc mesenteroides(HK 11), Streptococcus salivarius(HK 8). In order to check LAB which are showing a high survival rate in gut, we investigated three strains inhibiting both indicator microorganisms in artificial gastric acid and bile juice -all except HK8. The three strains mentioned above grew in extreme low acid conditions.

  11. Identification and Optimal Growth Conditions of Actinomycetes Isolated From Mangrove Environment

    International Nuclear Information System (INIS)

    Ameerah Abdul Hamid; Suhaidi Ariffin; Sharifah Aminah Syed Mohamad

    2015-01-01

    Actinomycetes are classified as saprophytes that play a significant role in the breakdown of organic matters into more readily assimilable nutrients. They are also known to produce a variety of secondary metabolites, which are useful for human, veterinary medicine and agriculture applications. The optimum growth conditions of actinomycetes isolated from mangrove environment at different temperature, pH and sodium chloride (NaCl) concentration were studied. The identification of the isolates by both biochemical test and 16S rRNA analysis was also done. All isolates were grown in ISP1 medium at different temperature (25, 30, 37 and 40 degree Celsius); pH (3.5, 5, 7 and 9); and salt concentration (0, 3, 5 and 7 % w/v). The optimum temperature for KMS1 and KMH2 isolates was at 25 degree Celsius. However, the optimum temperature for PCS1 and PMH1A was at 30 and 37 degree Celsius respectively. The optimum pH was pH 7 and the most preferred NaCl concentration of all isolates was at 3 % (w/v). All isolates showed negative result for carbohydrate fermentation except for KMS1 which showed utilisation of D-Fructose sugar with acid production. Only KMS1 and PCS1 exhibited amylolytic and proteolytic activity. The isolates were successfully identified as Streptomyces variables (KMS1), Streptomyces rubrolavendulae (KMH2), Streptomyces spp. CPE333 (PCS1) and Actinomycetales bacterium (PMH1A). (author)

  12. Isolation, Identification and Seasonal Distribution of Soilborne Fungi in Different Areas of Erbil Governorate

    Directory of Open Access Journals (Sweden)

    Farid M. Toma

    2012-10-01

    Full Text Available A survey study was undertaken for the isolation and identification of soilborne fungi inhabiting different areas in Erbil, Iraq. A total of 15 soil samples collected seasonally during August 2008-July 2009. Fungi were isolated from soil during this study's period, by soil dilution plate method on selective medium: Potato Dextrose Agar (PDA, eighty-one different genera of fungi were isolated during the four seasons; (30, 33, 70, and 53 genera isolated in (summer, autumn, winter and spring respectively, the most frequently isolated fungi during four seasons were Aspergillus sp. (539x103 colony forming units /g.soil, followed by Penicillium sp. (215x103 cfu/g.soil, Rhizopus spp. (115x103 cfu/g.soil, Emericella spp. (109x103 cfu/g.soil, Fusarium spp. (47x103 cfu/g.soil, and Ulocladium botrytis (47x103 cfu/g.soil, while the least frequently isolated fungal genera were Blakeslea tuningtam, Clasterosporium cyperi, Idriella sp., Naranus cryptomeriae and Torula alternata, (1x103 cfu/g.soil, for each one. Fungi isolated from soil by washing method counted for ninety three species belonging to fifty six genera, among them: Aspergillus sp., Circinella sp., Cunninghamella sp., Mucor spp., Mycelia sterilia, Rhizopus sp.

  13. Identification at Biovar Level of Brucella Isolates Causing Abortion in Small Ruminants of Iran

    Directory of Open Access Journals (Sweden)

    Ali Mohammad Behroozikhah

    2012-01-01

    Full Text Available To determine the most prevalent biovar responsible for brucellosis in sheep and goat populations of Iran, a cross-sectional study was carried out over 2 years in six provinces selected based on geography and disease prevalence. Specimens obtained from referred aborted sheep and goat fetuses were cultured on Brucella selective media for microbiological isolation. Brucellae were isolated from 265 fetuses and examined for biovar identification using standard microbiological methods. Results showed that 246 isolates (92.8% were B. melitensis biovar 1, 18 isolates (6.8% were B. melitensis biovar 2, and, interestingly, one isolate (0.4% obtained from Mazandaran province was B. abortus biovar 3. In this study, B. melitensis biovar 3 was isolated in none of the selected provinces, and all isolates from 3 provinces (i.e., Chehar-mahal Bakhtiari, Markazi, and Ilam were identified only as B. melitensis biovar 1. In conclusion, we found that B. melitensis biovar 1 remains the most prevalent cause of small ruminant brucellosis in various provinces of Iran.

  14. Detection, molecular typing and phylogenetic analysis of Leishmania isolated from cases of leishmaniasis among Syrian refugees in Lebanon

    Directory of Open Access Journals (Sweden)

    Tamara Salloum

    2016-06-01

    Two molecular typing methods of 39 FFPE Leishmania isolates were used: the ITS1-PCR RFLP and the nested ITS1-5.8S rDNA gene amplification followed by sequencing and phylogenetic analysis. The efficiency of these two techniques in Leishmania identification was compared and the phylogenetic relationships among these isolates were illustrated based on the neighbor-joining (NJ method. The results were statistically correlated with the parasitic index (PI. The DNA storage in formalin-fixed paraffin embedded (FFPE tissues was assessed as well. The parasites identified were all L. tropica as determined by both techniques. ITS1-5.8S rDNA gene based typing proved to be more sensitive in the detection of parasites (positive in 69.2% of the isolates as opposed to the ITS1-PCR RFLP method that was successful in identifying L. tropica in only 43.6% of the isolates. Sequencing and phylogenetic analysis revealed high levels of heterogeneity. A statistically significant correlation was observed between PI and the results of the nested ITS1-5.8S rDNA gene PCR. Genotyping at the species level is essential for monitoring the relative frequency of CL in the Mediterranean area that is correlated to three different Leishmania species (Leishmania infantum, Leishmania major and L. tropica, each characterized by distinct epidemiological features. The obtained results highlight the need to find a universally accepted diagnostic tool for Leishmania typing.

  15. White piedra: molecular identification of Trichosporon inkin in members of the same family.

    Science.gov (United States)

    Richini-Pereira, Virgínia Bodelão; Camargo, Rosângela Maria Pires de; Bagagli, Eduardo; Marques, Silvio Alencar

    2012-06-01

    White piedra is a superficial mycosis caused by the genus Trichosporon and characterized by nodules on hair shaft. The authors report a family referred to as pediculosis. Mycological culture on Mycosel® plus molecular identification was performed to precisely identify the etiology. A Trichosporon spp. infection was revealed. The molecular procedure identified the agent as Trichosporon inkin. White piedra and infection caused by T. inkin are rarely reported in Southern Brazil. The molecular tools are essentials on identifying the Trichosporon species.

  16. Species Identification, Strain Differentiation, and Antifungal Susceptibility of Dermatophyte Species Isolated From Clinically Infected Arabian Horses

    DEFF Research Database (Denmark)

    El Damaty, Hend M; Tartor, Yasmine H; Mahmmod, Yasser Saadeldien Ibrahim

    2017-01-01

    Arabian horses, the eldest equine breeds, have great economic and social significance for its long, unique, and storied history. Molecular characterization of dermatophyte species affecting Arabian horses is a crucial necessity for epidemiologic and therapeutic purposes. The objective of this study...... are more effective against T. mentagrophytes and T. verrucosum. In conclusion, PCR-RFLP technique is a reliable tool for the identification of dermatophyte species from Arabian horses. Internal transcribed spacer sequencing provides a precise and useful technique for the identification and differentiation...

  17. Performance of culture media for the isolation and identification of Staphylococcus aureus from bovine mastitis.

    Science.gov (United States)

    Bautista-Trujillo, G U; Solorio-Rivera, J L; Rentería-Solórzano, I; Carranza-Germán, S I; Bustos-Martínez, J A; Arteaga-Garibay, R I; Baizabal-Aguirre, V M; Cajero-Juárez, M; Bravo-Patiño, A; Valdez-Alarcón, J J

    2013-03-01

    Rapid isolation and identification of pathogens is a major goal of diagnostic microbiology. In order to isolate and identify Staphylococcus aureus, a number of authors have used a variety of selective and/or differential culture media. However, to date, there are no reports comparing the efficacy of selective and differential culture media for S. aureus isolation from bovine mastitis cases using the 16S rRNA (rrs) gene sequence as a gold standard test. In the present study, we evaluated the efficacy of four selective and/or differential culture media for the isolation of S. aureus from milk samples collected from cows suffering from bovine mastitis. Four hundred and forty isolates were obtained using salt-mannitol agar (SMA, Bioxon), Staphylococcus-110 agar (S110, Bioxon), CHROMAgar Staph aureus (CSA, BD-BBL) and sheep's blood agar (SBA, BD-BBL). All bacterial isolates were identified by their typical colony morphology in the respective media, by secondary tests (for coagulase and β-haemolysis) and by partial 16S rRNA (rrs) gene sequencing as a gold standard test. Sensitivity, positive predictive and negative predictive values were higher for SMA (86.96, 52.63 and 95.95%, respectively) compared with S110 (70.00, 23.73 and 90.91%, respectively), CSA (69.23, 28.13 and 95.74%, respectively) and SBA (68.75, 37.93 and 89.58%, respectively) while specificity values were similar for all media. Data indicated that the use of culture media for S. aureus isolation combined with determination of coagulase activity and haemolysis as secondary tests improved accuracy of the identification and was in accordance with rrs gene sequence-analysis compared with the use of the culture media alone.

  18. Molecular identification of Candida dubliniensis isolated from oral lesions of HIV-positive and HIV-negative patients in São Paulo, Brazil Identificação molecular de amostras de Candida dubliniensis isoladas de lesões orais de pacientes HIV positivos e negativos em São Paulo, Brasil

    Directory of Open Access Journals (Sweden)

    Jorge Kleber Chavasco

    2006-02-01

    Full Text Available Candida dubliniensis is a new, recently described species of yeast. This emerging oral pathogen shares many phenotypic and biochemical characteristics with C. albicans, making it hard to differentiate between them, although they are genotypically distinct. In this study, PCR (Polymerase Chain Reaction was used to investigate the presence of C. dubliniensis in samples in a culture collection, which had been isolated from HIV-positive and HIV-negative patients with oral erythematous candidiasis. From a total of 37 samples previously identified as C. albicans by the classical method, two samples of C. dubliniensis (5.4% were found through the use of PCR. This study underscores the presence of C. dubliniensis, whose geographical and epidemiological distribution should be more fully investigated.Candida dubliniensis é uma nova espécie recentemente descrita. Este patógeno oral emergente compartilha muitas características fenotípicas e bioquímicas com C. albicans dificultando assim a diferenciação entre elas. As mesmas, porém, mostram-se genotipicamente distintas. Este trabalho tem como objetivo identificar, pela técnica de PCR (Polymerase Chain Reaction, a possível presença de C. dubliniensis dentre amostras isoladas de candidose oral eritematosa, provenientes de pacientes HIV positivos e HIV negativos. Em um total de 37 amostras identificadas anteriormente, por método clássico, como C. albicans encontramos duas amostras de C. dubliniensis (5,4% utilizando a técnica do PCR. Esta técnica mostrou-se útil, prática e com identificação taxonômica mais acurada.

  19. Molecular epidemiology of Klebsiella pneumoniae K1 and K2 isolates in Japan.

    Science.gov (United States)

    Harada, Sohei; Ishii, Yoshikazu; Saga, Tomoo; Aoki, Kotaro; Tateda, Kazuhiro

    2018-03-20

    Although severe infections caused by hypervirulent Klebsiella pneumoniae isolates, such as K1 isolates belonging to sequence type (ST) 23, have been a significant problem in Asian countries, epidemiology of these isolates in Japan remains unclear. We performed a nationwide molecular epidemiological study of K. pneumoniae K1 and K2 isolates in Japan. Of the 259K. pneumoniae isolates collected, 14 and 16 isolates were identified as capsular genotypes K1 and K2, respectively. All K1 isolates were ST23 or its closely related clones and showed high genetic similarity by pulsed-field gel electrophoresis (PFGE) and the DiversiLab system (DL). K2 isolates, belonging to ST14, ST25, ST65, ST86, and ST110, were more genetically diverse than K1 isolates. Isolates belonging to a specific ST showed identical virulence gene profiles with a few exceptions. PFGE and DL results using K1 and K2 isolates were generally in agreement. Copyright © 2018. Published by Elsevier Inc.

  20. Morphological and molecular identification of fungi associated with ...

    African Journals Online (AJOL)

    Eneke

    Macro-propagation technology, which has the potential to produce 50-60 shoots per sucker ... Isolates were characterized using cultural, micro-morphological and ... The technology requires little .... and the colour tint on the surface was white,.

  1. Molecular Identification of Streptomyces producing antibiotics and their antimicrobial activities

    Directory of Open Access Journals (Sweden)

    Latifa A. Al_husnan

    2016-12-01

    Full Text Available Five strains of Streptomyces, namely S, N, W, E and C (designations should be mentioned in detail here isolated from the rhizosphere soil cultivated with palm Alajua (date, pressed dates, AlMedina city, Saudi Arabia, were induced to produce antibiotics. Antimicrobial activities were determined on solid medium supplemented with starch. The detection was based on the formation of transparent zones around colonies. The results indicated that isolates had antibacterial activities against Staphylococcus aureus, Bacillus cereus, B. subtilis, Pseudomonas aeruginosa and also showed antifungal activity against Candida albicans and Aspergillus niger. DNA extracted from five isolates was used as template for 16s rDNA gene amplification. The expected PCR size was 1.5 kbp;1.6 kbp; 1.25 kbp; 1.25kbp and 1.0 k bp for S, N, W, E and C isolates respectively using universal 16s rDNA gene primers using direct PCR. The isolates varied morphologically on the basis of spore color, aerial and substrate mycelium formation, and production of diffusible pigment. Isolates were tested under a microscope by using slide culture technique. The results indicate that the soil of this region is source of Streptomyces having antibacterial and antifungal activity and thus better utilization of these microorganisms as biological control agents.

  2. [Molecular epidemiology and antifungal susceptibility of Candida species isolated from urine samples of patients in intensive care unit].

    Science.gov (United States)

    Yüksekkaya, Serife; Fındık, Duygu; Arslan, Uğur

    2011-01-01

    The aims of this study were to analyse the amphotericin B and fluconazole susceptibility and molecular epidemiology of Candida strains (Candida albicans, Candida tropicalis and Candida glabrata) isolated from the urine samples of patients hospitalized in the intensive care unit. Identification of the isolates was done according to microscopic morphology (chlamydospor, blastospor, pseudohyphae and true hyphae) on cornmeal agar, germ tube formation and carbohydrate assimilation patterns (API ID 32C bioMérieux, France). Antifungal susceptibilities of the isolates were determined by in vitro broth microdilution method recommended by Clinical and Laboratory Standards Institute (CLSI). To investigate the clonal relationship of the isolates, randomly amplified polymorphic DNA (RAPD) analysis was performed by using Cnd3 primer. Of the 56 Candida isolates minimum inhibitory concentration (MIC) ranges, MIC50 and MIC90 values for amphotericin B were 0.125-1 µg/ml, 0.125 and 0.5 µg/ml for C.albicans, 0.125-1 µg/ml, 0.25 and 1 µg/ml for C.tropicalis and 0.125-1 µg/ml, 0.25 and 1 µg/ml for C.glabrata, respectively. Fluconazole MIC ranges, MIC50 and MIC90 values were 0.25-4 µg/ml, 0.25 and 0.5 µg/ml for C.albicans, 0.25-16 µg/ml, 0.5 and 1 µg/ml for C.tropicalis and 0.5-64 µg/ml, 8 and 16 µg/ml for C.glabrata, respectively. For amphotericin B, none of the isolates had high MIC values (MIC > 1 µg/ml). While one of the C.glabrata isolates was resistant to fluconazole (MIC ≥ 64 µg/ml), one C.tropicalis and two C.glabrata isolates were dose-dependent susceptible (MIC: 16-32 µg/ml). The results of RAPD analysis indicated an exogenous spread from two clones for C.albicans, one clone for C.glabrata and one clone for C.tropicalis. This study underlines the importance of molecular epidemiological analysis of clinical samples together with hospital environmental samples in terms of Candida spp. To determine the exogenous origin for the related strains and to prevent

  3. Molecular Typing and Antimicrobial Susceptibility of Methicillin-Resistant Staphylococcus aureus Isolated from Bovine Milk in Tanzania.

    Science.gov (United States)

    Mohammed, Jibril; Ziwa, Michael Henry; Hounmanou, Yaovi Mahuton Gildas; Kisanga, Adela; Tuntufye, Huruma Nelwike

    2018-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) in raw milk can be transmitted from animals to humans, and in Tanzania raw milk is sold in local markets and consumed as purchased. This study was performed to determine the molecular characteristics and antimicrobial susceptibility pattern of MRSA strains isolated from raw bovine milk sold at local markets in Tanzania. A total of 117 raw milk samples were cultured on Baird-Parker medium to isolate S. aureus and PCR was used for amplification of gltB gene for S. aureus identification and the presence of mecA gene for methicillin-resistant strains. Coagulase-negative (CN) S. aureus were reconfirmed using tube coagulase, DNase, and API Staph tests. MRSA isolates were spa typed whereas antimicrobial susceptibility testing was performed by the disc diffusion method. Forty-six coagulase positives (CP) and two CN S. aureus were identified. Most strains were resistant to penicillin (72%), and 3 isolates: 2 CN S. aureus and 1 coagulase-negative Staphylococci (CNS), were phenotypically resistant to vancomycin, oxacillin, and cefoxitin and were confirmed to carry mecA. Resistance to clindamycin, trimethoprim-sulfamethoxazole, and tetracycline was 23.9%, 30.4%, and 41.3%, respectively. Twelve isolates exhibited multidrug resistance; however, only one mecA positive strain among the three was typeable and belonged to spa type t2603. This study reports for the first time the presence of CN variant of MRSA, which was assigned the spa type t2603, and the presence of multidrug resistant S. aureus isolates from bovine milk in Morogoro, Tanzania.

  4. Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection.

    Directory of Open Access Journals (Sweden)

    Luis A Flores-Páez

    Full Text Available Staphylococcus epidermidis is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of S. epidermidis from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of S. epidermidis were used: one from ocular infection (n = 62, and another from healthy conjunctiva (n = 45. All isolates were genotyped by pulsed field gel electrophoresis (PFGE, multilocus sequence typing (MLST, staphylococcal cassette chromosome mec (SCCmec, detection of the genes icaA, icaD, IS256 and polymorphism type of agr locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%, while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%. By means of a principal coordinates analysis (PCoA and a discriminant analysis (DA it was found that ocular infection isolates had as discriminant markers agr III or agr II, SCCmec V or SCCmec I, mecA gene, resistance to tobramycin, positive biofilm, and IS256+. In contrast to the healthy conjunctiva isolates, the discriminating markers were agr I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66% had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva

  5. A rapid, one step molecular identification of Trichoderma citrinoviride and Trichoderma reesei.

    Science.gov (United States)

    Saroj, Dina B; Dengeti, Shrinivas N; Aher, Supriya; Gupta, Anil K

    2015-06-01

    Trichoderma species are widely used as production hosts for industrial enzymes. Identification of Trichoderma species requires a complex molecular biology based identification involving amplification and sequencing of multiple genes. Industrial laboratories are required to run identification tests repeatedly in cell banking procedures and also to prove absence of production host in the product. Such demands can be fulfilled by a brief method which enables confirmation of strain identity. This communication describes one step identification method for two common Trichoderma species; T. citrinoviride and T. reesei, based on identification of polymorphic region in the nucleotide sequence of translation elongation factor 1 alpha. A unique forward primer and common reverse primer resulted in 153 and 139 bp amplicon for T. citrinoviride and T. reesei, respectively. Simplification was further introduced by using mycelium as template for PCR amplification. Method described in this communication allows rapid, one step identification of two Trichoderma species.

  6. Isolation and identification of soil fungi isolates from forest soil for flooded soil recovery

    Science.gov (United States)

    Hazwani Aziz, Nor; Zainol, Norazwina

    2018-04-01

    Soil fungi have been evaluated for their ability in increasing and recovering nitrogen, phosphorus and potassium content in flooded soil and in promoting the growth of the host plant. Host plant was cultivated in a mixture of fertile forest soil (nutrient-rich soil) and simulated flooded soil (nutrient-poor soil) in an optimized soil condition for two weeks. The soil sample was harvested every day until two weeks of planting and was tested for nitrogen, phosphorus and potassium concentration. Soil fungi were isolated by using dilution plating technique and was identified by Biolog’s Microbial Systems. The concentration of nitrogen, phosphorus, and potassium was found to be increasing after two weeks by two to three times approximately from the initial concentration recorded. Two fungi species were identified with probability more than 90% namely Aspergillus aculeatus and Paecilomyces lilacinus. Both identified fungi were found to be beneficial in enhancing plant growth and increasing the availability of nutrient content in the soil and thus recovering the nutrient content in the flooded soil.

  7. Isolation and polymerase chain reaction-based identification of Riemerella anatipestifer from ducks in Kerala, India

    Directory of Open Access Journals (Sweden)

    Manju Soman

    2014-10-01

    Full Text Available Aim: The aim was to isolate and characterize Riemerella anatipestifer organisms from disease outbreaks in ducks in Kerala. Materials and Methods: Ducklings, suspected of Riemerella infection, were sacrificed and subjected to post-mortem examination. Heart blood smears and impression smears from liver and spleen were examined for the presence of pathogenic organisms. Heart blood, lung, liver, and spleen collected aseptically from the birds were subjected to isolation trials in brain heart infusion agar and 10% bovine blood agar. The isolates were characterized based on morphology, cultural characteristics and biochemical tests, and their identity were confirmed by polymerase chain reaction (PCR and the PCR amplified DNA was sequenced. The antibiotic sensitivity testing of the isolates were carried out using six antibiotics viz ciprofloxacin, chloramphenicol, enrofloxacin, amoxycillin, cotrimoxazole, and gentamicin. Results: Colonies suggestive of Riemerella organisms could be isolated on blood agar. Biochemical characterization and PCR confirmed the identity of isolates as R. anatipestifer. The nucleotide sequence of the PCR product showed 99% homology to the R. anatipestifer sequences in the NCBI. The antibiogram revealed that the organisms were sensitive to ciprofloxacin, enrofloxacin, and gentamicin. Conclusion: The present study suggests that the PCR assay can facilitate fast and proper identification of R. anatipestifer infection in ducks. The assay can also differentiate between R. anatipestifer and Pasteurella multocida and can replace the traditional methods of differentiation which are cumbersome and time-consuming.

  8. Identification of Candida species isolated from vulvovaginitis in Mashhad, Iran by Use of MALDI-TOF MS

    Directory of Open Access Journals (Sweden)

    Majid Alizadeh

    2017-12-01

     Of the 65 isolates analyzed, 61 (93.8% were recognised by MALDI-TOF mass spectrometry and for four isolates (6.1% only not relabile identifications were achieved. In this study, the most frequently isolated species were Candida albicans (58.5%, followed by Candida tropicalis (16.9%, Candida glabrata (7.7%, Candida parapsilosis (7.7% and Candida guillermondii (3.1%.  Conclusion presented results demonstrate that the MALDI TOF mass spectrometry is a fast and reliable technique, and has the potential to replace conventional phenotypic identification of Candida species and other yeast strains routinely isolated in clinical microbiology laboratories.

  9. Molecular identification of marine symbiont bacteria of gastropods from the waters of the Krakal coast Yogyakarta and its potential as a Multi-Drug Resistant (MDR) antibacterial agent

    Science.gov (United States)

    Bahry, Muhammad Syaifudien; Pringgenies, Delianis; Trianto, Agus

    2017-01-01

    The resistance of pathogenic bacteria may occur to many types of antibiotics, especially in cases of non-compliance use of antibiotics, which likely to allow the evolution of Multi-Drug Resistant (MDR) bacteria. Gastropods seas are marine invertebrates informed capable of production of secondary metabolites as antibacterial MDR. The purpose of the study was the isolation and identification of gastropod symbiont bacteria found in the waters of Krakal, Gunung Kidul, Yogyakarta, which has the ability to produce antibacterial compounds against MDR(Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, MRSA (methicillin-Resistant Staphylococcus aureus), Staphylococcus aureus, and Staphylococcus homunis) molecular. Stages of this research began with the isolation of bacteria, bacteria screening for anti-MDR compound, mass culture, and extraction, antibacterial activity test, DNA extraction, amplification by PCR 16S rDNA and sequencing. The results of the study showed that 19 isolates of bacteria were isolated from three species of gastropods namely Littorina scabra, Cypraea moneta and Conus ebraeus. Among them, 4 isolates showed activity against MDR test bacteria (E. coli, E. cloacae, K. pneumoniae, S. aureus and S. homunis). The highest activity was displayed by code LS.G1.8 isolate with the largest inhibition zone 15.47±0.45mm on S. humonis at 250 µg/disk concentration. Isolate CM.G2.1 showed largest inhibition zone, with 21.5±0.07mm on MRSA at 1000 µg/disk concentration and isolate the largest inhibition zone CM.G2.5 14.37±0.81mm on MRSA 14.37±0.81mm at concentrations 1000 µg/disk. The molecular identification of isolates LS.G1.8 has 99% homology with Bacillus subtilis and isolates CM.G2.1 has 99% homology with Bacillus pumillus.

  10. Isolation and identification of curcumin and bisacurone from rhizome extract of temu glenyeh (Curcuma soloensis. Val)

    Science.gov (United States)

    Vitasari, Rista A.; Wibowo, Fajar R.; Marliyana, Soerya D.; Widyo Wartono, M.

    2016-02-01

    Temu glenyeh (Curcuma soloensis. Val) is one of the medicinal plants that grow in Surakarta. This plant is similar with C. longa and C. Xanthoriza. Chemical constituents from an extract of the plant have never been studied. In this paper, we report the isolation of a terpenoid and curcumin from the rhizome of C. soloensis. The isolation was employed by soxhlet apparatus using acetone as solvent. The fractionation and purification of the compound from the acetone extracts were undertaken by vacuum liquid chromatography and flash chromatography. Identification of compounds used spectroscopy methods, such as FTIR, NMR (1H NMR, 13C NMR, COSY, HSQC and HMBC) and GC-MS. Isolated compounds were identified as curcumin (1) and bisacurone (2).

  11. A simple and rapid molecular method for Leptospira species identification

    NARCIS (Netherlands)

    Ahmed, Ahmed; Anthony, Richard M.; Hartskeerl, Rudy A.

    2010-01-01

    Serological and DNA-based classification systems only have little correlation. Currently serological and molecular methods for characterizing Leptospira are complex and costly restricting their world-wide distribution and use. Ligation mediated amplification combined with microarray analysis

  12. Molecular identification of endophytic fungi from Aquilaria sinensis ...

    African Journals Online (AJOL)

    Molecular phylogenetic analysis demonstrated that Botryosphaeria, Colletotrichum gloeosporioides, Phomopsis and Cylindrocladium species are members of the agarwood-producing wounded tree, while Phoma, Mycosphaerella, Sagenomella, Alternaria and Ramichloridium species is able to colonize the non-resinous ...

  13. Identification of a myometrial molecular profile for dystocic labor.

    LENUS (Irish Health Repository)

    Brennan, Donal J

    2011-01-01

    The most common indication for cesarean section (CS) in nulliparous women is dystocia secondary to ineffective myometrial contractility. The aim of this study was to identify a molecular profile in myometrium associated with dystocic labor.

  14. Genomic, transcriptomic, and proteomic approaches towards understanding the molecular mechanisms of salt tolerance in Frankia strains isolated from Casuarina trees.

    Science.gov (United States)

    Oshone, Rediet; Ngom, Mariama; Chu, Feixia; Mansour, Samira; Sy, Mame Ourèye; Champion, Antony; Tisa, Louis S

    2017-08-18

    Soil salinization is a worldwide problem that is intensifying because of the effects of climate change. An effective method for the reclamation of salt-affected soils involves initiating plant succession using fast growing, nitrogen fixing actinorhizal trees such as the Casuarina. The salt tolerance of Casuarina is enhanced by the nitrogen-fixing symbiosis that they form with the actinobacterium Frankia. Identification and molecular characterization of salt-tolerant Casuarina species and associated Frankia is imperative for the successful utilization of Casuarina trees in saline soil reclamation efforts. In this study, salt-tolerant and salt-sensitive Casuarina associated Frankia strains were identified and comparative genomics, transcriptome profiling, and proteomics were employed to elucidate the molecular mechanisms of salt and osmotic stress tolerance. Salt-tolerant Frankia strains (CcI6 and Allo2) that could withstand up to 1000 mM NaCl and a salt-sensitive Frankia strain (CcI3) which could withstand only up to 475 mM NaCl were identified. The remaining isolates had intermediate levels of salt tolerance with MIC values ranging from 650 mM to 750 mM. Comparative genomic analysis showed that all of the Frankia isolates from Casuarina belonged to the same species (Frankia casuarinae). Pangenome analysis revealed a high abundance of singletons among all Casuarina isolates. The two salt-tolerant strains contained 153 shared single copy genes (most of which code for hypothetical proteins) that were not found in the salt-sensitive(CcI3) and moderately salt-tolerant (CeD) strains. RNA-seq analysis of one of the two salt-tolerant strains (Frankia sp. strain CcI6) revealed hundreds of genes differentially expressed under salt and/or osmotic stress. Among the 153 genes, 7 and 7 were responsive to salt and osmotic stress, respectively. Proteomic profiling confirmed the transcriptome results and identified 19 and 8 salt and/or osmotic stress-responsive proteins in the

  15. A rapid PCR-based approach for molecular identification of filamentous fungi.

    Science.gov (United States)

    Chen, Yuanyuan; Prior, Bernard A; Shi, Guiyang; Wang, Zhengxiang

    2011-08-01

    In this study, a novel rapid and efficient DNA extraction method based on alkaline lysis, which can deal with a large number of filamentous fungal isolates in the same batch, was established. The filamentous fungal genomic DNA required only 20 min to prepare and can be directly used as a template for PCR amplification. The amplified internal transcribed spacer regions were easy to identify by analysis. The extracted DNA also can be used to amplify other protein-coding genes for fungal identification. This method can be used for rapid systematic identification of filamentous fungal isolates.

  16. Molecular identification of tsetse fly ( Diptera: Glossinidae ) species ...

    African Journals Online (AJOL)

    Inspite of the few mixed clusters, the pattern produced in the phylogenetic trees can provide a good guide to support any other method of Glossina identification. It was recommended that evaluations be made to validate other genetic markers that can produce better resolutions to identify tsetse fly species using phylogenetic ...

  17. Seasonal abundance and molecular identification of West Nile virus ...

    African Journals Online (AJOL)

    Methods: Mosquitoes belonging to the Culex pipens complex were captured in three different locations located within Abeokuta Metropolis between March 2012 and January 2013. Individual species were identified using morphometric methods. Amplification of the Ace2 gene by PCR confirmed morphormetric identification ...

  18. MOLECULAR GENETIC IDENTIFICATION OF THE SLOVENE HOME GUARDVICTIMS

    Directory of Open Access Journals (Sweden)

    Irena Zupanič Pajnič

    2008-11-01

    The research showed a high probability (from 99.9999 % to 99.999999 % that allthree victims of the killings under the Storžič Mountain are related to the living relatives,speaking in favour of the positive identification of the victims

  19. Prevalence, incidence and molecular identification of root-knot ...

    African Journals Online (AJOL)

    Tomato is a widely grown vegetable in Pakistan. However, its production is severely constrained by root knot nematodes (RKNs). Accurate identification of RKNs is essential for an appropriate control program. The current study evaluated the prevalence, incidence and diversity of RKNs of tomato crops grown in the Khyber ...

  20. Molecular characterization of Mycobacterium bovis strains isolated from cattle slaughtered at two abattoirs in Algeria

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    Ouzrout Rachid

    2009-01-01

    Full Text Available Abstract Background Bovine Tuberculosis is prevalent in Algeria despite governmental attempts to control the disease. The objective of this study was to conduct, for the first time, molecular characterization of a population sample of Mycobacterium bovis strains isolated from slaughter cattle in Algeria. Between August and November 2007, 7250 animals were consecutively screened at the abattoirs of Algiers and Blida. In 260 animals, gross visible granulomatous lesions were detected and put into culture. Bacterial isolates were subsequently analysed by molecular methods. Results Altogether, 101 bacterial strains from 100 animals were subjected to molecular characterization. M. bovis was isolated from 88 animals. Other bacteria isolated included one strain of M. caprae, four Rhodococcus equi strains, three Non-tuberculous Mycobacteria (NTM and five strains of other bacterial species. The M. bovis strains isolated showed 22 different spoligotype patterns; four of them had not been previously reported. The majority of M. bovis strains (89% showed spoligotype patterns that were previously observed in strains from European cattle. Variable Number of Tandem Repeat (VNTR typing supported a link between M. bovis strains from Algeria and France. One spoligotype pattern has also been shown to be frequent in M. bovis strains from Mali although the VNTR pattern of the Algerian strains differed from the Malian strains. Conclusion M. bovis infections account for a high amount of granulomatous lesions detected in Algerian slaughter cattle during standard meat inspection at Algiers and Blida abattoir. Molecular typing results suggested a link between Algerian and European strains of M. bovis.

  1. [Isolation and identification of aerobic and facultative anaerobic bacteria in the oral cavity].

    Science.gov (United States)

    Lu, Wenxin; Wu, Fanzi; Zhou, Xinxuan; Wu, Lan; Li, Mingyun; Ren, Biao; Guo, Qiang; Huang, Ruijie; Li, Jiyao; Xiao, Liying; Li, Yan

    2015-12-01

    To establish a systematic method for isolation and identification of aerobic and facultative anaerobic bacteria in the oral cavity. Samples of the saliva, dental plaque and periapical granulation tissue were collected from 20 subjects with healthy oral condition and from 8 patients with different oral diseases. The bacteria in the samples were identified by morphological identification, VITEK automatic microorganism identification and 16s rRNA gene sequencing. VITEK automatic microorganism identification and 16s rRNA gene sequencing showed an agreement rate of 22.39% in identifying the bacteria in the samples. We identified altogether 63 bacterial genus (175 species), among which Streptococcus, Actinomyces and Staphylococcus were the most common bacterial genus, and Streptococcus anginosus, Actinomyces oris, Streptococcus mutans and Streptococcus mitis were the most common species. Streptococcus anginosus was commonly found in patients with chronic periapical periodontitis. Streptococcus intermedius and Staphylococcus aureus were common in patients with radiation caries, and in patients with rampant caries, Streptococcus mutans was found at considerably higher rate than other species. Aerobic and facultative anaerobic bacteria are commonly found in the oral cavity, and most of them are gram-positive. 16s rRNA gene sequencing is more accurate than VITEK automatic microorganism identification in identifying the bacteria.

  2. Molecular identification of Giardia and Cryptosporidium from dogs and cats

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    Sotiriadou Isaia

    2013-01-01

    Full Text Available The aim of the present study was to diagnose the presence of Giardia cysts and Cryptosporidium oocysts in household animals using nested polymerase chain reaction (PCR and sequence analysis. One hundred faecal samples obtained from 81 dogs and 19 cats were investigated. The Cryptosporidium genotypes were determined by sequencing a fragment of the small subunit (SSU rRNA gene, while the Giardia Assemblages were determined through analysis of the glutamate dehydrogenase (GDH locus. Isolates from five dogs and two cats were positive by PCR for the presence of Giardia, and their sequences matched the zoonotic Assemblage A of Giardia. Cryptosporidium spp. isolated from one dog and one cat were both found to be C. parvum. One dog isolate harboured a mixed infection of C. parvum and Giardia Assemblage A. These findings support the growing evidence that household animals are potential reservoirs of the zoonotic pathogens Giardia spp. and Cryptosporidium spp. for infections in humans.

  3. Identification of lactic acid bacteria isolated from Tarhana, a traditional Turkish fermented food

    DEFF Research Database (Denmark)

    Sengun, Ilkin Yucel; Nielsen, Dennis Sandris; Karapinar, Mehmet

    2009-01-01

    Tarhana is a traditional fermented product produced from a mixture of spontaneously fermented yogurt and wheat flour in Turkey. The aims of the present study were to enumerate and identify for the first time by molecular biology-based methods predominant lactic acid bacteria (LAB) isolated during...... processing of Tarhana. Samples were collected from eight different regions of Turkey. In order to explore the relationship between raw material and the microbiology of Tarhana, yogurt and wheat flour were also analyzed. A total of 226 Gram-positive and catalase-negative isolates were obtained from MRS, M17...... and S. thermophilus was found to be the yogurt....

  4. [Identification of Candida dubliniensis strains using heat tolerance tests, morphological characteristics and molecular methods].

    Science.gov (United States)

    Arikan, Sevtap; Darka, Ozge; Hasçelik, Gülşen; Günalp, Ayfer

    2003-01-01

    Described in 1995, Candida dubliniensis is a novel Candida species closely related to Candida albicans due primarily to its ability to produce germ tube and chlamydospores. Given these phenotypic similarities between the two species, C. dubliniensis cannot be readily distinguished from Candida albicans by routine laboratory work-up. We explored the frequency of isolation of C. dubliniensis among 213 strains previously defined as C. albicans based on their ability to produce germ tube. The test isolates were initially examined for their morphological features on cornmeal tween 80 agar, inability to grow at 45 degrees C, and the biochemical assimilation profile (ID 32C system, bioMerieux, France). Among all, 2 (0.9%) of the isolates were identified as C. dubliniensis based on the production of numerous chlamydospores in chains on cornmeal tween 80 agar and the lack of growth at 45 degrees C. The assimilation profile of these isolates was found to be in accordance with this identification. In an effort to confirm the identification, polymerase chain reaction (PCR) studies were carried out by using the C. dubliniensis specific primer set, DUBF and DUBR. Both of the isolates yielded C. dubliniensis-specific 288 base pair amplification products, confirming the previous identification obtained with the initial screening tests. The isolates were found to be susceptible to fluconazole and itraconazole, and generated amphotericin B minimal inhibitory concentrations of 0.5-1 microgram/ml by NCCLS M27-A2 microdilution method. These data suggest that the isolation rate of C. dubliniensis among our clinical isolates is low. The morphological features on cornmeal tween 80 agar and the lack of ability to grow at 45 degrees C appear as reliable, cheap, and practical screening tests in initial identification of C. dubliniensis among germ tube-producing Candida strains.

  5. Molecular Methods Used for the Identification of Potentially Probiotic Lactobacillus reuteri Strains

    OpenAIRE

    Weiss, Agnes; Lettner, Hans Peter; Kramer, Walter; Mayer, Helmut Karl; Kneifel, Wolfgang

    2005-01-01

    Forty potentially probiotic Lactobacillus strains as well as reference strains of different genera were grown under standardised conditions. Cell masses were harvested and DNA was isolated. For identification, all strains were subjected to genus-specific polymerase chain reaction (PCR), and the affiliation with the genus Lactobacillus was confirmed for all isolates. Using two species-specific primer-pairs for Lactobacillus reuteri, specific amplicons were observed for eight of the forty inves...

  6. Identification of Group G Streptococcal Isolates from Companion Animals in Japan and Their Antimicrobial Resistance Patterns.

    Science.gov (United States)

    Tsuyuki, Yuzo; Kurita, Goro; Murata, Yoshiteru; Goto, Mieko; Takahashi, Takashi

    2017-07-24

    In this study, we conducted a species-level identification of group G streptococcal (GGS) isolates from companion animals in Japan and analyzed antimicrobial resistance (AMR) patterns. Strains were isolated from sterile and non-sterile specimens collected from 72 animals with clinical signs or symptoms in April-May, 2015. We identified the strain by 16S rRNA sequencing, mass spectrometry (MS), and an automated method based on their biochemical properties. Antimicrobial susceptibility was determined using the broth microdilution method and E-test. AMR determinants (erm(A), erm(B), mef(A), tet(M), tet(O), tet(K), tet(L), and tet(S)) in corresponding resistant isolates were amplified by PCR. The 16S rRNA sequencing identified the GGS species as Streptococcus canis (n = 68), Streptococcus dysgalactiae subsp. equisimilis (n = 3), and S. dysgalactiae subsp. dysgalactiae (n = 1). However, there were discrepancies between the sequencing data and both the MS and automated identification data. MS and the automated biochemical technique identified 18 and 37 of the 68 sequencing-identified S. canis strains, respectively. The AMR rates were 20.8% for tetracycline and 5.6% for clarithromycin, with minimum inhibitory concentrations (MIC) 50 -MIC 90 of 2-64 and ≤ 0.12-0.25μg/mL, respectively. AMR genotyping showed single or combined genotypes: erm(B) or tet(M)-tet(O)-tet(S). Our findings show the unique characteristics of GGS isolates from companion animals in Japan in terms of species-level identification and AMR patterns.

  7. [Principles for molecular identification of traditional Chinese materia medica using DNA barcoding].

    Science.gov (United States)

    Chen, Shi-Lin; Yao, Hui; Han, Jian-Ping; Xin, Tian-Yi; Pang, Xiao-Hui; Shi, Lin-Chun; Luo, Kun; Song, Jing-Yuan; Hou, Dian-Yun; Shi, Shang-Mei; Qian, Zhong-Zhi

    2013-01-01

    Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.

  8. Phenotypic and molecular characterization of Klebsiella spp. isolates causing community-acquired infections

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    U. Garza-Ramos

    2018-05-01

    Full Text Available Klebsiella spp. isolates from community-acquired infections were characterized. A total of 39 Klebsiella spp. isolates were obtained from outpatients at four rural hospitals in Mexico (2013–2014. The biochemical tests identified all as being K. pneumoniae. The molecular multiplex-PCR test identified 36 (92.4% K. pneumoniae isolates and one (2.5% K. variicola isolate, and phylogenetic analysis of the rpoB gene identified two isolates (5.1% belonging to K. quasipneumoniae subsp. quasipneumoniae and K. quasivariicola. The last one was confirmed by phylogenetic analysis of six-loci concatenated genes. Mostly the isolates were multidrug resistant; however, a minority were extended-spectrum β-lactamase producing (10.2%. The extended-spectrum β-lactamase CTX-M-15 gene was identified in these isolates. Analysis of biofilm production and the hypermucoviscosity phenotype showed a total of 35 (92.3% and seven (17.9% of the isolates were positive for these phenotypes respectively. The K2 (4/39, 10.2%, K5 (2/39, 5.1% and K54 (1/39, 2.5% serotypes were identified in seven (17.9% of the isolates, and only 28.5% (2/7 hypermucoviscous isolates were positive for the K2 and K5 serotypes. In general, the sequence type (ST analysis and phylogenetic analysis of seven multilocus sequence typing loci were heterogeneous; however, ST29 was the most prevalent ST in the analysed isolates, accounting for 19% (4/21 of the total isolates. Two of the four ST29 isolates had the hypermucoviscosity phenotype. The virulence factors for fimbriae were the most prevalent, followed by siderophores. Community-acquired infections are caused by various species from Klebsiella genus, with different profiles of antibiotic resistance and heterogeneous virulence factors. Keywords: Antimicrobial susceptibility, Bacterial resistance, Cephalosporin resistance, Community infection, ESBL, Hypermucoviscosity

  9. Morphological and Molecular Identification of Colletotrichum acutatum from Tomato Fruit

    OpenAIRE

    Svetlana Živković; Saša Stojanović; Žarko IVanović; Nenad Trkulja; Nenad Dolovac; Goran Aleksić; Jelica Balaž

    2010-01-01

    Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum coccodes, and Colletotrichum dematium are the four main species of Colletotrichum that cause tomato anthracnose. In Serbia, the occurrence of anthracnose on tomato fruit has been recorded during the last several years. Typical fruit symptoms include dark, sunken, and circular lesion with orange conidial masses. Pathogen isolates were obtained from a diseased tomato fruits, on PDA medium...

  10. Molecular Identification of Bacteria Involved in Degradation of Crude ...

    African Journals Online (AJOL)

    ISSN: 0189 1731. Available online at http://www.ajol.info/index.php/njb/index ... activities is a main cause of water and soil pollution. One of the ... 60% reduction in household food security and were capable of .... isolates to degrade crude oil, mineral salt media was .... to help maintain the cold chain and prevent the DNA ...

  11. Molecular genetic identification and extracellular laccase activity of ...

    African Journals Online (AJOL)

    ajl yemi

    2012-01-12

    Jan 12, 2012 ... that were used for inoculation of 50 mL of PDB in 250 mL flasks, which were incubated ... addition of100 µL of. 5% trichloracetic acid (v/v in water). .... induction of propagation structures in the isolates was beyond the scope of ...

  12. Molecular characterization of the first leptospires isolated from goats in Brazil

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    Walter Lilenbaum

    2014-12-01

    Full Text Available Two Leptospira sp. isolates were obtained by the first time from goats in Brazil and characterized by sequencing rrs, rpoB and secY genes, PFGE and typing with monoclonal antibodies. Both isolates are identical and belong to Leptospira santarosai. Analysis of the rrs and the rpoB genes sequences revealed 100% identity between the goat isolates and the Bananal reference strain. When secY sequences of the two isolates were compared to each other, it was observed that they had identical sequences. However, when compared to that of the Bananal reference strain, there were 15 mismatches along the 549 bp secY sequence. In conclusion, molecular methods are increasingly useful for the characterization of leptospires and allowed to identify those isolates of caprine origin as closely related but not identical to serovar Bananal, and constitute a new type named Carioca.

  13. Biological and molecular characterization of Brazilian isolates of Zucchini yellow mosaic virus

    Directory of Open Access Journals (Sweden)

    David Marques de Almeida Spadotti

    2015-02-01

    Full Text Available Zucchini yellow mosaic virus (ZYMV causes substantial economic losses in cucurbit crops. Although ZYMV has been present in Brazil for more than 20 years, there is little information about the biological and molecular characteristics of the isolates found in the country. This study aimed to characterize the experimental hosts, pathotypes and genetic diversity of a collection of eleven Brazilian ZYMV isolates within the coat protein gene. For biological analysis, plant species from Amaranthaceae, Chenopodiaceae, Cucurbitaceae, Fabaceae, Solanaceae, and Pedaliaceae were mechanically inoculated and pathotypes were identified based on the reaction of a resistant Cucumis melo, accession PI414723. All of the cucurbit species/varieties and Sesamum indicum were systemically infected with all isolates. The nucleotide sequence variability of the coat protein gene ranged from 82 % to 99 % compared to the corresponding sequences of ZYMV isolates from different geographical locations. No recombination event was detected in the coat protein gene of the isolates.

  14. Pathogenicity and molecular analysis of an infectious bursal disease virus isolated from Malaysian village chickens.

    Science.gov (United States)

    Tan, D Y; Hair-Bejo, M; Omar, A R; Aini, I

    2004-01-01

    The characteristics of the pathogenic infectious bursal disease virus (IBDV) that infected avian species other than commercial chickens were largely unknown. In this study, by using in vivo and molecular methods, we had characterized an IBDV isolate (named 94268) isolated from an infectious bursal disease (IBD) outbreak in Malaysian village chickens--the adulterated descendant of the Southeast Asian jungle fowl (Gallus bankiva) that were commonly reared in the backyard. The 94268 isolate was grouped as the very virulent IBDV (vvIBDV) strain because it caused severe lesions and a high mortality rate in village chickens (>88%) and experimentally infected specific-pathogen-free chickens (>66%). In addition, it possessed all of the vvIBDV molecular markers in its VP2 gene. Phylogenetic analysis using distance, maximum parsimony, and maximum likelihood methods revealed that 94268 was monophyletic with other vvIBDV isolates and closely related to the Malaysian vvIBDV isolates. Given that the VP2 gene of 94268 isolate was almost identical and evolutionarily closely related to other field IBDV isolates that affected the commercial chickens, we therefore concluded that IBD infections had spread across the farm boundary. IBD infection in the village chicken may represent an important part of the IBD epidemiology because these birds could harbor the vvIBDV strain and should not be overlooked in the control and prevention of the disease.

  15. Molecular characterization of Salmonella isolates by REP-PCR and RAPD analysis.

    Science.gov (United States)

    Albufera, U; Bhugaloo-Vial, P; Issack, M I; Jaufeerally-Fakim, Y

    2009-05-01

    Eighteen Salmonella isolates from both human and food (non-human) sources (fish, meat, and poultry) were characterized using conventional culture methods, biochemical, serological, and molecular analyses. REP-PCR and RAPD produced DNA profiles for differentiation purposes. Enterobacterial repetitive intergenic consensus (ERIC), repetitive extragenic palindronic (REP) and BOXAIR primers were selected for REP-PCR and two arbitrary primers, namely OPP-16 and OPS-11 were used for RAPD to generate DNA fingerprints from the Salmonella isolates. REP-PCR method showed greater discriminatory power in differentiating closely related strains of the related strains of Salmonella and produced more complex banding patterns as compared with RAPD. A dendogram was constructed with both sets of profiles using SPSS Version 13.0 computer software and showed that most human isolates were separately clustered from the non-human isolates. Two of the human isolates were closely related to some of the non-human isolates. A good correlation was also observed between the serogrouping of the O antigen and the molecular profiles obtained from REP-PCR and RAPD data of the Salmonella isolates. The results of a principal coordinate analysis (PCA) corresponded to the clustering in the dendrogram.

  16. Morphological and molecular identification of ticks infesting Boa constrictor (Squamata, Boidae in Manaus (Central Brazilian Amazon

    Directory of Open Access Journals (Sweden)

    Leonardo Costa Fiorini

    2014-12-01

    Full Text Available The Boa constrictor is one of the world's largest vertebrate carnivores and is often found in urban areas in the city of Manaus, Brazil. The morphological identification of ticks collected from 27 snakes indicated the occurrence of Amblyomma dissimile Koch 1844 on all individuals sampled. In contrast, Amblyomma rotundatum Koch was found on only two snakes. An analysis of the 16S rRNA molecular marker confirmed the morphological identification of these ectoparasites.

  17. Molecular identification of Coccidioides spp. in soil samples from Brazil

    OpenAIRE

    de Macêdo, Regina CL; Rosado, Alexandre S; da Mota, Fabio F; Cavalcante, Maria AS; Eulálio, Kelsen D; Filho, Antônio D; Martins, Liline MS; Lazéra, Márcia S; Wanke, Bodo

    2011-01-01

    Abstract Background Since 1991 several outbreaks of acute coccidioidomycosis (CM) were diagnosed in the semi-arid Northeast of Brazil, mainly related to disturbance of armadillo burrows caused by hunters while digging them for the capture of these animals. This activity causes dust contaminated with arthroconidia of Coccidioides posadasii, which, once inhaled, cause the mycosis. We report on the identification of C. posadasii in soil samples related to outbreaks of CM. Results Twenty four soi...

  18. Molecular subgroups of medulloblastoma identification using noninvasive magnetic resonance spectroscopy.

    Science.gov (United States)

    Blüml, Stefan; Margol, Ashley S; Sposto, Richard; Kennedy, Rebekah J; Robison, Nathan J; Vali, Marzieh; Hung, Long T; Muthugounder, Sakunthala; Finlay, Jonathan L; Erdreich-Epstein, Anat; Gilles, Floyd H; Judkins, Alexander R; Krieger, Mark D; Dhall, Girish; Nelson, Marvin D; Asgharzadeh, Shahab

    2016-01-01

    Medulloblastomas in children can be categorized into 4 molecular subgroups with differing clinical characteristics, such that subgroup determination aids in prognostication and risk-adaptive treatment strategies. Magnetic resonance spectroscopy (MRS) is a widely available, noninvasive tool that is used to determine the metabolic characteristics of tumors and provide diagnostic information without the need for tumor tissue. In this study, we investigated the hypothesis that metabolite concentrations measured by MRS would differ between molecular subgroups of medulloblastoma and allow accurate subgroup determination. MRS was used to measure metabolites in medulloblastomas across molecular subgroups (SHH = 12, Groups 3/4 = 17, WNT = 1). Levels of 14 metabolites were analyzed to determine those that were the most discriminant for medulloblastoma subgroups in order to construct a multivariable classifier for distinguishing between combined Group 3/4 and SHH tumors. Medulloblastomas across molecular subgroups revealed distinct spectral features. Group 3 and Group 4 tumors demonstrated metabolic profiles with readily detectable taurine, lower levels of lipids, and high levels of creatine. SHH tumors showed prominent choline and lipid with low levels of creatine and little or no evidence of taurine. A 5-metabolite subgroup classifier inclusive of creatine, myo-inositol, taurine, aspartate, and lipid 13a was developed that could discriminate between Group 3/4 and SHH medulloblastomas with excellent accuracy (cross-validated area under the curve [AUC] = 0.88). The data show that medulloblastomas of Group 3/4 differ metabolically as measured using MRS when compared with SHH molecular subgroups. MRS is a useful and accurate tool to determine medulloblastoma molecular subgroups. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Comparison phenotypic and genotypic identification of Staphylococcus species isolated from bovine mastitis

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    Felipe Freitas Guimarães

    Full Text Available ABSTRACT: In addition to Staphylococcus aureus nowadays other coagulase-positive staphylococci (CoPS and coagulase-negative staphylococci (CoNS, earlier considered of minor importance, are now accepted as relevant pathogens for humans and animals. The involvement of these microorganisms in bovine mastitis etiology and the possibility their transmission through milk to humans justify the requirement of developing reliable methods for identification of the most frequent species among them. The purpose of this study was to compare the phenotypic techniques with the genotypic method carried out by sequencing of the rpoB gene in identification of several species of the genus Staphylococcus isolated from bovine mastitis. A total of 300 staphylococci isolates of bovine mastitis cases from several Brazilian dairy herds were studied by phenotypic and genotypic techniques, respectively: 150 CoPS and 150 CoNS strains. A total of 18 CoNS different species and 4 CoPS species were identified. Among the CoNS the following species were recognized: 48 (32% Staphylococcus warneri, 22(15% S. epidermidis, 20(13% S. hyicus, 10(7% S. xylosus, 7(5% S. haemolyticus, 6(4% S. simulans, 6(4% S. schleiferi subsp schleiferi, 6(4% S. hominis, 5(3% S. pasteuri, 4(2.7% S. cohnii, 3(2% S. saprophyticus subsp. saprophyticus 3(2% S. chromogenes 3(2% S. sciuri, 2(1% S. saccharolyticus, 2(1% S. lugdunensi, 1(0,7% S. auricularis, 1(70% S. saprophyticus subsp. bovis, 1(0.7% S. capitis. And among the 150 CoPS were identified respectively: 105 (70% S. aureus, 21(14%, S. hyicus, 19(13% S. intermedius e 5(3% S. schleiferi subsp coagulans. Considering the 150 CoNS isolates, the identifications performed by phenotypic and genotypic tests presented 96.7% of concordance, kappa coefficient of agreement = 0.933, SE (standard error of kappa=0.021 (95% confidence interval: 0.893 to 0.974, Pearson’s correlation coefficient (r = 0.9977, (confidence interval 95%: 0.9938 a 0.9992 and in relation

  20. Molecular characterization of NDM-1 producing Enterobacteriaceae isolates in Singapore hospitals

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    Raymond Lin

    2012-03-01

    Full Text Available Objective: In this study, we molecularly characterized 12 NDM-1 producing clinical Enterobacteriaceae (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae isolates that were part of a collection of non-carbapenem susceptible isolates obtained during a one-year period. These isolates were obtained from four local general hospitals in Singapore.Methods: Polymerase chain reaction (PCR assays and sequencing was used to determine the presence of β-lactamase encoding genes (bla including blaNDM-1 and plasmid-mediated quinolone and aminoglycoside resistance determinants. Conjugation experiments were performed to determine the transferability of blaNDM-1. Isolate relatedness was determined by multilocus sequence typing (MLST.Results: The isolates were completely resistant to the second- and third-generation cephalosporins tested as well as carbapenems. Susceptibility profiling of the isolates indicated that 100% retained susceptibility to tigecycline while 11/12 (91.7% were susceptible to colistin. The blaNDM-1 gene was encoded on plasmids that were easily transferable. None of the patients had a travel history to countries where NDM-1 has been reported. The isolates appear clonally unrelated with MLST, revealing a diversity of clonal types among the K. pneumoniae and E. coli isolates.Conclusion: The ease of NDM-1 plasmid transmissibility may help their dissemination among the Enterobacteriaceae. Although it appears that the isolates are clonally unrelated, epidemiological links cannot be fully excluded without further research.

  1. Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China

    Science.gov (United States)

    Yang, Yongchun; Liu, Yinglong; Ding, Yunlei; Yi, Li; Ma, Zhe; Fan, Hongjie; Lu, Chengping

    2013-01-01

    One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae. PMID:23874442

  2. Morphological and molecular characterization, sexual reproduction, and pathogenicity of Setosphaeria rostrata isolates from rice leaf spot.

    Science.gov (United States)

    Kusai, Nor Azizah; Azmi, Madihah Mior Zakuan; Zainudin, Nur Ain Izzati Mohd; Yusof, Mohd Termizi; Razak, Azmi Abd

    2016-09-01

    Setosphaeria rostrata, a common plant pathogen causing leaf spot disease, affects a wide range of plant species, mainly grasses. Fungi were isolated from brown spots on rice leaves throughout Peninsular Malaysia, and 45 isolates were identified as Setosphaeria rostrata The isolates were then characterized using morphological and molecular approaches. The mating type was determined using PCR amplification of the mating type alleles, and isolates of opposite mating types were crossed to examine sexual reproduction. Based on nuclear ribosomal DNA ITS1-5.8S-ITS2 region (ITS) and beta-tubulin (BT2) sequences, two phylogenetic trees were constructed using the maximum likelihood method; S. rostrata was clustered in one well-supported clade. Pathogenicity tests showed that S. rostrata isolates are pathogenic, suggesting that it is the cause of the symptoms. Mating-type analyses indicated that three isolates carried the MAT1-1 allele, and the other 42 isolates carried MAT1-2 After isolates with opposite mating types were crossed on Sach's medium and incubated for 3 wk, six crosses produced pseudothecia that contained eight mature ascospores, and 12 other crosses produced numerous pseudothecia with no ascospores. To our knowledge, this is the first report on S. rostrata isolated from leaf spots on rice. © 2016 by The Mycological Society of America.

  3. 16S rRNA gene sequencing in routine identification of anaerobic bacteria isolated from blood cultures

    DEFF Research Database (Denmark)

    Justesen, Ulrik Stenz; Skov, Marianne Nielsine; Knudsen, Elisa

    2010-01-01

    A comparison between conventional identification and 16S rRNA gene sequencing of anaerobic bacteria isolated from blood cultures in a routine setting was performed (n = 127). With sequencing, 89% were identified to the species level, versus 52% with conventional identification. The times...

  4. Isolation and identification of halotolerant soil bacteria from coastal Patenga area.

    Science.gov (United States)

    Rahman, Shafkat Shamim; Siddique, Romana; Tabassum, Nafisa

    2017-10-30

    Halotolerant bacteria have multiple uses viz. fermentation with lesser sterility control and industrial production of bioplastics. Moreover, it may increase the crop productivity of coastal saline lands in Bangladesh by transferring the salt tolerant genes into the plants. The study focused on the isolation and identification of the halotolerant bacteria from three soil samples, collected from coastal Patenga area. The samples were inoculated in nutrient media containing a wide range of salt concentrations. All the samples showed 2, 4 and 6% (w/v) salt tolerance. The isolates from Patenga soil (4, 6%) and beach soil (2%) showed catalase activity and all the isolates showed negative results for oxidase activity, indole production, lactose and motility. All the samples provided positive results for dextrose fermentation. Other tests provided mixed results. Based on the morphological characteristics, biochemical tests and ABIS software analysis the isolates fall within the Enterobacteriaceae, Clostridium and Corynebacterium, with a predominance of Vibrios. Overall the isolates can be considered as mild halotolerant, with the best growth observed at lower salinities and no halophilism detected. Among many possibilities, the genes responsible for the salt tolerant trait in these species can be identified, extracted and inserted into the crop plants to form a transgenic plant to result in higher yield for the rest of the year.

  5. Identification of chitinolytic bacteria isolated from shrimp pond sediment and characterization of their chitinase encoding gene

    Science.gov (United States)

    Triwijayani, A. U.; Puspita, I. D.; Murwantoko; Ustadi

    2018-03-01

    Chitinolytic bacteria are a group of bacteria owning enzymes that able to hydrolyze chitin. Previously, we isolated chitinolytic bacteria from shrimp pond sediment in Bantul, Yogyakarta, and obtained five isolates showing high chitinolytic index named as isolate PT1, PT2, PT5, PT6 and PB2. The aims of this study were to identify chitinolytic bacteria isolated from shrimp pond sediment and to characterize the chitinase encoding gene from each isolate. The molecular technique was performed by amplification of 16S rDNA, amplification of chitinase encoding gene and sequence analysis. Two chitinolytic bacteria of PT1 and PT2 were similar to Aeromonas bivalvium strain D15, PT5 to Pseudomonas stutzeri strain BD-2.2.1, PT6 to Serratia marcescens strain FZSF02 and PB2 to Streptomyces misionensis strain OsiRt-1. The comparison of chitinase encoding gene between three isolates with those in Gen Bank shows that PT1 had similar sequences with the chi1 gene in Aeromonas sp. 17m, PT2 with chi1 gene in A. caviae (CB101) and PT6 with chiB gene in S. Marcescens (BJL200).

  6. Molecular characterization of KPC-producing Klebsiella pneumoniae isolated from patients in a Public Hospital in Caracas, Venezuela.

    Science.gov (United States)

    Falco Restrepo, Aura Dayana; Velásquez Nieves, Mariel Alexandra; Takiff, Howard

    Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria are amongst the most important causative agents of nosocomial infections worldwide. Isolates of this bacterium have been identified in Venezuela but little is known about their local spread. The aim of this study was to perform the molecular characterization of KPC-producing strains isolated from 2012 to 2013 in a public hospital in Caracas, Venezuela. Twenty-two K. pneumoniae clinical isolates phenotypically classified as KPC producing were subjected to PCR screening for the presence of bla KPC genes and their location within transposon Tn4401. The bla KPC PCR product was sequenced to identify the KPC alleles. Genotypic analysis was performed by means of repetitive extragenic palindromic PCR (rep-PCR) and Multi Locus Sequence Typing (MLST). Finally, conjugation and electroporation assays were used to determine whether the bla KPC genes were found in plasmids. All isolates contained the bla KPC-2 variant, and 21 of the 22 were associated with the Tn4401b isoform. The strains were distributed in 8 sequence types (ST), three of which were new. Conjugation and electroporation assays indicated that 95.5% (n=21/22) of the isolates contained the bla KPC gene in plasmids. This study on circulating bacterial strains and the identification of KPC alleles may help to understand the routes of dissemination and control their spread within this hospital. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  7. Identification of Clinical Staphylococcal Isolates from Humans by Internal Transcribed Spacer PCR

    Science.gov (United States)

    Couto, Isabel; Pereira, Sandro; Miragaia, Maria; Sanches, Ilda Santos; de Lencastre, Hermínia

    2001-01-01

    The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems. PMID:11526135

  8. Identification of clinical staphylococcal isolates from humans by internal transcribed spacer PCR.

    Science.gov (United States)

    Couto, I; Pereira, S; Miragaia, M; Sanches, I S; de Lencastre, H

    2001-09-01

    The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems.

  9. Sponge cell culture? A molecular identification method for sponge cells

    NARCIS (Netherlands)

    Sipkema, D.; Heilig, G.H.J.; Akkermans, A.D.L.; Osinga, R.; Tramper, J.; Wijffels, R.H.

    2003-01-01

    Dissociated sponge cells are easily confused with unicellular organisms. This has been an obstacle in the development of sponge-cell lines. We developed a molecular detection method to identify cells of the sponge Dysidea avara in dissociated cell cultures. The 18S ribosomal RNA gene from a Dysidea

  10. molecular identification of rotavirus strains associated with diarrhea

    African Journals Online (AJOL)

    DR. AMINU

    ABSTRACT. The study was carried out to determine the molecular characteristics of the rotavirus strains associated with diarrhea among children in Kwara state, Nigeria. A total of 150 stool samples were collected from diarrheic children. The stool samples were screened for rotavirus,using Enzyme linked Immunosorbent ...

  11. Molecular identification of rotavirus strains associated with diarrhea ...

    African Journals Online (AJOL)

    The study was carried out to determine the molecular characteristics of the rotavirus strains associated with diarrhea among children in Kwara state, Nigeria. A total of 150 stool samples were collected from diarrheic children. The stool samples were screened for rotavirus,using Enzyme linked Immunosorbent assay (ELISA).

  12. Molecular study for the sex identification in Japanese quails ...

    African Journals Online (AJOL)

    use

    2011-12-14

    Dec 14, 2011 ... The aim of this study was to determine the sex of. Japanese quail (Coturnix japonica) by DNA analysis. Chromo ... M, 100 bp DNA ladder (Fermentas, Germany); lane 1, negative control. (without DNA) .... low cost and reduced risk of contamination, and preven- .... Improving the reliability of molecular sexing.

  13. Identification of new molecular alterations in Fatal Familial Insomnia.

    Science.gov (United States)

    Fatal familial insomnia (FFI) is an autosomal dominant prion disease caused by a D178N mutation in PRNP in combination with methionine (Met) at codon 129 in the mutated allele of the same gene (D178N-129M haplotype). The present study analyzes pathological and molecular features in seven FFI cases c...

  14. Molecular identification of the insect adipokinetic hormone receptors

    DEFF Research Database (Denmark)

    Staubli, Frank; Jørgensen, Thomas J D; Cazzamali, Giuseppe

    2002-01-01

    identified the first insect AKH receptors, namely those from the fruitfly Drosophila melanogaster and the silkworm Bombyx mori. These results represent a breakthrough for insect molecular endocrinology, because it will lead to the cloning of all AKH receptors from all model insects used in AKH research, and...

  15. Molecular Detection of Inducible Clindamycin Resistance among Staphylococcal Strains Isolated from Hospital Patients

    Directory of Open Access Journals (Sweden)

    Shadiyeh Abdollahi

    2013-04-01

    Full Text Available Background & Objectives: Macrolide, lincosamide and streptogramin B (MLSB antimicrobial agents are used in the treatment of staphylococcal infections. They prevent the microbial protein synthesis system through binding to 23 S rRNA. The aim of this study was to apply molecular methods to detect inducible clindamycin resistance genes among staphylococcal strains isolated from clinical specimens.   Methods : Two hundred staphylococcus strains were isolated from nose and throat swabs of patients in Toohid and Besat hospitals in Sanandaj . Antimicrobial susceptibilities of isolates were determined using disc diffusion method, agar screen test and D-Test. A multiplex PCR was performed using primers specific for erm (A, B, C, TR genes.   Results: Out of 200 isolates, 18.5 % were MRSA and 32% were MRCNS (methicillin resistant coagulase negative staphylococci. Of 80 erythromycin resistant isolates, 48 were coagulase negative and 32 were S. aureus. Among the 48 coagulase negative staphylococci (CONS isolates, 11.63% expressed the MLSB-inducible phenotypes. Using PCR, the frequency of different genes in the collection of isolates were as follows: ermA 5.41 % , erm B 5.41 % , and erm C 3.13%. The ermTR gene was negative in all isolates. Among the 32 S. aureus isolates, 9.38% expressed the MLSB-nducible phenotype. Using PCR, these isolates harbored erm A (2.22%, ermB (2.22%, ermC (2.22% and ermTR (2.22% .   Conclusion: This is the first study to show the rate of inducible clindamycin clinical isolates of staphylococci harboring erm genes in Sananadaj. It also demonstrated the frequency of erm genes was higher among CONS isolates than S. aureus. This data suggested the transfer of resistance gene from nonpathogenic to pathogenic strains is likely to happen. Therefore, screening and control of these resistance genes is recommended at clinical laboratories.

  16. Isolation and molecular characterization of Clostridium perfringens from healthy Merino lambs in Patagonia region, Argentina

    DEFF Research Database (Denmark)

    Mignaqui, A. C.; Marcellino, R. B.; Ronco, Troels

    2017-01-01

    The presence and molecular characterization of Clostridium perfringens in healthy Merino lambs over a six-month period was investigated in this study. Overall, a high prevalence of C. perfringens was detected, even in day-old lambs. Even though the majority of the isolates were characterized...

  17. Isolation of Candida Species from Gastroesophageal Lesions among Pediatrics in Isfahan, Iran: Identification and Antifungal Susceptibility Testing of Clinical Isolates by E-test

    Directory of Open Access Journals (Sweden)

    Fatemeh Salehi

    2017-01-01

    Full Text Available Background: Candida species can become opportunistic pathogens causing local or systemic invasive infections. Gastroesophageal candidiasis may depend on the Candida colonization and local damage of the mucosal barrier. Risk factors are gastric acid suppression, diabetes mellitus, chronic debilitating states such as carcinomas, and the use of systemic antibiotics and corticosteroids. The aim of this study is collection and molecular identification of Candida species from gastroesophageal lesions among pediatrics in Isfahan, and determination of minimum inhibitory concentration (MIC ranges for clinical isolates. Materials and Methods: A total of 200 patients underwent endoscopy (130 specimens from gastritis and 70 samples from esophagitis were included in this study between April 2015 and November 2015. All specimens were subcultured on sabouraud dextrose agar, and genomic DNA of all strains was extracted using boiling method. Polymerase chain reaction and DNA sequencing of the ITS1-5.8SrDNA-ITS2 region were used for the identification of all Candida strains. MIC ranges were determined for itraconazole (ITC, amphotericin B (AmB, and fluconazole (FLU by E-test. Results: Twenty of 200 suspected patients (10% were positive by direct microscopy and culture. Candida albicans was the most common species (60% followed by Candida glabrata (30%, Candida parapsilosis (5%, and Candida kefyr (5%. MIC ranges were determined for FLU (0.125–8 μg/mL, ITC (0.008–0.75 μg/mL, and AmB (0.008–0.75 μg/mL, respectively. Conclusion: Every colonization of Candida species should be considered as a potentially factor of mucocutaneous candidiasis and should be treated with antifungal drugs.

  18. Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

    Directory of Open Access Journals (Sweden)

    Lista Florigio

    2011-12-01

    Full Text Available Abstract Background The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays. Results In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences. Conclusions MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.

  19. Advances in Dendrobium molecular research: Applications in genetic variation, identification and breeding.

    Science.gov (United States)

    Teixeira da Silva, Jaime A; Jin, Xiaohua; Dobránszki, Judit; Lu, Jiangjie; Wang, Huizhong; Zotz, Gerhard; Cardoso, Jean Carlos; Zeng, Songjun

    2016-02-01

    Orchids of the genus Dendrobium are of great economic importance in global horticultural trade and in Asian traditional medicine. For both areas, research yielding solid information on taxonomy, phylogeny, and breeding of this genus are essential. Traditional morphological and cytological characterization are used in combination with molecular results in classification and identification. Markers may be useful when used alone but are not always reliable in identification. The number of species studied and identified by molecular markers is small at present. Conventional breeding methods are time-consuming and laborious. In the past two decades, promising advances have been made in taxonomy, phylogeny and breeding of Dendrobium species due to the intensive use of molecular markers. In this review, we focus on the main molecular techniques used in 121 published studies and discuss their importance and possibilities in speeding up the breeding of new cultivars and hybrids. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. [Comparison between conventional methods, ChromAgar Candida® and PCR method for the identification of Candida species in clinical isolates].

    Science.gov (United States)

    Estrada-Barraza, Deyanira; Dávalos Martínez, Arturo; Flores-Padilla, Luis; Mendoza-De Elias, Roberto; Sánchez-Vargas, Luis Octavio

    2011-01-01

    The increase in the incidence of yeast species causing fungemia in susceptible immunocompromised patients in the last two decades and the low sensitivity of conventional blood culture has led to the need to develop alternative approaches for the early detection and identification of causative species. The aim of this study was to compare the usefulness of molecular testing by the polymerase chain reaction (PCR) and conventional methods to identify clinical isolates of different species, using the ID32C ATB system (bioMérieux, France), chromogenic culture Chromagar Candida® (CHROMagar, France) and morphogenesis in corn meal agar. We studied 79 isolates, in which the most prevalent species using the system ID32C and PCR was C. albicans, followed by C. tropicalis, C. glabrata and C .krusei. PCR patterns obtained for the identification of clinical isolates were stable and consistent in the various independent studies and showed good reproducibility, concluding that PCR with species-specific primers that amplify genes ITS1 and ITS2 for rRNA or topoisomerase II primers is a very specific and sensitive method for the identification of C. glabrata, C. krusei, C. albicans, and with less specificity for C. tropicalis. Copyright © 2010 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  1. Molecular Identification of Mycobacterium Tuberculosis and Analysis of Its Resistance to Rifampin in Sputa from Tuberculosis Suspected Patients

    International Nuclear Information System (INIS)

    Syaifudin, M.

    2010-01-01

    An accurate identification of different species of Mycobacterium provides to allow appropriate treatment for Mycobacterium tuberculosis infection. Beside that, drug resistance of M. tuberculosis strains to rifampin is not clearly understood in contributing to the spread of tuberculosis in Indonesia. To assess the molecular mechanism of rifampin resistance, a number of clinical specimens of M. tuberculosis were analyzed their molecular nature of a part of the rpoB gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) methods. DNA's extracted from sputum samples were amplified and 32 P-labeled by PCR with the specific primers and the product was analyzed their mutation conferring resistance by MDE gel electrophoresis. Of the 70 specimens tested, 57 specimens were positive for M. tuberculosis organism only, three specimens contained a mixture of M. tuberculosis and non tuberculosis mycobacteria (NTM), and 10 specimens were negative approved by Duplex PCR. Of these sixty DNA positive samples (thus the sensitivity of PCR was 85.71%), 5 (8.3%) of them suspected to contain mutations in rpoB which were associated with rifampin resistance. Even though the frequency of mutation was low, the results from our study clearly indicate that the molecular mechanism of rifampin resistance in M. tuberculosis isolates from Indonesia involves alterations in the rpoB gene. Molecular diagnosis by PCR which is fast and easy to perform is useful for early and rapid detection of TB in sputum specimen. (author)

  2. Molecular epidemiology of group A streptococcus from pharyngeal isolates in Auckland, New Zealand, 2013.

    Science.gov (United States)

    Williamson, Deborah A; Moreland, Nicole J; Carter, Philip; Upton, Arlo; Morgan, Julie; Proft, Thomas; Lennon, Diana; Baker, Michael G; Dunbar, Rod; Fraser, John D

    2014-01-24

    To describe the molecular epidemiology of emm types associated with circulating pharyngeal group A streptococcus (GAS) isolates in Auckland, New Zealand. GAS isolates were collected over a 10-day period from a community pathology provider in Auckland. PCR analysis and sequencing of the emm gene was performed at the Institute of Environmental Science and Research. A total of 52 emm types were identified from 278 GAS isolates. The three most common emm types were emm1, emm89 and emm12. Overall, the experimental 30-valent GAS M protein vaccine covered 19 / 52 (37%) of emm types in our study. Our study provides baseline data on the circulating pharyngeal GAS emm types in Auckland. Future clinical and molecular surveillance of GAS pharyngitis is essential in the context of ongoing GAS vaccine development.

  3. A molecular method for typing Herpes simplex virus isolates as an alternative to immunofluorescence methods

    Directory of Open Access Journals (Sweden)

    Abraham A

    2009-01-01

    Full Text Available Background: Typing of Herpes simplex virus (HSV isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF assays and polymerase chain reaction (PCR. This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. Objectives: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb based IF test. Study design : This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase ( pol gene of HSV isolates. Results: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42 of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42 were HSV-2, and two (4.8% were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30, whereas the cost per MAb IF test is about Rs 1,500 (USD 35 including all overheads (reagents, instruments, personnel time, and consumables. Conclusion: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers is now more widespread than fluorescence microscopes in a country like India.

  4. An application of coherence resonances in molecular transition identification

    International Nuclear Information System (INIS)

    Alekseev, V.A.; Salomaa, R.

    1978-01-01

    In Λ-type three level configurations having long lived lower levels extremely sharp two photon resonances occur. We want to draw attention to the use of these resonances for distinguishing the hyperfine splitting of lower and upper set of levels of molecular transitions. A new feature in the theoretical model is that the saturator and probe beams are coupled to both transitions rendering possible the appearance of interference between the resonances. (author)

  5. Molecular Detection and Characterization of Goat Isolate of Taenia hydatigena in Turkey

    Directory of Open Access Journals (Sweden)

    Armagan Erdem Utuk

    2012-01-01

    Full Text Available The aim of this study was to provide molecular detection and characterization of the goat isolate of Taenia hydatigena from Ankara province of Turkey. For this purpose, PCR amplification of small subunit ribosomal RNA (rrnS and partial sequencing of mitochondrial cytochrome c oxidase subunit 1 (mt-CO1 genes were performed in a one-month-old dead goat. According to rrnS-PCR results, parasites were identified as Taenia spp., and partial sequence of mt-CO1 gene was corresponding to T. hydatigena. At the end of the study, we concluded that molecular tools can be used to define species of parasites in cases where the key morphologic features cannot be detected. Nucleotide sequence data of Turkish goat isolate of T. hydatigena was submitted to GenBank for other researchers interested in this subject. By this study, molecular detection and characterization of T. hydatigena was done for the first time in Turkey.

  6. Isolation and identification of entomopathogenic nematodes from citrus orchards in South Africa and their biocontrol potential against false codling moth.

    Science.gov (United States)

    Malan, Antoinette P; Knoetze, Rinus; Moore, Sean D

    2011-10-01

    A survey was conducted to determine the diversity and frequency of endemic entomopathogenic nematodes (EPN) in citrus orchards in the Western Cape, Eastern Cape and Mpumalanga provinces of South Africa. The main aim of the survey was to obtain nematodes as biological control agents against false codling moth (FCM), Thaumatotibia leucotreta, a key pest of citrus in South Africa. From a total of 202 samples, 35 (17%) tested positive for the presence of EPN. Of these, four isolates (11%) were found to be steinernematids, while 31 (89%) were heterorhabditids. Sequencing and characterisation of the internal transcribed spacer (ITS) region was used to identify all nematode isolates to species level. Morphometrics, morphology and biology of the infective juvenile (IJ) and the first-generation male were used to support molecular identification and characterisation. The Steinernema spp. identified were Steinernema khoisanae, Steinernema yirgalemense and Steinernema citrae. This is the first report of S. yirgalemense in South Africa, while for S. citrae it is the second new steinernematid to be identified from South Africa. Heterorhabditis species identified include Heterorhabditis bacteriophora, Heterorhabditis zealandica and an unknown species of Heterorhabditis. Laboratory bioassays, using 24-well bioassay disks, have shown isolates of all six species found during the survey, to be highly virulent against the last instar of FCM larvae. S. yirgalemense, at a concentration of 50IJs/FCM larva caused 100% mortality and 74% at a concentration of 200IJs/pupa. Using a sand bioassay, S. yirgalemense gave 93% control of cocooned pupae and emerging moths at a concentration of 20IJs/cm(2). This is the first report on the potential use of EPN to control the soil-borne life stages of FCM, which includes larvae, pupae and emerging moths. It was shown that emerging moths were infected with nematodes, which may aid in control and dispersal. Copyright © 2011 Elsevier Inc. All rights

  7. Methodology for Isolation, Identification and Characterization of Microvesicles in Peripheral Blood

    Science.gov (United States)

    Jayachandran, Muthuvel; Miller, Virginia M.; Heit, John A.; Owen, Whyte G.

    2011-01-01

    Rationale Analyses of circulating cell membrane-derived microvesicles (MV) have come under scrutiny as potential diagnostic and prognostic biomarkers of disease. However, methods to isolate, label and quantify MV have been neither systematized nor validated. Objective To determine how pre-analytical, analytical and post-analytical factors affect plasma MV counts, markers for cell of origin and expression of procoagulant surface phosphatidylserine. Methods and Results Peripheral venous blood samples were collected from healthy volunteers and patients with cardiovascular disease and/or diabetes. Effects of blood sample collection, anticoagulant and sample processing to platelet free plasma (PFP), and MV isolation, staining and storage (freeze-thaw) and cytometer design were evaluated with replicate samples from these populations. The key finding is that use of citrate or EDTA anticoagulants decreases or eliminates microvesicles from plasma by inducing adhesion of the microvesicles to platelets or other formed elements. Protease inhibitor anticoagulants, including heparin, preserve MV counts. A centrifugation protocol was developed in which recovery of isolated MV was high with resolution down to the equivalent light scatter of 0.2 micron latex beads. Each procedure was systematically evaluated for its impact on the MV counts and characteristics. Conclusion This study provides a systematic methodology for MV isolation, identification and quantification, essential for development of MV as diagnostic and prognostic biomarkers of disease. PMID:22075275

  8. Identification of novel anti-inflammatory probiotic strains isolated from pulque.

    Science.gov (United States)

    Torres-Maravilla, Edgar; Lenoir, Marion; Mayorga-Reyes, Lino; Allain, Thibault; Sokol, Harry; Langella, Philippe; Sánchez-Pardo, María E; Bermúdez-Humarán, Luis G

    2016-01-01

    Probiotics are live microorganisms which when administered in adequate amounts, confer health benefits on the host. Their use is more and more widespread for both prevention and treatment of diseases, including traveler’s diarrhea and inflammatory bowel diseases (IBDs). In this work, we isolated and characterized novel candidate probiotic strains from pulque (xaxtle), a traditional Mexican alcoholic fermented beverage. A total of 14 strains were obtained from xaxtle samples isolated from three different Mexican regions. Species identification was performed by biochemical methods and 16S rRNA gene targeted PCR. The isolates belonged to the Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus brevis, and Lactobacillus composti phylogenetic groups, with L. brevis being the most dominant group. Bacteria were tested for lysozyme, low pH, and bile acid resistance. Moreover, the strains were tested for adherence to human intestinal epithelial cells and screened for their immunomodulatory properties using a cellular model. Selected bacterial strains with anti-inflammatory properties were then tested in vivo in a dinitro-benzene sulfonic acid (DNBS)-induced chronic colitis mouse model, and weight loss, gut permeability, and cytokine profiles were measured as readouts of inflammation. One of the selected strains, Lactobacillus sanfranciscensis LBH1068, improved mice health as observed by a reduction of weight loss, significant decreases in gut permeability, and cytokine modulation. Altogether, our results highlighted the potential of lactobacilli isolated from pulque and in particular the strain L. sanfranciscensis LBH1068 as a novel probiotic to treat IBD.

  9. Isolation and serological identification of enteropathogenic Escherichia coli in pasteurized milk in Brazil

    Directory of Open Access Journals (Sweden)

    Zoraide N da Silva

    2001-08-01

    Full Text Available OBJECTIVE: To evaluate the microbiological quality of pasteurized milk commercialized in Rio de Janeiro, Brazil, and determine serologically enteropathogenic Escherichia coli (EPEC strains in E. coli isolates obtained from milk samples. METHODS: Ninety samples of pasteurized milk -- types B and C -- of three different commercial brands, purchased in supermarkets and bakeries in Rio de Janeiro, were examined. The amount of total and fecal coliform bacteria was estimated using the Most Probable Number technique. Mesophilic, psychrotrophic, and thermoduric microorganism counts were determined by the Standard Plate Count technique. Isolation and identification of E. coli were carried out using conventional physiological tests. Commercial antisera were used for serological characterization of EPEC. RESULTS: The three milk brands analyzed revealed bacterial counts above the regulated values of the Brazilian government. It was found that among 208 strains of E. coli isolated, 46 (22.1% were serologically classified as EPEC. The most common EPEC serogroup was O55 (15.2%. CONCLUSIONS: Though recent studies on virulence factors indicate that not all strains serologically classified as EPEC are able to attaching/effacing lesion, it is believed that the isolation of EPEC serogroups from pasteurized milk represent a potential risk for children, as well as an indicative of the presence of other enteropathogens.

  10. Molecular Methods To Improve Diagnosis and Identification of Mucormycosis▿

    Science.gov (United States)

    Hammond, Sarah P.; Bialek, Ralf; Milner, Danny A.; Petschnigg, Eva M.; Baden, Lindsey R.; Marty, Francisco M.

    2011-01-01

    Mucormycosis is difficult to diagnose. Samples from suspected cases often fail to grow Mucorales in microbiologic cultures. We identified all hematologic malignancy and stem cell transplant patients diagnosed with proven mucormycosis between 2001 and 2009 at Brigham and Women's Hospital/Dana-Farber Cancer Institute. Seminested PCR targeting Mucorales 18S ribosomal DNA and sequencing were performed on formalin-fixed paraffin-embedded tissue samples. Of 29 cases of mucormycosis, 27 had tissue samples available for PCR and sequencing. Mucorales PCR was positive in 22. Among 12 culture-positive cases, 10 were PCR positive and sequencing was concordant with culture results to the genus level in 9. Among 15 culture-negative cases, PCR was positive and sequencing allowed genus identification in 12. Mucorales PCR is useful for confirmation of the diagnosis of mucormycosis and for further characterization of the infection in cases where cultures are negative. PMID:21508149

  11. Molecular methods to improve diagnosis and identification of mucormycosis.

    Science.gov (United States)

    Hammond, Sarah P; Bialek, Ralf; Milner, Danny A; Petschnigg, Eva M; Baden, Lindsey R; Marty, Francisco M

    2011-06-01

    Mucormycosis is difficult to diagnose. Samples from suspected cases often fail to grow Mucorales in microbiologic cultures. We identified all hematologic malignancy and stem cell transplant patients diagnosed with proven mucormycosis between 2001 and 2009 at Brigham and Women's Hospital/Dana-Farber Cancer Institute. Seminested PCR targeting Mucorales 18S ribosomal DNA and sequencing were performed on formalin-fixed paraffin-embedded tissue samples. Of 29 cases of mucormycosis, 27 had tissue samples available for PCR and sequencing. Mucorales PCR was positive in 22. Among 12 culture-positive cases, 10 were PCR positive and sequencing was concordant with culture results to the genus level in 9. Among 15 culture-negative cases, PCR was positive and sequencing allowed genus identification in 12. Mucorales PCR is useful for confirmation of the diagnosis of mucormycosis and for further characterization of the infection in cases where cultures are negative.

  12. Molecular Identification of Adult and Juvenile Linyphiid and Theridiid Spiders in Alpine Glacier Foreland Communities

    Science.gov (United States)

    Raso, Lorna; Sint, Daniela; Rief, Alexander; Kaufmann, Rüdiger; Traugott, Michael

    2014-01-01

    In glacier forelands spiders constitute a large proportion of the invertebrate community. Therefore, it is important to be able to determine the species that can be found in these areas. Linyphiid and theridiid spider identification is currently not possible in juvenile specimens using traditional morphological based methods, however, a large proportion of the population in these areas are usually juveniles. Molecular methods permit identification of species at different life stages, making juvenile identification possible. In this study we tested a molecular tool to identify the 10 most common species of Linyphiidae and Theridiidae found in three glacier foreland communities of the Austrian Alps. Two multiplex PCR systems were developed and over 90% of the 753 field-collected spiders were identified successfully. The species targeted were found to be common in all three valleys during the summer of 2010. A comparison between the molecular and morphological data showed that although there was a slight difference in the results, the overall outcome was the same independently of the identification method used. We believe the quick and reliable identification of the spiders via the multiplex PCR assays developed here will aid the study of these families in Alpine habitats. PMID:25050841

  13. Molecular and Clinical Epidemiology of Salmonella Paratyphi A Isolated from Patients with Bacteremia in Nepal.

    Science.gov (United States)

    Sherchan, Jatan Bahadur; Morita, Masatomo; Matono, Takashi; Izumiya, Hidemasa; Ohnishi, Makoto; Sherchand, Jeevan B; Tandukar, Sarmila; Laghu, Ujjwal; Nagamatsu, Maki; Kato, Yasuyuki; Ohmagari, Norio; Hayakawa, Kayoko

    2017-12-01

    Little is known about the epidemiology of typhoid and paratyphoid fever in Nepal. We aimed to elucidate the molecular and clinical epidemiology of Salmonella Paratyphi A in Nepal. Isolates were collected from 23 cases of bacteremia due to S. Paratyphi A between December 2014 and October 2015. Thirteen patients (57%) were male, and the median age was 21 years. None of the patients had an underlying chronic disease. All S. Paratyphi A isolates were sensitive to ampicillin, trimethoprim/sulfamethoxazole, ceftriaxone, and chloramphenicol. All isolates were resistant to nalidixic acid and were categorized as intermediately susceptible to levofloxacin. Phylogenetic analysis revealed close relatedness among the isolates, including several clonal groups, suggesting local spread. Patients with bacteremia due to S. Paratyphi A in Kathmandu, Nepal, were relatively young and nondebilitated. Improving control of S . Paratyphi infections should focus on effective infection control measures and selection of empirical therapy based on current resistance patterns.

  14. Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Bermuda.

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    Patrick Eberechi Akpaka

    Full Text Available Molecular characteristics of vancomycin resistant enterococci isolates from Bermuda Island is currently unknown. This study was conducted to investigate phenotypic and genotypic characteristics of VRE isolates from Bermuda Island using the chromogenic agar, E-tests, polymerase chain reaction (PCR, pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST. Eighteen E. faecium isolates were completely analyzed and were all resistant to vancomycin, susceptible to linezolid and quinupristin/dalfopristin, positive for vanA and esp genes. The MLST analysis confirmed most isolates were of the sequence types linked to clonal complex 17 (CC17 that is widely associated with outbreaks in hospitals. Infection control measures, antibiotic stewardship, and surveillance activities will continue to be a priority in hospital on the Island.

  15. Identification of some Rice Mutants using Morphological and Molecular Methods

    International Nuclear Information System (INIS)

    Sobieh, S.E.S.

    2006-01-01

    This investigation was conducted at the experimental farm of plant research department, nuclear research center, atomic energy authority, abu zaabal in order to verify four rice genotypes i.e sakha 102, giza 178, high yielding mutant (Ms 6) and high yielding mutant (MG 16). the (UPOV) rice descriptor was used to identify the germplasm morphologically .Molecular RAPD-PCR was used to identify genetic variability on the molecular level for the tested genotypes. 1- the results indicated that according to (UPOV) rice descriptor eight morphological traits were completely different between mutant Ms 6 in comparison with the parent sakha 102 and mut. MG 16 in comparison parent giza 178, the traits were ; stem thickness, stem length, panicle length, 1000-grain weight, grain length, grain width decorticated grain length and decorticated grain width. 2- using 10 arbitrary primers, through four rice genotypes on the molecular level using RAPD markers. the size of the amplified fragments were ranged from 0.201 to 2.036 k bp. two primers OPB-13 and OPB-16 showed no polymorphism among genotypes tested. 3- the total number of amplicons produced by the 8 polymorphic RAPD profiels was 66. the total number of monomorphic amplicons was 32. however, the total number of polymorphic amplicons was 34. 4- the percentage of polymorphism ranged from (22.22%) for primer OPA-18 to (90%) for primer OPB-11. 5-the highest genetic similarity (90.3%) was between sakha 102 and high yielding mut. (Ms 6). the genetic similarity (75.5%) was between giza 178 and high yielding mut.(MG 16). 6- one positive unique marker amplified by OPA-18 Primer identified the high yielding mutant Ms 6 but three positive unique markers amplified by OPB-17 primer and OPA-18 primer identified the high yielding mutant MG 16

  16. Molecular techniques for the identification and detection of microorganisms relevant for the food industry

    NARCIS (Netherlands)

    Klijn, N.

    1996-01-01

    The research described in this thesis concerns the development and application in food microbiology of molecular identification and detection techniques based on 16S rRNA sequences. The technologies developed were applied to study the microbial ecology of two groups of bacteria, namely

  17. Isolation, identification, and complete genome sequence of a bovine adenovirus type 3 from cattle in China

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    Zhu Yuan-Mao

    2011-12-01

    Full Text Available Abstract Background Bovine adenovirus type 3 (BAV-3 belongs to the Mastadenovirus genus of the family Adenoviridae and is involved in respiratory and enteric infections of calves. The isolation of BAV-3 has not been reported prior to this study in China. In 2009, there were many cases in cattle showing similar clinical signs to BAV-3 infection and a virus strain, showing cytopathic effect in Madin-Darby bovine kidney cells, was isolated from a bovine nasal swab collected from feedlot cattle in Heilongjiang Province, China. The isolate was confirmed as a bovine adenovirus type 3 by PCR and immunofluorescence assay, and named as HLJ0955. So far only the complete genome sequence of prototype of BAV-3 WBR-1 strain has been reported. In order to further characterize the Chinese isolate HLJ0955, the complete genome sequence of HLJ0955 was determined. Results The size of the genome of the Chinese isolate HLJ0955 is 34,132 nucleotides in length with a G+C content of 53.6%. The coding sequences for gene regions of HLJ0955 isolate were similar to the prototype of BAV-3 WBR-1 strain, with 80.0-98.6% nucleotide and 87.5-98.8% amino acid identities. The genome of HLJ0955 strain contains 16 regions and four deletions in inverted terminal repeats, E1B region and E4 region, respectively. The complete genome and DNA binding protein gene based phylogenetic analysis with other adenoviruses were performed and the results showed that HLJ0955 isolate belonged to BAV-3 and clustered within the Mastadenovirus genus of the family Adenoviridae. Conclusions This is the first study to report the isolation and molecular characterization of BAV-3 from cattle in China. The phylogenetic analysis performed in this study supported the use of the DNA binding protein gene of adenovirus as an appropriate subgenomic target for the classification of different genuses of the family Adenoviridae on the molecular basis. Meanwhile, a large-scale pathogen and serological epidemiological

  18. Molecular Characterization of Mycobacterium massiliense and Mycobacterium bolletii in Isolates Collected from Outbreaks of Infections after Laparoscopic Surgeries and Cosmetic Procedures▿

    Science.gov (United States)

    Viana-Niero, Cristina; Lima, Karla Valéria Batista; Lopes, Maria Luiza; da Silva Rabello, Michelle Christiane; Marsola, Lourival Rodrigues; Brilhante, Vânia Cristina Ribeiro; Durham, Alan Mitchel; Leão, Sylvia Cardoso

    2008-01-01

    An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Belém, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak, all of which were originally attributed to a single strain of M. abscessus. PMID:18174307

  19. Occurrence and molecular characterization of cultivable mesophilic and thermophilic obligate anaerobic bacteria isolated from paper mills.

    Science.gov (United States)

    Suihko, Maija-Liisa; Partanen, Laila; Mattila-Sandholm, Tiina; Raaska, Laura

    2005-08-01

    The aim of this work was to characterize the cultivable obligate anaerobic bacterial population in paper mill environments. A total of 177 anaerobically grown bacterial isolates were screened for aerotolerance, from which 67 obligate anaerobes were characterized by automated ribotyping and 41 were further identified by partial 16S rDNA sequencing. The mesophilic isolates indicated 11 different taxa (species) within the genus Clostridium and the thermophilic isolates four taxa within the genus Thermoanaerobacterium and one within Thermoanaerobacter (both formerly Clostridium). The most widespread mesophilic bacterium was closely related to C. magnum and occurred in three of four mills. One mill was contaminated with a novel mesophilic bacterium most closely related to C. thiosulfatireducens. The most common thermophile was T. thermosaccharolyticum, occurring in all four mills. The genetic relationships of the mill isolates to described species indicated that most of them are potential members of new species. On the basis of identical ribotypes clay could be identified to be the contamination source of thermophilic bacteria. Automated ribotyping can be a useful tool for the identification of clostridia as soon as comprehensive identification libraries are available.

  20. Molecular Identification of Cryptosporidium Species from Pet Snakes in Thailand.

    Science.gov (United States)

    Yimming, Benjarat; Pattanatanang, Khampee; Sanyathitiseree, Pornchai; Inpankaew, Tawin; Kamyingkird, Ketsarin; Pinyopanuwat, Nongnuch; Chimnoi, Wissanuwat; Phasuk, Jumnongjit

    2016-08-01

    Cryptosporidium is an important pathogen causing gastrointestinal disease in snakes and is distributed worldwide. The main objectives of this study were to detect and identify Cryptosporidium species in captive snakes from exotic pet shops and snake farms in Thailand. In total, 165 fecal samples were examined from 8 snake species, boa constrictor (Boa constrictor constrictor), corn snake (Elaphe guttata), ball python (Python regius), milk snake (Lampropeltis triangulum), king snake (Lampropeltis getula), rock python (Python sebae), rainbow boa (Epicrates cenchria), and carpet python (Morelia spilota). Cryptosporidium oocysts were examined using the dimethyl sulfoxide (DMSO)-modified acid-fast staining and a molecular method based on nested-PCR, PCR-RFLP analysis, and sequencing amplification of the SSU rRNA gene. DMSO-modified acid-fast staining revealed the presence of Cryptosporidium oocysts in 12 out of 165 (7.3%) samples, whereas PCR produced positive results in 40 (24.2%) samples. Molecular characterization indicated the presence of Cryptosporidium parvum (mouse genotype) as the most common species in 24 samples (60%) from 5 species of snake followed by Cryptosporidium serpentis in 9 samples (22.5%) from 2 species of snake and Cryptosporidium muris in 3 samples (7.5%) from P. regius.

  1. Molecular Genotype Identification of Different Chickens: Major Histocompatibility Complex

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    Hongzhi Wang

    2014-09-01

    Full Text Available Chicken is a main poultry in China. Molecular breeding for disease resistance plays an important role in the control of diseases, especially infectious diseases. Choice of genes for disease resistance is the key technology of molecular breeding. The major histocompatibility complex (MHC is of great interest to poultry breeding scientists for its extraordinary polymorphism and close relation with traits of resistance against infectious diseases. The MHC-B haplotype plays an important role in the study of disease resistance in chicken. The traditional chicken MHC-B haplotype is commonly defined by serologic reactions of erythrocytes and the majority of studies have been conducted in Leghorn and broiler but study about other chicken breeds is little. In this study, firstly, the microsatellite marker LEI0258 which is located within the MHC was sequenced by using target sequence capture assay in different chicken breeds, and then according to the number of repeated structures and polymorphic sequences in microsatellite, sequence information for the region defined by LEI0258 was obtained for different haplotypes. Afterwards, we identified the relation between MHC-B haplotypes and disease resistance. Collectively, these observed results provided the reference data for disease-resistant breeding association with blood type and for further study of MHC gene function in poultry.

  2. Molecular genetic identification of some wheat cultivars in the sudan

    International Nuclear Information System (INIS)

    Mekki, I. I; El Amin, H. B.

    2002-01-01

    Four wheat (Triticum aestivum L.) cultivars, namely condor, El-Nellene, Wadi El Neil and Debeira were characterized on biochemical and molecular bases. The biochemical ones were protein-banding patterns, using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and isozymes to identify the biochemical genetic fingerprint of the four cultivars. Water-soluble protein-banding pattern showed no polymorphisms among the tested cultivars. The data from starch gel electrophoresis of enzymes, malate dehydrogenase (MDH), esterase (EST) and acid phosphate (ACPH) showed that the cultivars are monomorphic. Further trials to identify the molecular genetic fingerprints of the studied cultivars were carried out using RAPD-PCR twenty-five primers were tested to perform. RAPD-PCR analysis. From the PCR products, a phylogenetic map, i.e, dendrogram, was constructed for the studied cultivars which depicted tow groups. The first group contained Wadi El Neil and Deberia with 48.4% similarity, and the second group contained Condor and El Neileen with 100% similarity. There was no similarity between Condor and Debeira (100% dissimilarity). Therefor, these data can be used subsequently for genetic engineering research and for wheat breeding programmes in the Sudan.(Author)

  3. Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

    Science.gov (United States)

    Pestel-Caron, Martine; Graff, Gabriel; Berthelot, Gilles; Pons, Jean-Louis; Lemeland, Jean-François

    1999-01-01

    Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships. PMID:10405383

  4. Molecular identification of catalases from Nicotiana plumbaginifolia (L.).

    Science.gov (United States)

    Willekens, H; Villarroel, R; Van Montagu, M; Inzé, D; Van Camp, W

    1994-09-19

    We have isolated three different catalase cDNAs from Nicotiana plumbaginifolia (cat1, cat2, and cat3) and a partial sequence of a fourth catalase gene (cat4) that shows no discernible expression based on Northern analysis. The catalase sequences were used to determine the similarity with other plant catalases and to study the transcriptional response to paraquat, 3-aminotriazole, and salicylic acid. 3-Aminotriazole induces mRNA levels of cat1, cat2 and cat3, indicating that a reduction in catalase activity positively affects catalase mRNA abundance. Salicylic acid that binds catalase in vitro, had no effect on catalase transcript levels at physiological concentrations. Paraquat resulted in the induction of cat1.

  5. Staphylococcus aureus Clinical Isolates: Antibiotic Susceptibility, Molecular Characteristics, and Ability to Form Biofilm

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    N. Indrawattana

    2013-01-01

    Full Text Available Periodic monitoring of Staphylococcus aureus characteristics in a locality is imperative as their drug-resistant variants cause treatment problem. In this study, antibiograms, prevalence of toxin genes (sea-see, seg-ser, seu, tsst-1, eta, etb, and etd, PFGE types, accessory gene regulator (agr groups, and ability to form biofilm of 92 S. aureus Thailand clinical isolates were investigated. They were classified into 10 drug groups: groups 1–7 (56 isolates were methicillin resistant (MRSA and 8–10 (36 isolates were methicillin sensitive (MSSA. One isolate did not have any toxin gene, 4 isolates carried one toxin gene (seq, and 87 isolates had two or more toxin genes. No isolate had see, etb, or tsst-1; six isolates had eta or etd. Combined seg-sei-sem-sen-seo of the highly prevalent egc locus was 26.1%. The seb, sec, sel, seu, and eta associated significantly with MSSA; sek was more in MRSA. The sek-seq association was 52.17% while combined sed-sej was not found. Twenty-three PFGE types were revealed, no association of toxin genes with PFGE types. All four agr groups were present; agr group 1 was predominant (58.70% but agr group 2 strains carried more toxin genes and were more frequent toxin producers. Biofilm formation was found in 72.83% of the isolates but there was no association with antibiograms. This study provides insight information on molecular and phenotypic markers of Thailand S. aureus clinical isolates which should be useful for future active surveillance that aimed to control a spread of existing antimicrobial resistant bacteria and early recognition of a newly emerged variant.

  6. Molecular Identification of Paramecium bursaria Syngens and Studies on Geographic Distribution using Mitochondrial Cytochrome C Oxidase Subunit I (COI).

    Science.gov (United States)

    Zagata, Patrycja; Greczek-Stachura, Magdalena; Tarcz, Sebastian; Rautian, Maria

    2015-01-01

    Paramecium bursaria is composed of five syngens that are morphologically indistinguishable but sexually isolated. The aim of the present study was to confirm by molecular methods (analyses of mitochondrial COI) the identification of P. bursaria syngens originating from different geographical locations. Phylograms constructed using both the neighbor-joining and maximum-likelihood methods based on a comparison of 34 sequences of P. bursaria strains and P. multimicronucleatum, P. caudatum and P.calkinsi strains used as outgroups revealed five clusters which correspond to results obtained previously by mating reaction. Our analysis shows the existence of 24 haplotypes for the COI gene sequence in the studied strains. The interspecies haplotype diversity was Hd = 0.967. We confirmed genetic differentiation between strains of P. bursaria and the occurrence of a correlation between geographical distribution and the correspondent syngen.

  7. Staphylococcus aureus carriage in older populations in community residential care homes: Prevalence and molecular characterization of MRSA isolates.

    Science.gov (United States)

    Galán-Sánchez, Fátima; Pérez-Eslava, Maria; Machuca, Jesús; Trujillo-Soto, Teresa; Arca-Suarez, Jorge; Rodríguez-Iglesias, Manuel

    2018-06-20

    The epidemiology of S. aureus depends on conditions in specific populations. Few studies of S. aureus colonization in the older population have been performed in Spain. The aim of this study was to determine the prevalence of methicillin-resistant S. aureus (MRSA) colonization and its molecular epidemiological characteristics in an institutionalized population in community residential care homes in Cadiz, Spain. A cross-sectional epidemiological study was conducted in three residential care homes for older people. Axilla and nostril samples were tested. Identification of S. aureus and antimicrobial susceptibility testing were by MALDI-TOF and MicroScan panels. MRSA strains were subjected to SCCmec typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The presence of Panton-Valentine leukocidin (PVL) genes was determined by PCR in all S. aureus strains. A total of 293 residents were included. Fifty-one residents (17.4%) were colonized with methicillin-sensitive S. aureus (MSSA) and 11 (3.8%) with MRSA. Resistance to at least two aminoglycosides was observed in 25.4% of MSSA and 90.9% and of MRSA isolates, and resistance to levofloxacin in 80.3% of MSSA and 100% of MRSA isolates. SCCmecIV was detected in all isolates and all except one (ST-125) were ST-8. None of the S. aureus isolates were positive for PVL. A low rate of S. aureus carriage was detected and the prevalence of MRSA was very low. ST8-MRSA-IVc was the dominant clone, and only one strain belonged to ST125-MRSA-IVc. We found MRSA transmission within the residential care homes and a very high rate of quinolone resistance in MSSA and MRSA. Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  8. Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array.

    Science.gov (United States)

    Aydin, Muhsin; Carter-Conger, Jacqueline; Gao, Ning; Gilmore, David F; Ricke, Steven C; Ahn, Soohyoun

    2018-04-01

    Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

  9. A molecular genetic approach to roebuck individual identification in the case of poaching in Serbia

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    Dimitrijević Vladimir

    2013-01-01

    Full Text Available Application of the molecular genetic methods in forensic cases dealing with wild animals has significantly increased recently. These techniques are practically used in order to help solving four key problems : determination of kind of the wild animal, geographic origin, kinship ties and individual identification. In this work the first case of introducing the examination of polimorphism of microsatelite genetic markers within forensic analysis in the cases of poaching in Serbia is presented. The objectives of this forensic analysis was to determine if the meat confiscated during house search of the suspect comes from roebuck origin (Capreolus capreolus, which remains had been found by a game warden in the field during closed season, where the suspect denied the offense, claiming that the meat comes from other roebuck that had been shot during the previous hunting season. DNK was isolated from the skin and fur samples taken from the roebuck corpse found in the woods, as well as from the frozen meat found in the suspect’s house. Both amplification and polimorphism examination of the eight microsatelite markers (ROE01, NVHRT21, NVHRT24, NVHRT48, NVHRT73, RT7 AND RT27 were carried out. In all the examined samples, the same pattern of variability of the tested microsatelites was determined, that is it was proved that DNK profiles of the samples taken from roebuck corpse were identical to DNK profile of the meat sample found in the suspect’s house. This result clearly indicates that all the examined biological samples originate from the same animal, and consequently represents forensically valid evidence in the case of roebuck poaching. [Projekat Ministarstva nauke Republike Srbije, br. III46002

  10. Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques

    Science.gov (United States)

    Baldi, Paolo; La Porta, Nicola

    2017-01-01

    In the never ending struggle against plant pathogenic bacteria, a major goal is the early identification and classification of infecting microorganisms. Xylella fastidiosa, a Gram-negative bacterium belonging to the family Xanthmonadaceae, is no exception as this pathogen showed a broad range of vectors and host plants, many of which may carry the pathogen for a long time without showing any symptom. Till the last years, most of the diseases caused by X. fastidiosa have been reported from North and South America, but recently a widespread infection of olive quick decline syndrome caused by this fastidious pathogen appeared in Apulia (south-eastern Italy), and several cases of X. fastidiosa infection have been reported in other European Countries. At least five different subspecies of X. fastidiosa have been reported and classified: fastidiosa, multiplex, pauca, sandyi, and tashke. A sixth subspecies (morus) has been recently proposed. Therefore, it is vital to develop fast and reliable methods that allow the pathogen detection during the very early stages of infection, in order to prevent further spreading of this dangerous bacterium. To this purpose, the classical immunological methods such as ELISA and immunofluorescence are not always sensitive enough. However, PCR-based methods exploiting specific primers for the amplification of target regions of genomic DNA have been developed and are becoming a powerful tool for the detection and identification of many species of bacteria. The aim of this review is to illustrate the application of the most commonly used PCR approaches to X. fastidiosa study, ranging from classical PCR, to several PCR-based detection methods: random amplified polymorphic DNA (RAPD), quantitative real-time PCR (qRT-PCR), nested-PCR (N-PCR), immunocapture PCR (IC-PCR), short sequence repeats (SSRs, also called VNTR), single nucleotide polymorphisms (SNPs) and multilocus sequence typing (MLST). Amplification and sequence analysis of specific

  11. Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques

    Directory of Open Access Journals (Sweden)

    Paolo Baldi

    2017-06-01

    Full Text Available In the never ending struggle against plant pathogenic bacteria, a major goal is the early identification and classification of infecting microorganisms. Xylella fastidiosa, a Gram-negative bacterium belonging to the family Xanthmonadaceae, is no exception as this pathogen showed a broad range of vectors and host plants, many of which may carry the pathogen for a long time without showing any symptom. Till the last years, most of the diseases caused by X. fastidiosa have been reported from North and South America, but recently a widespread infection of olive quick decline syndrome caused by this fastidious pathogen appeared in Apulia (south-eastern Italy, and several cases of X. fastidiosa infection have been reported in other European Countries. At least five different subspecies of X. fastidiosa have been reported and classified: fastidiosa, multiplex, pauca, sandyi, and tashke. A sixth subspecies (morus has been recently proposed. Therefore, it is vital to develop fast and reliable methods that allow the pathogen detection during the very early stages of infection, in order to prevent further spreading of this dangerous bacterium. To this purpose, the classical immunological methods such as ELISA and immunofluorescence are not always sensitive enough. However, PCR-based methods exploiting specific primers for the amplification of target regions of genomic DNA have been developed and are becoming a powerful tool for the detection and identification of many species of bacteria. The aim of this review is to illustrate the application of the most commonly used PCR approaches to X. fastidiosa study, ranging from classical PCR, to several PCR-based detection methods: random amplified polymorphic DNA (RAPD, quantitative real-time PCR (qRT-PCR, nested-PCR (N-PCR, immunocapture PCR (IC-PCR, short sequence repeats (SSRs, also called VNTR, single nucleotide polymorphisms (SNPs and multilocus sequence typing (MLST. Amplification and sequence analysis of

  12. Molecular Typing of Neisseria gonorrhoeae Isolates by Opa-Typing and Ribotyping in New Delhi, India

    Directory of Open Access Journals (Sweden)

    Pejvak Khaki

    2009-01-01

    Full Text Available Control and preventive measures for gonococcal infections are based on precise epidemiological characteristics of N. gonorrhoeae isolates. In the present study the potential utility of opa-typing and ribotyping for molecular epidemiological study of consecutive gonococcal strains was determined. Sixty gonococcal isolates were subjected to ribotyping with two restriction enzymes, AvaII and HincII, and opa-typing with TaqI and HpaII for epidemiological characterization of gonococcal population. Ribotyping with AvaII yielded 6 ribotype patterns while twelve RFLP patterns were observed with HincII. Opa-typing of the 60 isolates revealed a total 54 opa-types, which 48 were unique and 6 formed clusters. Fifty-two opa-types were observed with TaqI-digested PCR product while opa-typing with HpaII demonstrated 54 opa-types. The opa-types from isolates that were epidemiologically unrelated were distinct, whereas those from the sexual contacts were identical. The results showed that opa-typing is highly useful for characterizing gonococcal strains from sexual contacts and has more discriminatory than ribotyping that could differentiate between gonococci of the same ribotype. The technique even with a single restriction enzyme has a high level of discrimination (99.9% between epidemiologically unrelated isolates. In conclusion, the molecular methods such as opa-typing and ribotyping can be used for epidemiological characterization of gonococcal strains.

  13. Molecular characterization of Acanthamoeba strains isolated from domestic dogs in Tenerife, Canary Islands, Spain.

    Science.gov (United States)

    Valladares, María; Reyes-Batlle, María; Martín-Navarro, Carmen M; López-Arencibia, Atteneri; Dorta-Gorrín, Alexis; Wagner, Carolina; Martínez-Carretero, Enrique; Piñero, José E; Valladares, Basilio; Lorenzo-Morales, Jacob

    2015-06-01

    The present study describes two cases of Acanthamoeba infections (keratitis and ascites/peritonitis) in small breed domestic dogs in Tenerife, Canary Islands, Spain. In both cases, amoebic trophozoites were observed under the inverted microscope and isolated from the infected tissues and/or fluids, without detecting the presence of other viral, fungal or bacterial pathogens. Amoebae were isolated using 2 % non-nutrient agar plates and axenified for further biochemical and molecular analyses. Osmotolerance and thermotolerance assays revealed that both isolates were able to grow up to 37 °C and 1 M of mannitol and were thus considered as potentially pathogenic. Moreover, the strains were classified as highly cytotoxic as they cause more than 75 % of toxicity when incubated with two eukaryotic cell lines. In order to classify the strains at the molecular level, the diagnostic fragment 3 (DF3) region of the 18S rDNA of Acanthamoeba was amplified and sequenced, revealing that both isolates belonged to genotype T4. In both cases, owners of the animals did not allow any further studies or follow-up and therefore the current status of these animals is unknown. Furthermore, the isolation of these pathogenic amoebae should raise awareness with the veterinary community locally and worldwide.

  14. Molecular Genetic Analysis of Multi-drug Resistance in Indian Isolates of Mycobacterium tuberculosis

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    Noman Siddiqi

    1998-09-01

    Full Text Available A total of 116 isolates from patients attending the out-patient department at the All India Institute of Medical Sciences, New Delhi and the New Delhi Tuberculosis Centre, New Delhi, India were collected. They were analyzed for resistance to drugs prescribed in the treatment for tuberculosis. The drug resistance was initially determined by microbiological techniques. The Bactec 460TB system was employed to determine the type and level of resistance in each isolate. The isolates were further characterized at molecular level. The multi-drug loci corresponding to rpo b, gyr A, kat G were studied for mutation(s by the polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP technique. The SSCP positive samples were sequenced to characterize the mutations in rpo b, and gyr A loci. While previously reported mutations in the gyr A and rpo b loci were found to be present, several novel mutations were also scored in the rpo b locus. Interestingly, analysis of the gyr A locus showed the presence of point mutation(s that could not be detected by PCR-SSCP. Furthermore, rifampicin resistance was found to be an important marker for checking multi-drug resistance (MDR in clinical isolates of Mycobacterium tuberculosis. This is the first report on molecular genetic analysis of MDR tuberculosis one from India, highlights the increasing incidence of MDR in the Indian isolates of M. tuberculosis.

  15. Molecular Typing of Neisseria gonorrhoeae Isolates by Opa-Typing and Ribotyping in New Delhi, India

    Science.gov (United States)

    Khaki, Pejvak; Bhalla, Preena; Fayaz, Ahmad Mir; Moradi Bidhendi, Sohiela; Esmailzadeh, Majid; Sharma, Pawan

    2009-01-01

    Control and preventive measures for gonococcal infections are based on precise epidemiological characteristics of N. gonorrhoeae isolates. In the present study the potential utility of opa-typing and ribotyping for molecular epidemiological study of consecutive gonococcal strains was determined. Sixty gonococcal isolates were subjected to ribotyping with two restriction enzymes, AvaII and HincII, and opa-typing with TaqI and HpaII for epidemiological characterization of gonococcal population. Ribotyping with AvaII yielded 6 ribotype patterns while twelve RFLP patterns were observed with HincII. Opa-typing of the 60 isolates revealed a total 54 opa-types, which 48 were unique and 6 formed clusters. Fifty-two opa-types were observed with TaqI-digested PCR product while opa-typing with HpaII demonstrated 54 opa-types. The opa-types from isolates that were epidemiologically unrelated were distinct, whereas those from the sexual contacts were identical. The results showed that opa-typing is highly useful for characterizing gonococcal strains from sexual contacts and has more discriminatory than ribotyping that could differentiate between gonococci of the same ribotype. The technique even with a single restriction enzyme has a high level of discrimination (99.9%) between epidemiologically unrelated isolates. In conclusion, the molecular methods such as opa-typing and ribotyping can be used for epidemiological characterization of gonococcal strains. PMID:20016674

  16. Molecular Analysis of Vancomycin-Resistant Enterococci Isolated from Regional Hospitals in Trinidad and Tobago

    Directory of Open Access Journals (Sweden)

    Patrick E. Akpaka

    2016-01-01

    Full Text Available Geographic spread of vancomycin-resistant enterococci (VRE clones in cities, countries, or even continents has been identified by molecular techniques. This study aimed at characterizing virulent genes and determining genetic relatedness of 45 VRE isolates from Trinidad and Tobago using molecular tools, including polymerase chain reaction, pulsed-field gel electrophoresis (PFGE, and Random Amplification Polymorphic DNA (RAPD. The majority (84% of the isolates were Enterococcus faecium possessing vanA gene while the rest (16% were Enterococcus faecalis possessing vanB. The esp gene was found in all 45 VRE isolates while hyl genes were found only in E. faecium species. The E. faecium species expressed five distinct PFGE patterns. The predominant clones with similar or common patterns belonged to clones one and three, and each had 11 (29% of the VRE isolates. Plasmid content was identified in representative isolates from each clonal group. By contrast, the E. faecalis species had one PFGE pattern suggesting the presence of an occult and limited clonal spread. The emergence of VRE in the country seems to be related to intra/interhospital dissemination of an epidemic clone carrying the vanA element. Therefore, infection control measures will be warranted to prevent any potential outbreak and spread of VRE in the country.

  17. Molecular identification and characterization of prohibitin from Echinococcus granulosus.

    Science.gov (United States)

    Zhong, Xiuqin; Song, Xingju; Wang, Ning; Hu, Dandan; Liu, Tinayu; Wang, Tao; Gu, Xiaobin; Lai, Weimin; Peng, Xuerong; Yang, Guangyou

    2016-02-01

    Prohibitin (PHB) is a widely distributed protein that functions as a molecular chaperone, is involved in the regulation of cell cycle, and maintains mitochondrial structure and functions of the anti-apoptosis, senescence, and proliferation. The aim of this study was to characterize PHB in Echinococcus granulosus (EgPHB), a harmful cestode parasite of humans, many livestock species, and wild animals. We found that EgPHB is a conserved SPFH (stomatin, prohibitin, flotillin, and HflK/C) domain-containing protein, consisting of 289 amino acids, which shares 42.66-99.31% identity with PHBs from other parasites and mammals. EgPHB was located mainly in the tegument issue of protoscoleces, in the inner body of adult worms, and was expressed widely in the germinal layer. This is the first report on prohibitin from E. granulosus, and EgPHB is considered to be a valuable protein to study more in the future.

  18. Molecular Identification of Gambierdiscus and Fukuyoa (Dinophyceae from Environmental Samples

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    Kirsty F. Smith

    2017-08-01

    Full Text Available Ciguatera Fish Poisoning (CFP is increasing across the Pacific and the distribution of the causative dinoflagellates appears to be expanding. Subtle differences in thecal plate morphology are used to distinguish dinoflagellate species, which are difficult to determine using light microscopy. For these reasons we sought to develop a Quantitative PCR assay that would detect all species from both Gambierdiscus and Fukuyoa genera in order to rapidly screen environmental samples for potentially toxic species. Additionally, a specific assay for F. paulensis was developed as this species is of concern in New Zealand coastal waters. Using the assays we analyzed 31 samples from three locations around New Zealand and the Kingdom of Tonga. Fourteen samples in total were positive for Gambierdiscus/Fukuyoa and two samples were also positive using the F. paulensis assay. Samples from the Kermadec Islands were further characterized using high-throughput sequencing metabarcoding. The majority of reads corresponded to Gambierdiscus species with three species identified at all sites (G. australes, G. honu and G. polynesiensis. This is the first confirmed identification of G. polynesiensis, a known ciguatoxin producer, in New Zealand waters. Other known toxin-producing genera were also detected, included Alexandrium, Amphidinium, Azadinium, Dinophysis, Ostreopsis, and Prorocentrum.

  19. Identification of a myometrial molecular profile for dystocic labor

    Science.gov (United States)

    2011-01-01

    Background The most common indication for cesarean section (CS) in nulliparous women is dystocia secondary to ineffective myometrial contractility. The aim of this study was to identify a molecular profile in myometrium associated with dystocic labor. Methods Myometrial biopsies were obtained from the upper incisional margins of nulliparous women undergoing lower segment CS for dystocia (n = 4) and control women undergoing CS in the second stage who had demonstrated efficient uterine action during the first stage of labor (n = 4). All patients were in spontaneous (non-induced) labor and had received intrapartum oxytocin to accelerate labor. RNA was extracted from biopsies and hybridized to Affymetrix HuGene U133A Plus 2 microarrays. Internal validation was performed using quantitative SYBR Green Real-Time PCR. Results Seventy genes were differentially expressed between the two groups. 58 genes were down-regulated in the dystocia group. Gene ontology analysis revealed 12 of the 58 down-regulated genes were involved in the immune response. These included (ERAP2, (8.67 fold change (FC)) HLA-DQB1 (7.88 FC) CD28 (2.60 FC), LILRA3 (2.87 FC) and TGFBR3 (2.1 FC)) Hierarchical clustering demonstrated a difference in global gene expression patterns between the samples from dystocic and non-dystocic labours. RT-PCR validation was performed on 4 genes ERAP2, CD28, LILRA3 and TGFBR3 Conclusion These findings suggest an underlying molecular basis for dystocia in nulliparous women in spontaneous labor. Differentially expressed genes suggest an important role for the immune response in dystocic labor and may provide important indicators for new diagnostic assays and potential intrapartum therapeutic targets. PMID:21999197

  20. Identification of a Myometrial Molecular Profile for Dystocic Labor

    LENUS (Irish Health Repository)

    Brennan, Donal J

    2011-10-16

    Abstract Background The most common indication for cesarean section (CS) in nulliparous women is dystocia secondary to ineffective myometrial contractility. The aim of this study was to identify a molecular profile in myometrium associated with dystocic labor. Methods Myometrial biopsies were obtained from the upper incisional margins of nulliparous women undergoing lower segment CS for dystocia (n = 4) and control women undergoing CS in the second stage who had demonstrated efficient uterine action during the first stage of labor (n = 4). All patients were in spontaneous (non-induced) labor and had received intrapartum oxytocin to accelerate labor. RNA was extracted from biopsies and hybridized to Affymetrix HuGene U133A Plus 2 microarrays. Internal validation was performed using quantitative SYBR Green Real-Time PCR. Results Seventy genes were differentially expressed between the two groups. 58 genes were down-regulated in the dystocia group. Gene ontology analysis revealed 12 of the 58 down-regulated genes were involved in the immune response. These included (ERAP2, (8.67 fold change (FC)) HLA-DQB1 (7.88 FC) CD28 (2.60 FC), LILRA3 (2.87 FC) and TGFBR3 (2.1 FC)) Hierarchical clustering demonstrated a difference in global gene expression patterns between the samples from dystocic and non-dystocic labours. RT-PCR validation was performed on 4 genes ERAP2, CD28, LILRA3 and TGFBR3 Conclusion These findings suggest an underlying molecular basis for dystocia in nulliparous women in spontaneous labor. Differentially expressed genes suggest an important role for the immune response in dystocic labor and may provide important indicators for new diagnostic assays and potential intrapartum therapeutic targets.