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.

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 ...

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.

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.

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.

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.

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

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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.

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

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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.

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.

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.

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

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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.

Isolation and identification of Staphylococcus sp. in powdered infant milk

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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.

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

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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

Isolation and identification of Pseudomonas azotoformans for induced calcite precipitation.

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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.

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.

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

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.

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

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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.

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)

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

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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.

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)

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

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

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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.

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

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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

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

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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.

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.

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.

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

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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

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

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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

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

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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

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

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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.

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

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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.

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

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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.

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

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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.

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

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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,...

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

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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.

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

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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.

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

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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

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

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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.

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...

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

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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.

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

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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.

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

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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.

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.

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.

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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.

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

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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.

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.

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

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

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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.

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.

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

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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.

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

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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.

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

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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%.

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.

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

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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.

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.

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

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

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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.

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

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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.

Molecular DNA Analysis in Forensic Identification.

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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.

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

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

Identification of clinically relevant Corynebacterium strains by Api Coryne, MALDI-TOF-mass spectrometry and molecular approaches.

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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.

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.

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...

Molecular characterization of lactobacilli isolated from fermented idli batter

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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.

Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates.

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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.

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).

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

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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.

ISOLATION AND IDENTIFICATION OF FUNGI RESPONSIBLE FOR ...

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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 ...

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.

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

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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

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

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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.

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

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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.

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

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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.

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 ...

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

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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.

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

Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

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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.

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.

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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.

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.

Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry.

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Lagrain, Bert; Brunnbauer, Markus; Rombouts, Ine; Koehler, Peter

2013-01-01

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for

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

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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.

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

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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.

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

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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.

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

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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.

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

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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.

Isolation and Identification of the Chitinolytic Bacteria from Rumen Ecosystem

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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

Testing of DNA isolation for the identification of hemp

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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.

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

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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.

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

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 ...

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

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

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

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.

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.

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

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

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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.

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.

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.

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

Molecular identification of Coccidioides spp. in soil samples from Brazil

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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

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 ...

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.

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

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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.

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

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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.

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

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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.

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.

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.

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.

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

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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

[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.

Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry.

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Bert Lagrain

Full Text Available The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS, the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC, and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%, the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and

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.

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 ...

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.

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.

Identification of mucorales from clinical specimens: a 4-year experience in a single institution.

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Yang, Mina; Lee, Jang Ho; Kim, Young Kwon; Ki, Chang Seok; Huh, Hee Jae; Lee, Nam Yong

2016-01-01

Mucormycosis, a fatal opportunistic infection in immunocompromised hosts, is caused by fungi belonging to the order Mucorales. Early diagnosis based on exact identification and multidisciplinary treatments is critical. However, identification of Mucorales fungi is difficult and often delayed, resulting in poor prognosis. This study aimed to compare the results of phenotypic and molecular identification of 12 Mucorales isolates collected from 4-yr-accumulated data. All isolates were identified on the basis of phenotypic characteristics such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing were performed to target internal transcribed spacer (ITS) and/or D1/D2 regions. Target DNA sequencing identified five Lichtheimia isolates, two Rhizopus microsporus isolates, two Rhizomucor pusillus isolates, one Cunninghamella bertholletiae isolate, one Mucor fragilis isolate, and one Syncephalastrum racemosum isolate. Five of the 12 (41.7%) isolates were incorrectly identified on the basis of phenotypic identification. DNA sequencing showed that of these five isolates, two were Lichtheimia isolates, one was Mucor isolate, one was Rhizomucor isolate, and one was Rhizopus microspores. All the isolates were identified at the species level by ITS and/or D1/D2 analyses. Phenotypic differentiation and identification of Mucorales is difficult because different Mucorales share similar morphology. Our results indicate that the molecular methods employed in this study are valuable for identifying Mucorales.

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.

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 ...

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...

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

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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

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)

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

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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.

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

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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.

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

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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.

Molecular analysis of clinical isolates previously diagnosed as Mycobacterium intracellulare reveals incidental findings of "Mycobacterium indicus pranii" genotypes in human lung infection.

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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".

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.

Spa typing and identification of pvl genes of meticillin-resistant Staphylococcus aureus isolated from a Libyan hospital in Tripoli.

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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.

Identification of some human pathogenic fungi using four DNA ...

African Journals Online (AJOL)

Stocks from pathogenic fungi isolated from infected areas on different patients, around Lagos-Nigeria were analysed using molecular methods (DNA extraction, PCR-RFLP and DNA sequencing). Four DNA extraction protocols were employed in the identification of the fungal isolates. Sixteen different fungal isolates were ...

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

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

Molecular characterization of Cryptosporidium isolates from humans in Equatorial Guinea.

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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.

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.

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

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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…

Evaluation of the Vitek 2 ANC card for identification of clinical isolates of anaerobic bacteria.

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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).

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

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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.

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

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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.

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

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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.

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-...

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 ...

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.

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.

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

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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.

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

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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.

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

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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 �

Molecular identification of Lodoicea maldivica (coco de mer seeds

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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.

Molecular Characterization of Clostridium difficile Isolates in China From 2010 to 2015

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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.

A rapid PCR-based approach for molecular identification of filamentous fungi.

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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.

Molecular identification of Nocardia species using the sodA gene

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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.

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 ...

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 ...

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

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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.

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

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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.

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

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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.

Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

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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

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

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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.

Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

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Seng, Piseth; Abat, Cedric; Rolain, Jean Marc; Colson, Philippe; Lagier, Jean-Christophe; Gouriet, Frédérique; Fournier, Pierre Edouard; Drancourt, Michel; La Scola, Bernard; Raoult, Didier

2013-07-01

During the past 5 years, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.

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

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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

9 CFR 130.15 - User fees for veterinary diagnostic isolation and identification tests performed at NVSL...

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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...

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...

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

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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.

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.

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

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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.

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

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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.

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.

[Principles for molecular identification of traditional Chinese materia medica using DNA barcoding].

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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.

Identification and antimicrobial susceptibility patterns of Staphylococcus spp. isolated from canine chronic otitis externa

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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.

Rapid molecular identification of human taeniid cestodes by pyrosequencing approach.

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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.

Performance Assessment of the CapitalBio Mycobacterium Identification Array System for Identification of Mycobacteria

Science.gov (United States)

Liu, Jingbo; Yan, Zihe; Han, Min; Han, Zhijun; Jin, Lingjie; Zhao, Yanlin

2012-01-01

The CapitalBio Mycobacterium identification microarray system is a rapid system for the detection of Mycobacterium tuberculosis. The performance of this system was assessed with 24 reference strains, 486 Mycobacterium tuberculosis clinical isolates, and 40 clinical samples and then compared to the “gold standard” of DNA sequencing. The CapitalBio Mycobacterium identification microarray system showed highly concordant identification results of 100% and 98.4% for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM), respectively. The sensitivity and specificity of the CapitalBio Mycobacterium identification array for identification of Mycobacterium tuberculosis isolates were 99.6% and 100%, respectively, for direct detection and identification of clinical samples, and the overall sensitivity was 52.5%. It was 100% for sputum, 16.7% for pleural fluid, and 10% for bronchoalveolar lavage fluid, respectively. The total assay was completed in 6 h, including DNA extraction, PCR, and hybridization. The results of this study confirm the utility of this system for the rapid identification of mycobacteria and suggest that the CapitalBio Mycobacterium identification array is a molecular diagnostic technique with high sensitivity and specificity that has the capacity to quickly identify most mycobacteria. PMID:22090408

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.

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....

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.

Identification of Candida species isolated from vulvovaginitis in Mashhad, Iran by Use of MALDI-TOF MS

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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.

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.

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

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

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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.

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.

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 ...

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.

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.

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.

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

Molecular characterization and susceptibility to antimicrobial drugs of isolated bacterials from shrimps (“Litopenaeus vannamei” Caracterização molecular e susceptibilidade aos antimicrobianos de isolados bacterianos de camarões

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Ricardo Castelo Branco Albinati

2010-06-01

Full Text Available The objective was to isolate bacteria from gut of shrimps from Litopenaeus vannamei, by biochemical characterization and molecular identification, inhibition activity in vitro of Bacillus cereus and sensitivity pattern determination. The bacterial species isolated were: Aeromonas caviae (n = 7, Alcaligenes denitrificans (n = 1, Bacillus cereus (n = 1 and Enterobacter spp. (n = 3. Bacillus cereus isolated in this study did not have inhibitory activity to other shrimps isolated bacteria evaluated. In the susceptibility to antimicrobial drug test, it were observed 68,7% to erythromycin, 50% to tetracycline, 81,2% to trimethoprim:sulfamethoxazole, neomycin and estreptomycin, 12,5% to lincomycin and ampicillin, 87,5% to enrofloxacin and nitrofurantoin, 93,7% to ceftriaxone, 100% to norfloxacin and nalidix acid. The characterization molecular is important on identifying the microrganisms studied. The nalidixic acid and norfloxacin are antimicrobial drugs with high sensitivity for bacteria isolated from shrimps.Objetivou-se isolar bactérias provenientes do trato intestinal de camarões da espécie Litopenaeus vannamei, por meio da caracterização bioquímica e molecular, atividade de inibição in vitro do Bacillus cereus e perfil de sensibilidade aos antimicrobianos. As espécies bacterianas identificadas foram Aeromonas caviae (n = 7, Alcaligenes denitrificans (n = 1, Bacillus cereus (n = 1 e Enterobacter spp. (n = 3. Bacillus cereus obtido neste estudo não apresentou atividade de inibição frente às demais bactérias isoladas de camarões. Quanto ao perfil de sensibilidade aos antimicrobianos, foram observados 68,7% de eritromicina, 50% de tetraciclina, 81,2% de sulfametoxazol/trimetoprina, neomicina e estreptomicina, 12,5% de lincomicina e ampicilina, 87,5% de enrofloxacina e nitrofurantoína, 93,7% de ceftriaxona, 100% de norfloxacina e ácido nalidíxico. A caracterização molecular é útil para identificação dos microrganismos estudados

[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.

Genetic variability within n-rDNA region of ectomycorrhizal isolates ...

African Journals Online (AJOL)

In view of this, this study aimed at confirming the earlier identification of these fungi, which was based on morphological characters, and also to find suitable molecular restriction fragment length polymorphism (RFLP) markers for the identification of ECM fungi up to the level of species or isolates as part of an expandable ...

Identification of genus Acinetobacter: Standardization of in-house PCR and its comparison with conventional phenotypic methods.

Science.gov (United States)

Kulkarni, Sughosh S; Madalgi, Radhika; Ajantha, Ganavalli S; Kulkarni, Raghavendra D

2017-01-01

Acinetobacter is grouped under nonfermenting Gram-negative bacilli. It is increasingly isolated from pathological samples. The ability of this genus to acquire drug resistance and spread in the hospital settings is posing a grave problem in healthcare. Specific treatment protocols are advocated for Acinetobacter infections. Hence, rapid identification and drug susceptibility profiling are critical in the management of these infections. To standardize an in-house polymerase chain reaction (PCR) for identification of genus Acinetobacter and to compare PCR with two protocols for its phenotypic identification. A total of 96 clinical isolates of Acinetobacter were included in the study. An in-house PCR for genus level identification of Acinetobacter was standardized. All the isolates were phenotypically identified by two protocols. The results of PCR and phenotypic identification protocols were compared. The in-house PCR standardized was highly sensitive and specific for the genus Acinetobacter . There was 100% agreement between the phenotypic and molecular identification of the genus. The preliminary identification tests routinely used in clinical laboratories were also in complete agreement with phenotypic and molecular identification. The in-house PCR for genus level identification is specific and sensitive. However, it may not be essential for routine identification as the preliminary phenotypic identification tests used in the clinical laboratory reliably identify the genus Acinetobacter .

[Evaluation of common commercial systems for the identification of yeast isolates in microbiology laboratories: a multicenter study].

Science.gov (United States)

Karabıçak, Nilgün; Uludağ Altun, Hatice; Karatuna, Onur; Hazırolan, Gülşen; Aksu, Neriman; Adiloğlu, Ali; Akyar, Işın

2015-04-01

Accurate and rapid identification of yeast isolates have become important in recent years for not only antifungal susceptibility testing due to the species-specific clinical resistance breakpoints but also early initiation of appropriate antifungal therapy. In clinical microbiology laboratories species identification of yeasts is often performed with several commercial systems based on biochemical properties and rarely according to the physiological and morphological characteristics. The aim of this study was to compare the two common commercial systems, VITEK 2 YST ID Card (Vitek; bioMérieux, France) and API 20C AUX (API; bioMérieux, France) with conventional mycological methods. A total of 473 clinical yeast strains isolated from clinical specimens in different university and training/research hospitals and identified by Vitek system were included in the study. The isolates were re-identified with API and conventional methods including morphological identification in the Mycology Reference Laboratory of the Public Health Institute of Turkey. Candida dubliniensis MYA 583, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019, Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 32268 were used as quality control strains and those standard strains were studied consecutively 10 days with both of the methods. The results of identification by Vitek and API were compared with the results of conventional methods for those 473 yeast isolates [6 genus (Candida, Cryptococcus, Blastoshizomyces, Rhodotorula, Saccharomyces, Trichosporon), 17 species (5 common and 12 rarely isolated)]. The performances of the systems were better (Vitek: 95%; API: 96%) for the commonly detected species (C.albicans, C.parapsilosis, C.glabrata, C.tropicalis and C.krusei) than those for rarely detected species (Vitek: 78.4%; API: 71.6%) (p= 0.155). Misidentification or unidentification were mostly detected for C.parapsilosis (Vitek: 6/87; API: 7/87) and C.glabrata (Vitek: 9/104; API

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

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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.

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

Molecular and Pathotype Identification of Potato Cyst Nematodes

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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

Isolation and identification of mold and yeast in medombae, a rice wine starter culture from Kompong Cham Province, Cambodia

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Chay, C.,

2017-07-01

Full Text Available Medombae is a dried starter culture used for traditional rice wine processing in Cambodia. However, studies on the role of mold and yeast present and their efficacy for rice wine fermentation are still limited. Cultural and morphological tests revealed that the isolated representative mold strains were isolated based on the method of identification used as Mucor spp and Rhizopus oryzae. On the other hand, the biochemical properties of the first yeast isolate using the Vitek 2 identification system and YST Card identification suggests its identity as Candida tropicalis. The second yeast strain examined for its morphological and cultural characteristic using agar slide technique, and its protein profile which was compared to the reference and sample protein masses using Biomerieux Vitek MS (MALD-TOF showed the presence of Saccharomyces cerevisiae. The biochemical characteristics and cellular characteristics of the third yeast isolate as described by Lodder (1970 and Kreger-Van Rij (1984 confirmed its identity as Saccharomycopsis spp. The DNA test of identification of the isolates should be conducted to further confirm the identity of the isolates.

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.

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.

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.

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.

Isolation and identification of marine fish tumour (odontoma associated bacteria

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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.

Isolation & characterization of Brucella melitensis isolated from patients suspected for human brucellosis in India

Science.gov (United States)

Barua, Anita; Kumar, Ashu; Thavaselvam, Duraipandian; Mangalgi, Smita; Prakash, Archana; Tiwari, Sapana; Arora, Sonia; Sathyaseelan, Kannusamy

2016-01-01

Background & objectives: Brucellosis is endemic in the southern part of India. A combination of biochemical, serological and molecular methods is required for identification and biotyping of Brucella. The present study describes the isolation and biochemical, molecular characterization of Brucella melitensis from patients suspected for human brucellosis. Methods: The blood samples were collected from febrile patients suspected to have brucellosis. A total of 18 isolates were obtained from 102 blood samples subjected to culture. The characterization of these 18 isolates was done by growth on Brucella specific medium, biochemical reactions, CO2 requirement, H2S production, agglutination with A and M mono-specific antiserum, dye sensitivity to basic fuchsin and thionin. Further, molecular characterization of the isolates was done by amplification of B. melitensis species specific IS711 repetitive DNA fragment and 16S (rRNA) sequence analysis. PCR-restriction fragment length polymorphism (RFLP) analysis of omp2 locus and IS711 gene was also done for molecular characterization. Results: All 102 suspected samples were subjected to bacteria isolation and of these, 18 isolates could be recovered on blood culture. The biochemical, PCR and PCR-RFLP and 16s rRNA sequencing revealed that all isolates were of B. melitensis and matched exactly with reference strain B. melitensis 16M. Interpretation & conclusions: The present study showed an overall isolation rate of 17.64 per cent for B. melitensis. There is a need to establish facilities for isolation and characterization of Brucella species for effective clinical management of the disease among patients as well as surveillance and control of infection in domestic animals. Further studies are needed from different geographical areas of the country with different level of endemicity to plan and execute control strategies against human brucellosis. PMID:27488010

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 .

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.

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

Identification of new IS711 insertion sites in Brucella abortus field isolates

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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.

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.

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)

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.

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...

Identification of Candida species isolated from vulvovaginitis using matrix assisted laser desorption ionization-time of flight mass spectrometry.

Science.gov (United States)

Alizadeh, Majid; Kolecka, Anna; Boekhout, Teun; Zarrinfar, Hossein; Ghanbari Nahzag, Mohamad A; Badiee, Parisa; Rezaei-Matehkolaei, Ali; Fata, Abdolmajid; Dolatabadi, Somayeh; Najafzadeh, Mohammad J

2017-12-01

Vulvovaginal candidiasis (VVC) is a common problem in women. The purpose of this study was to identify Candida isolates by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) from women with vulvovaginitis that were referred to Ghaem Hospital, Mashhad, Iran. This study was conducted on 65 clinical samples isolated from women that were referred to Ghaem Hospital. All specimens were identified using phenotyping techniques, such as microscopy and culture on Sabouraud dextrose agar and corn meal agar. In addition, all isolates were processed for MALDI-TOF MS identification. Out of the 65 analyzed isolates, 61 (94%) samples were recognized by MALDI-TOF MS. However, the remaining four isolates (6%) had no reliable identification. According to the results, C. albicans (58.5%) was the most frequently isolated species, followed by C. tropicalis (16.9%), C. glabrata (7.7%), C. parapsilosis (7.7%), and guilliermondii (3.1%). As the findings indicated, MALDI TOF MS was successful in the identification of clinical Candida species. C. albicans was identified as the most common Candida species isolated from the women with VVC. Moreover, C. tropicalis was the most common species among the non- albicans Candida species.

Molecular identification of Echinococcus granulosus isolates from ruminants in Greece.

Science.gov (United States)

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.

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 ...

The Identification of Complex Organic Molecules in the Interstellar Medium: Using Lasers and Matrix Isolation Spectroscopy to Simulate the Interstellar Environment

Science.gov (United States)

Stone, Bradley M.

1998-01-01

The Astrochemistry Group at NASA Ames Research Center is interested in the identification of large organic molecules in the interstellar medium Many smaller organic species (e.g. hydrocarbons, alcohols, etc.) have been previously identified by their radiofrequency signature due to molecular rotations. However, this becomes increasingly difficult to observe as the size of the molecule increases. Our group in interested in the identification of the carriers of the Diffuse Interstellar Bands (absorption features observed throughout the visible and near-infrared in the spectra of stars, due to species in the interstellar medium). Polycyclic Aromatic Hydrocarbons (PAHs) and related molecules are thought to be good candidates for these carriers. Laboratory experiments am performed at Ames to simulate the interstellar environment, and to compare spectra obtained from molecules in the laboratory to those derived astronomically. We are also interested in PAHs with respect to their possible connection to the UIR (Unidentified infrared) and ERE (Extended Red Emission) bands - emission features found to emanate from particular regions of our galaxy (e.g. Orion nebula, Red Rectangle, etc.). An old, "tried and proven spectroscopic technique, matrix isolation spectroscopy creates molecular conditions ideal for performing laboratory astrophysics.

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

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 ...

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.

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.

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 ...

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.

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.

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

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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.

Identification, Typing, Antifungal Resistance Profile, and Biofilm Formation of Candida albicans Isolates from Lebanese Hospital Patients

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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.

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...

[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

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.

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 ...

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.

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.

Gender Identification in Date Palm Using Molecular Markers.

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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.

Isolation and identification of aerobic polychlorinated biphenyls degrading bacteria

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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.

Isolation and identification of iron ore-solubilising fungus

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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.

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...

Isolation and identification of acetogenic bacteria obtained from deer rumen and their potential for methanogenesis inhibitor

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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.

Performance of matrix-assisted laser desorption-time of flight mass spectrometry for identification of clinical yeast isolates

DEFF Research Database (Denmark)

Rosenvinge, Flemming S; Dzajic, Esad; Knudsen, Elisa

2013-01-01

Accurate and fast yeast identification is important when treating patients with invasive fungal disease as susceptibility to antifungal agents is highly species related. Matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) provides a powerful tool with a clear potential...... spectra output, all 13 isolates were correctly identified, resulting in an overall identification performance of 92%. No misidentifications occurred with the two systems. Of the routine isolates one laboratory identified 99/99 (100%) and 90/99 (91%) to species level by Saramis/Axima and conventional...... identification, respectively, whereas the other laboratory identified 83/98 (85%) to species level by both BioTyper/Bruker and conventional identification. Both MALDI-TOF-MS systems are fast, have built-in databases that cover the majority of clinically relevant Candida species, and have an accuracy...

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

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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.

Prevalence, identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia.

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Hmaïed, F; Helel, S; Le Berre, V; François, J-M; Leclercq, A; Lecuit, M; Smaoui, H; Kechrid, A; Boudabous, A; Barkallah, I

2014-02-01

We aimed at evaluating the prevalence of Listeria species isolated from food samples and characterizing food and human cases isolates. Between 2005 and 2007, one hundred food samples collected in the markets of Tunis were analysed in our study. Five strains of Listeria monocytogenes responsible for human listeriosis isolated in hospital of Tunis were included. Multiplex PCR serogrouping and pulsed field gel electrophoresis (PFGE) applying the enzyme AscI and ApaI were used for the characterization of isolates of L. monocytogenes. We have developed a rapid microarray-based assay to a reliable discrimination of species within the Listeria genus. The prevalence of Listeria spp. in food samples was estimated at 14% by using classical biochemical identification. Two samples were assigned to L. monocytogenes and 12 to L. innocua. DNA microarray allowed unambiguous identification of Listeria species. Our results obtained by microarray-based assay were in accordance with the biochemical identification. The two food L. monocytogenes isolates were assigned to the PCR serogroup IIa (serovar 1/2a). Whereas human L. monocytogenes isolates were of PCR serogroup IVb, (serovars 4b). These isolates present a high similarity in PFGE. Food L. monocytogenes isolates were classified into two different pulsotypes. These pulsotypes were different from that of the five strains responsible for the human cases. We confirmed the presence of Listeria spp. in variety of food samples in Tunis. Increased food and clinical surveillance must be taken into consideration in Tunisia to identify putative infections sources. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

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.

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

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).

Molecular characterization of acquired enrofloxacin resistance in Mycoplasma synoviae field isolates.

Science.gov (United States)

Lysnyansky, I; Gerchman, I; Mikula, I; Gobbo, F; Catania, S; Levisohn, S

2013-07-01

The in vitro activity of enrofloxacin against 73 Mycoplasma synoviae field strains isolated in Israel and Europe was determined by broth microdilution. Decreased susceptibility to enrofloxacin was identified in 59% of strains, with the MICs ranging from 1 to >16 μg/ml. The estimated MIC50 and MIC90 values for enrofloxacin were 2 and 8 μg/ml, respectively. Moreover, this study showed that 92% of recent Israeli field isolates (2009 to 2011) of M. synoviae have MICs of ≥ 2 μg/ml to enrofloxacin. Comparison of the quinolone resistance-determining regions (QRDRs) in M. synoviae isolates revealed a clear correlation between the presence of one of the amino acid substitutions Asp79-Asn, Thr80-Ala/Ile, Ser81-Pro, and Asp84-Asn/Tyr/His of the ParC QRDR and decreased susceptibility to enrofloxacin (MIC, ≥ 1 μg/ml). Amino acid substitutions at positions GyrA 87, GyrB 401/402, and ParE 420/454 were also identified, but there was no clear-cut correlation with susceptibility to enrofloxacin. Comparison of vlhA molecular profiles revealed the presence of 9 different genotypes in the Israeli M. synoviae field isolates and 10 genotypes in the European isolates; only one vlhA genotype (type 4) was identified in both cohorts. Based on results of vlhA molecular typing, several mechanisms for emergence and dissemination of Israeli enrofloxacin-resistant M. synoviae isolates are suggested.

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

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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.

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...

Molecular identification of the Sporothrix schenckii complex.

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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.

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus

KAUST Repository

Hill-Cawthorne, Grant A.

2014-06-27

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. © 2014 Hill-Cawthorne et al.

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus.

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Grant A Hill-Cawthorne

Full Text Available Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec. We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA, a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4% of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus

KAUST Repository

Hill-Cawthorne, Grant A.; Hudson, Lyndsey O.; Abd El Ghany, Moataz; Piepenburg, Olaf; Nair, Mridul; Dodgson, Andrew; Forrest, Matthew S.; Clark, Taane G.; Pain, Arnab

2014-01-01

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. © 2014 Hill-Cawthorne et al.

Physiological and Molecular Characterization of Cephaleuros virescens Occurring in Mango Trees.

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Vasconcelos, Camila Vilela; Pereira, Fabíola Teodoro; Duarte, Elizabeth Amélia Alves; de Oliveira, Thiago Alves Santos; Peixoto, Nei; Carvalho, Daniel Diego Costa

2018-06-01

The objective of this work was to accomplish the isolation, molecular identification and characterizing the physiology of the causal agent of the algal spot in mango trees. For this purpose, the pathogen growth was assessed in different culture media, with subsequent observation and measurements of the filamentous cells. The molecular identification was made using mycelium obtained from leaf lesions and pure algae colonies grown in culture medium. Descriptions based on DNA sequencing indicated that the algae is Cephaleuros virescens . The algae must be isolated primarily in liquid medium for further pricking into agar medium. The highest mycelial growth average in Petri dishes occurred when the algae were grown in Trebouxia and BBM. Trebouxia enabled larger cells in the filamentous cells when compared to other culture media.

Effects of solid-medium type on routine identification of bacterial isolates by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

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Anderson, Neil W; Buchan, Blake W; Riebe, Katherine M; Parsons, Lauren N; Gnacinski, Stacy; Ledeboer, Nathan A

2012-03-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for the identification of bacteria. Factors that may alter protein profiles, including growth conditions and presence of exogenous substances, could hinder identification. Bacterial isolates identified by conventional methods were grown on various media and identified using the MALDI Biotyper (Bruker Daltonics, Billerica, MA) using a direct smear method and an acid extraction method. Specimens included 23 Pseudomonas isolates grown on blood agar, Pseudocel (CET), and MacConkey agar (MAC); 20 Staphylococcus isolates grown on blood agar, colistin-nalidixic acid agar (CNA), and mannitol salt agar (MSA); and 25 enteric isolates grown on blood agar, xylose lysine deoxycholate agar (XLD), Hektoen enteric agar (HE), salmonella-shigella agar (SS), and MAC. For Pseudomonas spp., the identification rate to genus using the direct method was 83% from blood, 78% from MAC, and 94% from CET. For Staphylococcus isolates, the identification rate to genus using the direct method was 95% from blood, 75% from CNA, and 95% from MSA. For enteric isolates, the identification rate to genus using the direct method was 100% from blood, 100% from MAC, 100% from XLD, 92% from HE, and 87% from SS. Extraction enhanced identification rates. The direct method of MALDI-TOF analysis of bacteria from selective and differential media yields identifications of varied confidence. Notably, Staphylococci spp. from CNA exhibit low identification rates. Extraction enhances identification rates and is recommended for colonies from this medium.

Phenotypic and molecular characterization of antimicrobial resistance and virulence factors in Pseudomonas aeruginosa clinical isolates from Recife, State of Pernambuco, Brazil

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Paula Regina Luna de Araújo Jácome

2012-12-01

Full Text Available INTRODUCTION: The emergence of carbapenem resistance mechanisms in Pseudomonas aeruginosa has been outstanding due to the wide spectrum of antimicrobial degradation of these bacteria, reducing of therapeutic options. METHODS: Sixty-one clinical strains of P. aeruginosa isolated from five public hospitals in Recife, Pernambuco, Brazil, were examined between 2006 and 2010, aiming of evaluating the profiles of virulence, resistance to antimicrobials, presence of metallo-β-lactamase (MBL genes, and clonal relationship among isolates. RESULTS: A high percentage of virulence factors (34.4% mucoid colonies; 70.5% pyocyanin; 93.4% gelatinase positives; and 72.1% hemolysin positive and a high percentage of antimicrobial resistance rates (4.9% pan-resistant and 54.1% multi-drug resistant isolates were observed. Among the 29 isolates resistant to imipenem and/or ceftazidime, 44.8% (13/29 were MBL producers by phenotypic evaluation, and of these, 46.2% (6/13 were positive for the blaSPM-1 gene. The blaIMP and blaVIM genes were not detected. The molecular typing revealed 21 molecular profiles of which seven were detected in distinct hospitals and periods. Among the six positive blaSPM-1 isolates, three presented the same clonal profile and were from the same hospital, whereas the other three presented different clonal profiles. CONCLUSIONS: These results revealed that P. aeruginosa is able to accumulate different resistance and virulence factors, making the treatment of infections difficult. The identification of blaSPM-1 genes and the dissemination of clones in different hospitals, indicate the need for stricter application of infection control measures in hospitals in Recife, Brazil, aiming at reducing costs and damages caused by P. aeruginosa infections.

Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolated in Nepal.

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Poudel, Ajay; Nakajima, Chie; Fukushima, Yukari; Suzuki, Haruka; Pandey, Basu Dev; Maharjan, Bhagwan; Suzuki, Yasuhiko

2012-06-01

Despite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance in Mycobacterium tuberculosis is required. In the present study, we investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 158 M. tuberculosis isolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) of rpoB were identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in the katG gene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in the inhA promoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance in M. tuberculosis in Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.

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.

Rapid and reliable MALDI-TOF mass spectrometry identification of Candida non-albicans isolates from bloodstream infections.

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Pulcrano, Giovanna; Iula, Dora Vita; Vollaro, Antonio; Tucci, Alessandra; Cerullo, Monica; Esposito, Matilde; Rossano, Fabio; Catania, Maria Rosaria

2013-09-01

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has recently become an effective instrument for rapid microbiological diagnostics and in particular for identification of micro-organisms directly in a positive blood culture. The aim of the study was to evaluate a collection of 82 stored yeast isolates from bloodstream infection, by MALDI-TOF MS; 21 isolates were identified also directly from positive blood cultures and in the presence of other co-infecting micro-organisms. Of the 82 isolates grown on plates, 64 (76%) were correctly identified by the Vitek II system and 82 (100%) by MALDI-TOF MS; when the two methods gave different results, the isolate was identified by PCR. MALDI-TOF MS was unreliable in identifying two isolates (Candida glabrata and Candida parapsilosis) directly from blood culture; however, direct analysis from positive blood culture samples was fast and effective for the identification of yeast, which is of great importance for early and adequate treatment. © 2013. Published by Elsevier B.V. All rights reserved.

Molecular characterization of KPC-producing Klebsiella pneumoniae isolated from patients in a Public Hospital in Caracas, Venezuela.

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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.

Isolation and identification of lactic acid bacteria from abalone (Haliotis asinina as a potential candidate of probiotic

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YAYAN SOFYAN

2010-01-01

Full Text Available Sarkono, Faturrahman, Sofyan Y. 2010. Isolation and identification of lactic acid bacteria from abalone (Haliotis asinina as a potential candidate of probiotic. Nusantara Bioscience 2: 38-42. The purpose of this study was to isolate, select and characterize lactic acid bacteria (LAB from abalone as a potential candidate probiotic in abalone cultivation system. Selective isolation of LAB performed using de Man Rogosa Sharpe medium. LAB isolate that potential as probiotics was screened. Selection was based on its ability to suppress the growth of pathogenic bacteria, bacterial resistance to acidic conditions and bacterial resistance to bile salts (bile. Further characterization and identification conducted to determine the species. The results showed that two of the ten isolates potential to be developed as probiotic bacteria that have the ability to inhibit several pathogenic bacteria such as Eschericia coli, Bacillus cereus dan Staphylococus aureus, able to grow at acidic condition and bile tolerance during the incubation for 24 hour. Based on the API test kit, the both of isolate identified as members of the species Lactobacillus paracasei ssp. paracasei.

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.

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

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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

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

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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

Identification of Microorganisms by Modern Analytical Techniques.

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Buszewski, Bogusław; Rogowska, Agnieszka; Pomastowski, Paweł; Złoch, Michał; Railean-Plugaru, Viorica

2017-11-01

Rapid detection and identification of microorganisms is a challenging and important aspect in a wide range of fields, from medical to industrial, affecting human lives. Unfortunately, classical methods of microorganism identification are based on time-consuming and labor-intensive approaches. Screening techniques require the rapid and cheap grouping of bacterial isolates; however, modern bioanalytics demand comprehensive bacterial studies at a molecular level. Modern approaches for the rapid identification of bacteria use molecular techniques, such as 16S ribosomal RNA gene sequencing based on polymerase chain reaction or electromigration, especially capillary zone electrophoresis and capillary isoelectric focusing. However, there are still several challenges with the analysis of microbial complexes using electromigration technology, such as uncontrolled aggregation and/or adhesion to the capillary surface. Thus, an approach using capillary electrophoresis of microbial aggregates with UV and matrix-assisted laser desorption ionization time-of-flight MS detection is presented.

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.

A rapid, one step molecular identification of Trichoderma citrinoviride and Trichoderma reesei.

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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.

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

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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.

Identification of clinical yeasts by Vitek MS system compared with API ID 32 C.

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Durán-Valle, M Teresa; Sanz-Rodríguez, Nuria; Muñoz-Paraíso, Carmen; Almagro-Moltó, María; Gómez-Garcés, José Luis

2014-05-01

We performed a clinical evaluation of the Vitek MS matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) system with the commercial database version 2.0 for rapid identification of medically important yeasts as compared with the conventional phenotypic method API ID 32 C. We tested 161 clinical isolates, nine isolates from culture collections and five reference strains. In case of discrepant results or no identification with one or both methods, molecular identification techniques were employed. Concordance between both methods was observed with 160/175 isolates (91.42%) and misidentifications by both systems occurred only when taxa were not included in the respective databases, i.e., one isolate of Candida etchellsii was identified as C. globosa by Vitek MS and two isolates of C. orthopsilosis were identified as C. parapsilosis by API ID 32 C. Vitek MS could not identify nine strains (5.14%) and API ID 32 C did not identify 13 (7.42%). Vitek MS was more reliable than API ID 32 C and reduced the time required for the identification of clinical isolates to only a few minutes.

16S-ARDRA and MALDI-TOF mass spectrometry as tools for identification of Lactobacillus bacteria isolated from poultry.

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Dec, Marta; Puchalski, Andrzej; Urban-Chmiel, Renata; Wernicki, Andrzej

2016-06-13

The objective of our study is to evaluate the potential use of Amplified 16S Ribosomal DNA Restriction Analysis (16S-ARDRA) and MALDI-TOF mass spectrometry (MS) as methods for species identification of Lactobacillus strains in poultry. A total of 80 Lactobacillus strains isolated from the cloaca of chicken, geese and turkeys were identified to the species level by MALDI-TOF MS (on-plate extraction method) and 16S-ARDRA. The two techniques produced comparable classification results, some of which were additionally confirmed by sequencing of 16S rDNA. MALDI-TOF MS enabled rapid species identification but produced more than one reliable identification result for 16.25 % of examined strains (mainly of the species L. johnsonii). For 30 % of isolates intermediate log(scores) of 1.70-1.99 were obtained, indicating correct genus identification but only presumptive species identification. The 16S-ARDRA protocol was based on digestion of 16S rDNA with the restriction enzymes MseI, HinfI, MboI and AluI. This technique was able to distinguish 17 of the 19 Lactobacillus reference species tested and enabled identification of all 80 wild isolates. L. salivarius dominated among the 15 recognized species, followed by L. johnsonii and L. ingluviei. The MALDI-TOF MS and 16S-ARDRA assays are valuable tools for the identification of avian lactobacilli to the species level. MALDI-TOF MS is a fast, simple and cost-effective technique, and despite generating a high percentage of results with a log(score) Lactobacillus bacteria from different habitats.

Rapid identification of Enterobacter hormaechei and Enterobacter cloacae genetic cluster III.

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Ohad, S; Block, C; Kravitz, V; Farber, A; Pilo, S; Breuer, R; Rorman, E

2014-05-01

Enterobacter cloacae complex bacteria are of both clinical and environmental importance. Phenotypic methods are unable to distinguish between some of the species in this complex, which often renders their identification incomplete. The goal of this study was to develop molecular assays to identify Enterobacter hormaechei and Ent. cloacae genetic cluster III which are relatively frequently encountered in clinical material. The molecular assays developed in this study are qPCR technology based and served to identify both Ent. hormaechei and Ent. cloacae genetic cluster III. qPCR results were compared to hsp60 sequence analysis. Most clinical isolates were assigned to Ent. hormaechei subsp. steigerwaltii and Ent. cloacae genetic cluster III. The latter was proportionately more frequently isolated from bloodstream infections than from other material (P < 0·05). The qPCR assays detecting Ent. hormaechei and Ent. cloacae genetic cluster III demonstrated high sensitivity and specificity. The presented qPCR assays allow accurate and rapid identification of clinical isolates of the Ent. cloacae complex. The improved identifications obtained can specifically assist analysis of Ent. hormaechei and Ent. cloacae genetic cluster III in nosocomial outbreaks and can promote rapid environmental monitoring. An association was observed between Ent. cloacae cluster III and systemic infection that deserves further attention. © 2014 The Society for Applied Microbiology.

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.

Molecular identification and phylogenetic study of Demodex caprae.

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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.

Control of electron localization to isolate and enhance molecular harmonic plateau in asymmetric HeH2+ system

International Nuclear Information System (INIS)

Lu, Ruifeng; Yu, Chao; Wang, Yunhui; Shi, Qi; Zhang, Yadong

2014-01-01

High-order harmonic generation from the asymmetric molecular ion HeH 2+ exposed to intense laser fields was investigated by quantum wave packet calculations in which the initial wave packet of HeH 2+ was prepared in the first excited 2pσ state. The calculated molecular harmonic plateau at low frequencies was effectively isolated and enhanced by adjusting the carrier-envelope phase (CEP) of the laser field. Furthermore, double-well model, time-dependent electronic density, electronic state population, and time-frequency analyses were presented to explain the underlying mechanism of the efficient isolated molecular plateau. By taking advantage of the CEP effect to control the electronic dynamics, this isolated molecular plateau can be used to generate high-intensity single attosecond pulses.

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

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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.

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

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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.

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

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.

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...

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.

Analysis of the epidemiological dynamics during the 1982-1983 epidemic of foot-and-mouth disease in Denmark based on molecular high-resolution strain identification

DEFF Research Database (Denmark)

Christensen, Laurids Siig; Normann, Preben; Thykier-Nielsen, Søren

2005-01-01

An epidemic of foot-and-mouth disease (FMD) causing a total of 23 cases in 1982-1983, primarily on the island of Funen, Denmark, was subjected to molecular epidemiological investigations. In an attempt to exploit the quasi-species nature of foot-and-mouth disease virus strains for molecular high......-resolution strain identification in order to analyse the dynamics of this epidemic, full-length VP1 coding regions were sequenced for 17 isolates collected at different farms during the epidemic. The sequence information together with epidemiological information gathered during the epidemic suggests......, and the prerequisite of co- or superinfection of animals with variant strains in turn implies that they have a common source or epidemiologically related sources originating from an area with endemic FMD....

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.

Molecular Detection and Characterization of Goat Isolate of Taenia hydatigena in Turkey

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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.

Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase and molecular sequencing of selected strains

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Mauro Cintra Giudice

2012-09-01

Full Text Available A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum. The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

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)

Molecular characterisation of Mycobacterium bovis isolated from African buffaloes (Syncerus caffer in Hluhluwe-iMfolozi Park in KwaZulu-Natal, South Africa

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Tiny M. Hlokwe

2011-06-01

Full Text Available Bovine tuberculosis (BTB, a chronic disease of mammals caused by Mycobacterium bovis, is a threat to South African wildlife. It has been reported that African buffaloes (Syncerus caffer are reservoir hosts of BTB in South African wildlife populations. This study reports on the molecular identification and typing of 31 M. bovis isolates collected between 1993 and 2008, mainly from buffaloes but also from two lions and a bush pig, in the Hluhluwe-iMfolozi Park (HiP in KwaZulu-Natal. To study the dynamics of BTB in the buffalo populations, 28 M. bovis isolates from the HiP and epidemiologically related parks were characterised using regions of difference deletion analysis for species identification and spoligotyping, variable number of tandem repeats (VNTR, polymorphic G–C-rich sequences and IS6110 restriction fragment length polymorphism (RFLP genotyping methods. At least three distinct M. bovis genotypes were found amongst HiP samples. The combination of VNTR typing (using a 16-loci panel and IS6110 RFLP revealed the presence of three additional genetic profiles in individual buffaloes, demonstrating that the highest level of discrimination was achieved by these typing methods. One of the observed spoligotypes (SB0130 was dominant and represented 75% of isolates from buffaloes. A novel M. bovis spoligotype (SB1474, which is reported for the first time in this study, was observed in 14.3% of isolates from buffaloes. Based on the observed genetic relationships, the findings suggest independent introductions from at least three unrelated sources. These findings improve the knowledge regarding the diversity of circulating M. bovis strains in the HiP.

Exploring MALDI-TOF MS approach for a rapid identification of Mycobacterium avium ssp. paratuberculosis field isolates.

Science.gov (United States)

Ricchi, M; Mazzarelli, A; Piscini, A; Di Caro, A; Cannas, A; Leo, S; Russo, S; Arrigoni, N

2017-03-01

The aim of the study was to explore the suitability of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and correct identification of Mycobacterium avium ssp. paratuberculosis (MAP) field isolates. MALDI-TOF MS approach is becoming one of the most popular tests for the identification of intact bacterial cells which has been shown to be fast and reliable. For this purpose, 36 MAP field isolates were analysed through MALDI-TOF MS and the spectra compared with two different databases: one provided by the vendor of the system employed (Biotyper ver. 3·0; Bruker Daltonics) and a homemade database containing spectra from both tuberculous and nontuberculous Mycobacteria. Moreover, principal component analysis procedure was employed to confirm the ability of MALDI-TOF MS to discriminate between very closely related subspecies. Our results suggest MAP can be differentiated from other Mycobacterium species, both when the species are very close (M. intracellulare) and when belonging to different subspecies (M. avium ssp. avium and M. avium ssp. silvaticum). The procedure applied is fast, easy to perform, and achieves an earlier accurate species identification of MAP and nontuberculous Mycobacteria in comparison to other procedures. The gold standard test for the diagnosis of paratuberculosis is still isolation of MAP by cultural methods, but additional assays, such as qPCR and subculturing for determination of mycobactin dependency are required to confirm its identification. We have provided here evidence pertaining to the usefulness of MALDI-TOF MS approach for a rapid identification of this mycobacterium among other members of M. avium complex. © 2016 The Society for Applied Microbiology.

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.

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).

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.

Identification and characterization of pathogenic Pestalotiopsis species to pecan tree in Brazil

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Marília Lazarotto

2014-06-01

Full Text Available The objective of this work was to characterize and cluster isolates of Pestalotiopsis species and to identify those that are pathogenic to pecan, based on morphological and molecular characters. Pestalotiopsis spp. isolates were identified by sequencing the internal transcribed spacer (ITS and β?tubulin regions. Identification methods were compared to indicate the key morphological characters for species characterization. Thirteen isolates were used for the pathogenicity tests. Morphological characterization was performed using the following variables: mycelial growth rate, sporulation, colony pigmentation, and conidial length and width. Ten pathogenic isolates were identified, three as -tubulin regions. Identification methods were compared to indicate the key morphological characters for species characterization. Thirteen isolates were used for the pathogenicity tests. Morphological characterization was performed using the following variables: mycelial growth rate, sporulation, colony pigmentation, and conidial length and width. Ten pathogenic isolates were identified, three as Pestalotiopsis clavispora and three as P. cocculi. The other isolates remained as an undefined species. The morphological characters were efficient for an initial separation of the isolates, which were grouped according to differences at species level, mainly colony diameter, which was identified as an important morphological describer. Beta-tubulin gene sequencing was less informative than the ITS region sequencing for species identification.

Detection, molecular typing and phylogenetic analysis of Leishmania isolated from cases of leishmaniasis among Syrian refugees in Lebanon

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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.

Molecular identification of Nocardia species using the sodA gene: Identificación molecular de especies de Nocardia utilizando el gen sodA.

Science.gov (United States)

Sánchez-Herrera, K; Sandoval, H; Mouniee, D; Ramírez-Durán, N; Bergeron, E; Boiron, P; Sánchez-Saucedo, N; Rodríguez-Nava, V

2017-09-01

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 sod A gene (encoding the enzyme superoxide dismutase) has had good results in identifying species of other Actinomycetes. In this study the sod A 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 sod A gene was amplified and sequenced, and a phylogenetic analysis was performed comparing the genes rrs (1171 bp), hsp 65 (401 bp), sec A1 (494 bp), gyr B (1195 bp) and rpo B (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 sod A 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 sod A 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.

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.

Isolation and identification of phytoestrogens and flavonoids in an Ayurvedic proprietary medicine using chromatographic and Mass Spectroscopic analysis

Institute of Scientific and Technical Information of China (English)

Sulaiman CT; Arun A; Anandan EM; Sandhya CR; Indira Balachandran

2015-01-01

Objective: To develop analytical methods for the isolation and structural identification of poly phenols including phytoestrogens in Mensokot tablet, a herbal proprietary medicine. Methods:Isolation consisted of an ultrasound-assisted extraction, followed by acid hydrolysis and a final liquid-liquid extraction step in diethyl ether. Identification and structural characterisation was done by liquid chromatography coupled with Q-TOF-ESI-MS/MS analysis. Results:Phytoestrogens such as Coumestrol, Genistein and Glycitein have been identified in Mensokot tablet along with several other flavonoids. Conclusion: In the present research, a rapid HPLC-MS/MS method has been developed for the identification of phytoestrogens and other flavonoids from an Ayurvedic proprietary medicine. Phytoestrogens are considered to play an important role in the prevention of cancers, heart disease, menopausal symptoms and osteoporosis.

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.

Morphological and molecular identification of potato cyst nematode populations in Serbia

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Oro Violeta

2010-01-01

Full Text Available Quarantine species such as potato cyst nematodes Globodera rostochiensis and G. pallida are present in Serbia since 1999 and 2005, respectively. These nematodes are sibling species and their morphological identification is complex due to their morphometric overlap. The cysts from the localities of Kladnica, Šanac, Gojna Gora and Milatovići were grown on susceptible potato varieties and their morphological differences have been discussed. To avoid ambiguities in species morphological designation a duplex PCR method was chosen for a rapid and accurate species identification. The whole procedure, from DNA extraction to DNA isolation, can be performed in a single day. .

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.

Use of matrix assisted laser desorption ionisation-time of flight mass spectrometry in a paediatric clinical laboratory for identification of bacteria commonly isolated from cystic fibrosis patients.

Science.gov (United States)

Desai, Ankita Patel; Stanley, Theresa; Atuan, Maria; McKey, Jonelle; Lipuma, John J; Rogers, Beverly; Jerris, Robert

2012-09-01

Matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) has been described as a rapid, accurate method for bacterial identification. To investigate the ability of the technique, using the unamended database supplied with the system, to identify bacteria commonly isolated in cystic fibrosis (CF) patients. Organisms commonly isolated from CF patients identified by MALDI-TOF MS were compared to conventional phenotypic and genotypic analyses. For MALDI-TOF MS, the direct colony technique was used routinely with one extraction procedure performed on a mucoid Pseudomonas aeruginosa. For 24 unique CF specimens, workload comparison and time to identification were assessed. Of 464 tested isolates, conventional (phenotypic and genotypic) identification compared to MALDI-TOF MS showed complete genus, species agreement in 92%, with genus agreement in 98%. This included 29 isolates within the Burkholderia cepacia complex. All 29 were correctly identified to the genus level and 24 of these were speciated. Time to identification with 47 bacterial isolates from 24 CF patients showed identification of 85% of isolates by MALDI-TOF MS at 48 h of incubation, compared to only 34% with conventional methods. Using the unamended database supplied with the system, MALDI-TOF MS provides rapid and reliable identification of bacteria isolated from CF specimens. Time to identification studies showed that the use of same day, same method for organism identification will decrease time to result and optimise microbiology workflow.

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.

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.

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.

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.

Molecular typing of mycobacterium tuberculosis isolates circulating in Jiangsu Province, China

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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

Application of MALDI-TOF MS fingerprinting as a quick tool for identification and clustering of foodborne pathogens isolated from food products.

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Elbehiry, Ayman; Marzouk, Eman; Hamada, Mohamed; Al-Dubaib, Musaad; Alyamani, Essam; Moussa, Ihab M; AlRowaidhan, Anhar; Hemeg, Hassan A

2017-10-01

Foodborne pathogens can be associated with a wide variety of food products and it is very important to identify them to supply safe food and prevent foodborne infections. Since traditional techniques are timeconsuming and laborious, this study was designed for rapid identification and clustering of foodborne pathogens isolated from various restaurants in Al-Qassim region, Kingdom of Saudi Arabia (KSA) using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Sixty-nine bacterial and thirty-two fungal isolates isolated from 80 food samples were used in this study. Preliminary identification was carried out through culture and BD Phoenix™ methods. A confirmatory identification technique was then performed using MALDI-TOF MS. The BD Phoenix results revealed that 97% (67/69 isolates) of bacteria were correctly identified as 75% Enterobacter cloacae, 95.45% Campylobacter jejuni and 100% for Escherichia coli, Salmonella enterica, Staphylococcus aureus, Acinetobacter baumannii, and Klebsiella pneumoniae. While 94.44% (29/32 isolates) of fungi were correctly identified as 77.77% Alternaria alternate, 88.88% Aspergillus niger and 100% for Aspergillus flavus, Penicillium digitatum, Candida albicans and Debaryomyces hansenii. However, all bacterial and fungal isolates were 100% properly identified by MALDI-TOF MS fingerprinting with a score value ≥2.00. A gel view illustrated that the spectral peaks for the identified isolates fluctuate between 3,000 and 10,000 Da. The results of main spectra library (MSP) dendrogram showed that the bacterial and fungal isolates matched with 19 and 9 reference strains stored in the Bruker taxonomy, respectively. Our results indicated that MALDI-TOF MS is a promising technique for fast and accurate identification of foodborne pathogens.

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates.

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Matthew L Faron

Full Text Available The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA System (Bruker Daltonics Inc, Billerica, MA for the identification of aerobic gram-negative bacteria as part of a 510(k submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263 to genus and 98.2% (2,222/2,263 to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria.

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates.

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Faron, Matthew L; Buchan, Blake W; Hyke, Josh; Madisen, Neil; Lillie, Jennifer L; Granato, Paul A; Wilson, Deborah A; Procop, Gary W; Novak-Weekley, Susan; Marlowe, Elizabeth; Cumpio, Joven; Griego-Fullbright, Christen; Kindig, Sandra; Timm, Karen; Young, Stephen; Ledeboer, Nathan A

2015-01-01

The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria.

Molecular, ultrastructural, and biological characterization of Pennsylvania isolates of Plum pox virus.

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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.

Genomic, transcriptomic, and proteomic approaches towards understanding the molecular mechanisms of salt tolerance in Frankia strains isolated from Casuarina trees.

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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

Rapid isolation of gene homologs across taxa: Efficient identification and isolation of gene orthologs from non-model organism genomes, a technical report

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Heffer Alison

2011-03-01

Full Text Available Abstract Background Tremendous progress has been made in the field of evo-devo through comparisons of related genes from diverse taxa. While the vast number of species in nature precludes a complete analysis of the molecular evolution of even one single gene family, this would not be necessary to understand fundamental mechanisms underlying gene evolution if experiments could be designed to systematically sample representative points along the path of established phylogenies to trace changes in regulatory and coding gene sequence. This isolation of homologous genes from phylogenetically diverse, representative species can be challenging, especially if the gene is under weak selective pressure and evolving rapidly. Results Here we present an approach - Rapid Isolation of Gene Homologs across Taxa (RIGHT - to efficiently isolate specific members of gene families. RIGHT is based upon modification and a combination of degenerate polymerase chain reaction (PCR and gene-specific amplified fragment length polymorphism (AFLP. It allows targeted isolation of specific gene family members from any organism, only requiring genomic DNA. We describe this approach and how we used it to isolate members of several different gene families from diverse arthropods spanning millions of years of evolution. Conclusions RIGHT facilitates systematic isolation of one gene from large gene families. It allows for efficient gene isolation without whole genome sequencing, RNA extraction, or culturing of non-model organisms. RIGHT will be a generally useful method for isolation of orthologs from both distant and closely related species, increasing sample size and facilitating the tracking of molecular evolution of gene families and regulatory networks across the tree of life.

Molecular epidemiology of group A streptococcus from pharyngeal isolates in Auckland, New Zealand, 2013.

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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.

Performance of culture media for the isolation and identification of Staphylococcus aureus from bovine mastitis.

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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.

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

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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.

Identification at Biovar Level of Brucella Isolates Causing Abortion in Small Ruminants of Iran

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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.

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

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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.

Identification of Pseudallescheria and Scedosporium Species by Three Molecular Methods▿

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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

Isolation of Cryptococcus gattii molecular type VGIII, from Corymbia ficifolia detritus in Colombia.

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Escandón, P; Sánchez, A; Firacative, C; Castañeda, E

2010-06-01

An environmental sampling survey was carried out in different areas of Bogotá, Colombia, to obtain isolates of members of the Cryptococcus neoformans/C. gattii species complex from Corymbia ficifolia trees. During a 6-month period in 2007, 128 samples consisting of bark, soil around trunk bases, detritus, seeds and flowers were collected from 91 trees and processed according to standard procedures. The molecular type was determined using URA5 restriction fragment length polymorphism (RFLP) analysis and the mating type was established by PCR using specific primers for Mfalpha and Mfa C. gattii was isolated from 15 of the 128 (11.7%) samples, of which three (20%) were recovered from the red flower extract and the remaining 12 from C. ficifolia detritus. URA5 RFLP analysis revealed that all 15 isolates belonged to the molecular type VGIII and mating type specific PCR revealed that all were mating type a. The isolation of C. gattii from C. ficifolia represents an important finding since this is the first report revealing C. ficifolia as a habitat for C. gattii and adds additional information to the ever growing spectrum of tree species from which C. gattii can be recovered.

Molecular Characterization and Antibiotic Susceptibility Pattern of Acinetobacter Baumannii Isolated in Intensive Care Unit Patients in Al-Hassa, Kingdom of Saudi Arabia.

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Alhaddad, Mohammed Sami; AlBarjas, Afnan Khalifah; Alhammar, Lolowah Ebraheem; Al Rashed, Abdullatif Sami; Badger-Emeka, Lorina Ineta

2018-01-01

Acinetobacter baumannii , is an emerging nosocomial multidrug resistance pathogen with the rapid spread of clones being reported in health-care settings and hospitals worldwide. Carbapenem resistance in this bacterium has been attributed to D OXA β-lactamases with OXA-51-like β-lactamase, being present in all A. baumannii isolate. The present study looks into the antibiotics susceptibility and molecular characterization of clinical A. baumannii isolates from Intensive Care Unit (ICU) samples in Al-Hofuf, South-eastern region of Saudi Arabia. Eleven strains of ICU A. baumanni i isolates were used for the investigation. Bacteria isolation was by basic microbiological techniques. Organisms identification and antibiogram susceptibility testing was by the BioMerieux VITEK 2 compact automated system (BioMerieux, Marcy I'Etoile France), according to the manufacturers guidelines. Confirmation of A. baumannii was by the presence of the OX-51 gene, also, carbapenemase encoding resistant genesbla OXA-23 , bla OXA-40 , and bla OXA-51 , were analyzed using multiplex PCR. The Student's t test was used to analyze the obtained data for between group comparisons with statistically significance level set at P < 0.05. Eight of the isolates were confirmed to be A. baumannii . Five of which were resistant to the carbapenems against which they had been tested. One isolate was resistant to tigecycline, whereas three tested intermediate to the drug. OXA-23 was detected in isolates 1, 4, 5, 6, and 7. It can, therefore, be concluded that the probable predominate carbapenems resistant genes in ICU isolates from the present investigation, are those associated with OXA-23.

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.

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.

Variation in extragenic repetitive DNA sequences in Pseudomonas syringae and potential use of modified REP primers in the identification of closely related isolates

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Elif Çepni

2012-01-01

Full Text Available In this study, Pseudomonas syringe pathovars isolated from olive, tomato and bean were identified by species-specific PCR and their genetic diversity was assessed by repetitive extragenic palindromic (REP-PCR. Reverse universal primers for REP-PCR were designed by using the bases of A, T, G or C at the positions of 1, 4 and 11 to identify additional polymorphism in the banding patterns. Binding of the primers to different annealing sites in the genome revealed additional fingerprint patterns in eight isolates of P. savastanoi pv. savastanoi and two isolates of P. syringae pv. tomato. The use of four different bases in the primer sequences did not affect the PCR reproducibility and was very efficient in revealing intra-pathovar diversity, particularly in P. savastanoi pv. savastanoi. At the pathovar level, the primer BOX1AR yielded shared fragments, in addition to five bands that discriminated among the pathovars P. syringae pv. phaseolicola, P. savastanoi pv. savastanoi and P. syringae pv. tomato. REP-PCR with a modified primer containing C produced identical bands among the isolates in a pathovar but separated three pathovars more distinctly than four other primers. Although REP-and BOX-PCRs have been successfully used in the molecular identification of Pseudomonas isolates from Turkish flora, a PCR based on inter-enterobacterial repetitive intergenic concensus (ERIC sequences failed to produce clear banding patterns in this study.

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.

Toxocariasis in Carnivora from Argentinean Patagonia: Species molecular identification, hosts, and geographical distribution

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R.M. Vega

2018-04-01

Full Text Available Twenty four specimens of seven species belonging to the families Felidae, Mustelidae, and Canidae were obtained in Lanín and Nahuel Huapi National Parks from March 1996 to April 2016. Specimens were processed by necropsy in order to contribute to the knowledge of toxocariasis in wild carnivores of Argentinean Patagonia. The only Puma concolor and the seven Leopardus geoffroyi were positive for Toxocara cati. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP of the ITS-1 region from larval and adult DNA was carried out to confirm parasite species identification. This is the first molecular determination of T. cati from wild felids in Argentina and the study also fill gaps about the spatial distribution and hosts for Toxocara cati. Keywords: Toxocara cati, Puma concolor, Leopardus geoffroyi, Molecular identification, Argentina

Performance of optimized McRAPD in identification of 9 yeast species frequently isolated from patient samples: potential for automation.

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Trtkova, Jitka; Pavlicek, Petr; Ruskova, Lenka; Hamal, Petr; Koukalova, Dagmar; Raclavsky, Vladislav

2009-11-10

Rapid, easy, economical and accurate species identification of yeasts isolated from clinical samples remains an important challenge for routine microbiological laboratories, because susceptibility to antifungal agents, probability to develop resistance and ability to cause disease vary in different species. To overcome the drawbacks of the currently available techniques we have recently proposed an innovative approach to yeast species identification based on RAPD genotyping and termed McRAPD (Melting curve of RAPD). Here we have evaluated its performance on a broader spectrum of clinically relevant yeast species and also examined the potential of automated and semi-automated interpretation of McRAPD data for yeast species identification. A simple fully automated algorithm based on normalized melting data identified 80% of the isolates correctly. When this algorithm was supplemented by semi-automated matching of decisive peaks in first derivative plots, 87% of the isolates were identified correctly. However, a computer-aided visual matching of derivative plots showed the best performance with average 98.3% of the accurately identified isolates, almost matching the 99.4% performance of traditional RAPD fingerprinting. Since McRAPD technique omits gel electrophoresis and can be performed in a rapid, economical and convenient way, we believe that it can find its place in routine identification of medically important yeasts in advanced diagnostic laboratories that are able to adopt this technique. It can also serve as a broad-range high-throughput technique for epidemiological surveillance.

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.

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

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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.

Selection, isolation, and identification of fungi for bioherbicide production

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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.

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.

[Isolation of Aspergillus tritici from internal environment (Chile): Ecological and clinical scope].

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Vieille Oyarzo, Peggy; Cruz Choappa, Rodrigo; Piontelli Laforet, Eduardo

2018-03-29

Indoor environments provide important protective habitats for humans, who live or work in them most of the time. Many of these environments lack ventilation, which affects the composition of microbial communities, especially that of the fungal community. The aim of this study is to report the isolation of Aspergillus section Candidi from indoor environments of the School of Medicine at Universidad de Valparaiso, Chile, and identification through morpho-physiological and molecular approaches. Their ecological and clinical features were highlighted. An environmental non-volumetric sampling was performed on PDA medium; 2 petri dishes were exposed in 10 different places to select the Aspergillus samples. Subcultures were performed on agar Czapek with yeast extract (CYA), malt extract agar (MEA) and creatin sacarose agar (CREA) media only for the morpho-physiological and later the molecular identification of white spore species. Of the 20 samples analyzed, one Aspergillus belonging to Candidi section was isolated. Based on its morphology and molecular features, it was classified as Aspergillustritici Mehrotra & Basu. Its ecology and medical relevance are reviewed and discussed. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

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.

Molecular prey identification in Central European piscivores.

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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.

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.

Molecular Identification of Mycobacterium Tuberculosis and Analysis of Its Resistance to Rifampin in Sputa from Tuberculosis Suspected Patients

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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)

Identification of novel anti-inflammatory probiotic strains isolated from pulque.

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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.

Speciation and susceptibility of Nocardia isolated from ocular infections.

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Reddy, A K; Garg, P; Kaur, I

2010-08-01

Twenty Nocardia spp. isolated from ocular infections were identified by 16S rRNA gene sequencing and susceptibility was determined using the E-test (AB Biodisk, Sweden). Species distribution among the 20 isolates was as follows: Nocardia levis (n = 7), Nocardia farcinica (n = 3), Nocardia abscessus (n = 2), Nocardia brasiliensis (n = 2), Nocardia amamiensis (n = 2), Nocardia puris (n = 1), Nocardia beijingensis (n = 1), Nocardia otitidiscaviarum (n = 1) and Nocardia thailandica (n = 1). All isolates were sensitive to amikacin. Eighteen (90%) isolates were sensitive to tobramycin, 11 (55%) to ciprofloxacin and gatifloxacin, and seven (35%) to azithromycin and clarithromycin. Molecular methods are useful for the identification and for the detection of Nocardia species that have not so far been reported in human infections.

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.

Molecular and phenotypic characterization of endophytic bacteria isolated from sulla nodules.

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Beghalem, Hamida; Aliliche, Khadidja; Chriki, Ali; Landoulsi, Ahmed

2017-10-01

In the current study, bacterial diversity was investigated in root nodules of Sulla pallida and Sulla capitata. The isolates were analyzed on the basis of their phenotypic and molecular characteristics. The phylogenetic analysis based on 16S rRNA and housekeeping genes (recA and atpD) showed that the isolated bacteria related to Sinorhizobium, Neorhizobium, Phyllobacterium, Arthrobacter, Variovorax and Pseudomonas genera. This is the first report of Neorhizobium genus associated with Hedysarum genus. Phenotypically, all strains tolerate the elevated temperature of 40 °C, and salt stress at a concentration of 2%. In addition, the isolates failed to induce nodulation on their original host; and the symbiotic genes could not be amplified, suggesting that these strains are endophytic bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular Identification of Paramecium bursaria Syngens and Studies on Geographic Distribution using Mitochondrial Cytochrome C Oxidase Subunit I (COI).

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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.

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

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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

Molecular identification of Echinococcus granulosus on the Tibetan Plateau using mitochondrial DNA markers.

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Hu, D; Song, X; Wang, N; Zhong, X; Wang, J; Liu, T; Jiang, Z; Dawa, T; Gu, X; Peng, X; Yang, G

2015-10-30

Cystic echinococcosis (CE) is an important worldwide zoonotic disease that causes large economic losses and human suffering. Echinococcus granulosus, the causative agent of CE, exhibits different genotypes in different locations. In order to identify its genotypes and analyze its genetic structure on the Tibetan Plateau, we collected 72 hydatid cysts from different intermediate hosts and amplified and sequenced their mitochondrial cytochrome c oxidase subunit 2 (cox2) genes. Seventy isolates were identified as the E. granulosus G1 genotype, while two isolates belonged to the G6 genotype. There were 18 haplotypes among the 70 E. granulosus isolates, which exhibited a star-like network pattern and shared a common haplotype (H1). There was little difference between geographical sub-populations. Our results suggest that a recent E. granulosus population expansion occurred on the Tibetan Plateau, suggesting that E. granulosus was introduced into China. This study increases the basic molecular data needed for the molecular diagnosis, epidemiology, prevention, and control of Echinococcus diseases.

The identification of carbapenemase types in Enterobacteriaceae by using molecular assay and phenotyping confirmation tests.

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Genc, Ozlem; Aksu, Evrim; Gulcan, Aynur

2016-06-01

In this study, we aimed to identify the molecular carbapenemase types of the Enterobacteriaceae isolates and to evaluate the performance of manually prepared and commercially available combination disc methods and the modified Hodge test. One hundred and forty carbapenemase producing isolates and 45 isolates as control group were included in our study. The Xpert CARBA-R test was used as the molecular method. Antibiotic susceptibility tests were performed using combined discs, manually prepared with APBA (3-aminophenyl boronic acid), DPA (dipicolinic acid), EDTA (Ethylene diamine tetra acetic acid), cloxacillin supplements and Mastdiscs Combi-D70C that includes four antibiotic discs with specific inhibitors and temocillin discs. The modified Hodge test was performed on all isolates. OXA-48 gene was identified in 129 isolates , the NDM gene was identified in 10 isolates and VIM in one isolate. Thirty inaccurate results (30/185, 16%) were detected by using the manually prepared confirmation test. The sensitivity and specificity of this test were identified respectively 85% and 73%. Also, the sensitivity and specificity of the Mastdiscs Combi-D70C were identified as 100%. Negative results were detected in 3 NDM isolates with the use of a modified Hodge test. Sensitivity and specificity were calculated for the modified Hodge test respectively 97% and 100%. Finally, molecular methods provide results rapidly but they are not always easily accessible. The modified Hodge test can be used only for screening as a first step test and is not one of the tests that can identify the type of the carbapenemase. When carbapenem-resistant Enterobacteriaceae are detected, a commercial kit like Mastdiscs Combi-D70 may be preferred instead of the manually prepared phenotypic verification tests. Copyright © 2016 Elsevier B.V. All rights reserved.

Occurrence and molecular characterization of cultivable mesophilic and thermophilic obligate anaerobic bacteria isolated from paper mills.

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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.

PRNP genetic variability and molecular typing of natural goat scrapie isolates in a high number of infected flocks.

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Fragkiadaki, Eirini G; Vaccari, Gabriele; Ekateriniadou, Loukia V; Agrimi, Umberto; Giadinis, Nektarios D; Chiappini, Barbara; Esposito, Elena; Conte, Michela; Nonno, Romolo

2011-09-30

One hundred and four scrapie positive and 77 negative goats from 34 Greek mixed flocks were analysed by prion protein gene sequencing and 17 caprine scrapie isolates from 11 flocks were submitted to molecular isolate typing. For the first time, the protective S146 variant was reported in Greece, while the protective K222 variant was detected in negative but also in five scrapie positive goats from heavily infected flocks. By immunoblotting six isolates, including two goat flockmates carrying the K222 variant, showed molecular features slightly different from all other Greek and Italian isolates co-analysed, possibly suggesting the presence of different scrapie strains in Greece.

PRNP genetic variability and molecular typing of natural goat scrapie isolates in a high number of infected flocks

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Fragkiadaki Eirini G

2011-09-01

Full Text Available Abstract One hundred and four scrapie positive and 77 negative goats from 34 Greek mixed flocks were analysed by prion protein gene sequencing and 17 caprine scrapie isolates from 11 flocks were submitted to molecular isolate typing. For the first time, the protective S146 variant was reported in Greece, while the protective K222 variant was detected in negative but also in five scrapie positive goats from heavily infected flocks. By immunoblotting six isolates, including two goat flockmates carrying the K222 variant, showed molecular features slightly different from all other Greek and Italian isolates co-analysed, possibly suggesting the presence of different scrapie strains in Greece.

Molecular characterization of Acanthamoeba strains isolated from domestic dogs in Tenerife, Canary Islands, Spain.

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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.

Molecular Analysis of Vancomycin-Resistant Enterococci Isolated from Regional Hospitals in Trinidad and Tobago

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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.

[Comparison of Phoenix™ Yeast ID Panel and API® ID 32C commercial systems for the identification of Candida species isolated from clinical samples].

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Gayibova, Ülkü; Dalyan Cılo, Burcu; Ağca, Harun; Ener, Beyza

2014-07-01

Opportunistic fungal pathogens are one of the important causes of nosocomial infections, and several different types of yeasts, especially Candida species are increasingly recovered from immunocompromised patients. Since many of the yeasts are resistant to the commonly used antifungal agents, the introduction of appropriate therapy depends on rapid and accurate identification. The aims of this study were to compare the commercial identification systems namely API® ID 32C (bioMerieux, France) and Phoenix™ Yeast ID Panel (Becton Dickinson Diagnostics, USA) for the identification of Candida species and to evaluate the effect of morphological findings in the identification process. A total of 211 yeast strains isolated from different clinical samples (111 urine, 34 blood/vascular catheter, 27 upper/lower respiratory tract, 16 abscess/pus, 13 throat/vagina swabs and 10 sterile body fluids) of 137 patients hospitalized in Uludag University Health and Research Center between October 2013 to January 2014, were included in the study. Samples were cultured on blood agar, chromogenic agar (CHROMagar Candida, BD, USA) and Saboraud's dextrose agar (SDA), and isolated yeast colonies were evaluated with germ tube test and morphological examination by microscopy on cornmeal/Tween-80 agar. The isolates were identified as well by two commercial systems according to the manufacturers' recommendations. Discrepant results between the systems were tried to be resolved by using morphological characteristics of the yeasts. Of the isolates 159 were identified identical by both of the systems, and the concordance between those systems were estimated as 75.4%. According to the concordant identification, the most frequently isolated species was C.albicans (44.1%) followed by C.tropicalis (9.9%), C.glabrata (9.5%), C.parapsilosis (8.5%) and C.kefyr (8.1%). The concordance rate was 81.7% in identification of frequently isolated species (C.albicans, C.tropicalis, C.parapsilosis, C.glabrata, C

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.

Molecular epidemiological study of Arctic rabies virus isolates from Greenland and comparison with isolates from throughout the Arctic and Baltic regions

DEFF Research Database (Denmark)

Mansfield, K.L.; Racloz, V.; McElhinney, L.M.

2006-01-01

We report a Molecular epidemiological study of rabies in Arctic Countries by comparing a panel of novel Greenland isolates to a larger cohort of viral sequences from both Arctic and Baltic regions. Rabies Virus isolates originating from wildlife (Arctic/red foxes, raccoon-dogs and reindeer), from...... sequences from the Arctic and Arctic-like viruses, which were distinct from rabies isolates originating ill the Baltic region of Europe, the Steppes in Russia and from North America. The Arctic-like group consist of isolates from India, Pakistan, southeast Siberia and Japan. The Arctic group...... in northeast Siberia and Alaska. Arctic 2b isolates represent a biotype, which is dispersed throughout the Arctic region. The broad distribution of rabies in the Arctic regions including Greenland, Canada and Alaska provides evidence for the movement of rabies across borders....

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

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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.

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...

Molecular epidemiology of Corynebacterium pseudotuberculosis isolated from horses in California.

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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.

Isolation, identification, characterization, and evaluation of cadmium removal capacity of Enterobacter species.

Science.gov (United States)

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

2014-12-01

This study focused on the isolation and characterization of high cadmium-resistant bacterial strains, possible exploitation of its cadmium-accumulation and cadmium-induced proteins. Cadmium-resistant bacterial strains designated as RZ1 and RZ2 were isolated from industrial wastewater of Penang, Malaysia. These isolates were identified as Enterobacter mori and Enterobacter sp. WS12 on the basis of phenotypic, biochemical and 16S rDNA sequence based molecular phylogenetic characteristics. Both isolates were Gram negative, cocci, and growing well in Lauria-Bertani broth medium at 35 °C temperature and pH 7.0. Results also indicated that Enterobacter mori and Enterobacter sp. WS12are capable to remove 87.75 and 85.11% of the cadmium from 100 µg ml(-1) concentration, respectively. This study indicates that these strains can be useful as an inexpensive and efficient bioremediation technology to remove and recover the cadmium from wastewater. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Isolation and identification of aerobic and facultative anaerobic bacteria in the oral cavity].

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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.

Pathogenicity and molecular analysis of an infectious bursal disease virus isolated from Malaysian village chickens.

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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.

Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk.

Science.gov (United States)

Shome, Bibek Ranjan; Bhuvana, Mani; Mitra, Susweta Das; Krithiga, Natesan; Shome, Rajeswari; Velu, Dhanikachalam; Banerjee, Apala; Barbuddhe, Sukhadeo B; Prabhudas, Krishnamshetty; Rahman, Habibar

2012-12-01

Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A-D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B

Towards a molecular identification and classification system of lepidopteran-specific baculoviruses

International Nuclear Information System (INIS)

Lange, Martin; Wang Hualin; Hu Zhihong; Jehle, Johannes A.

2004-01-01

Virus genomics provides novel approaches for virus identification and classification. Based on the comparative analyses of sequenced lepidopteran-specific baculovirus genomes, degenerate oligonucleotides were developed that allow the specific amplification of several regions of the genome using polymerase chain reaction (PCR) followed by DNA sequencing. The DNA sequences within the coding regions of three highly conserved genes, namely polyhedrin/granulin (polh/gran), late expression factor 8 (lef-8), and late expression factor 9 (lef-9), were targeted for amplification. The oligonucleotides were tested on viral DNAs isolated from historical field samples, and amplification products were generated from 12 isolated nucleopolyhedrovirus (NPV) and 8 granulovirus (GV) DNAs. The PCR products were cloned or directly sequenced, and phylogenetic trees were inferred from individual and combined data sets of these three genes and compared to a phylogeny, which includes 22 baculoviruses using a combined data set of 30 core genes. This method allows a fast and reliable detection and identification of lepidopteran-specific NPVs and GVs. Furthermore, a strong correlation of the base composition of these three genome areas with that of the complete virus genome was observed and used to predict the base composition of uncharacterized baculovirus genomes. These analyses suggested that GVs have a significantly higher AT content than NPVs

Molecular characterization and drug susceptibility profile of a Mycobacterium avium subspecies avium isolate from a dog with disseminated infection.

Science.gov (United States)

Armas, Federica; Furlanello, Tommaso; Camperio, Cristina; Trotta, Michele; Novari, Gianluca; Marianelli, Cinzia

2016-01-12

Mycobacterium avium complex (MAC) infections have been described in many mammalian species including humans and pets. We isolated and molecularly typed the causative agent of a rare case of disseminated mycobacteriosis in a dog. We identified the pathogen as a M. avium subspecies avium by sequencing the partial genes gyrB and rpsA. Considering the zoonotic potential of this infection, and in an attempt to ensure the most effective treatment for the animal, we also determined the drug susceptibility profile of the isolate to the most common drugs used to treat MAC disease in humans. The pathogen was tested in vitro against the macrolide clarithromycin, as well as against amikacin, ciprofloxacin, rifampicin, ethambutol and linezolid by the resazurin microdilution assay. It was found to be sensitive to all tested drugs save ethambutol. Despite the fact that the pathogen was sensitive to the therapies administered, the dog's overall clinical status worsened, and the animal died shortly after antimicrobial susceptibility results became available. Nucleotide sequencing of the embB gene, the target gene most commonly associated with ethambutol resistance, showed new missense mutations when compared to sequences available in public databases. In conclusion, we molecularly identified the MAC pathogen and determined its drug susceptibility profile in a relatively short period of time (seven days). We also characterized new genetic mutations likely to have been involved in the observed ethambutol resistance. Our results confirm the usefulness of both the gyrB and the rpsA genes as biomarkers for an accurate identification and differentiation of MAC pathogens.

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

Isolation of plasmodesmata from Arabidopsis suspension culture cells.

Science.gov (United States)

Grison, Magali S; Fernandez-Calvino, Lourdes; Mongrand, Sébastien; Bayer, Emmanuelle M F

2015-01-01

Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

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).

Morphological description and molecular detection of Pestalotiopsis sp. on hazelnut in Serbia

Energy Technology Data Exchange (ETDEWEB)

Vasić, T.; Jevremović, D.; Krnjaja, V.; Leposavić, A.; Anđelković, S.; Živković, S.; Paunović, S.

2017-07-01

In autumn 2015, hazelnut plants with leaf blight symptoms were noticed in a commercial plantation in the Province of Vojvodina, Serbia. Symptomatic samples were collected and submitted to laboratory analysis. Based on morphological characterization, the fungus isolated from the material was initially identified as Pestalotiopsis sp. Pathogenicity tests showed that two selected isolates infected hazelnut leaves and fruits that developed symptoms after artificial inoculation. The pathogen was re-isolated from diseased leaves and fruits, confirming Koch’s postulates. Molecular identification was performed with sequence and phylogenetic analysis of ITS, EF1-α, and TUB genomic regions. Phylogenetic analysis confirmed the results of the morphological identification. The detection of Pestalotiopsis sp., a causal agent of leaf blight on hazelnut in Serbia, is one of a few reports of these pathogenic fungi on hazelnut.

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.

Model-based evaluation of the use of polycyclic aromatic hydrocarbons molecular diagnostic ratios as a source identification tool

International Nuclear Information System (INIS)

Katsoyiannis, Athanasios; Breivik, Knut

2014-01-01

Polycyclic Aromatic Hydrocarbons (PAHs) molecular diagnostic ratios (MDRs) are unitless concentration ratios of pair-PAHs with the same molecular weight (MW); MDRs have long been used as a tool for PAHs source identification purposes. In the present paper, the efficiency of the MDR methodology is evaluated through the use of a multimedia fate model, the calculation of characteristic travel distances (CTD) and the estimation of air concentrations for individual PAHs as a function of distance from an initial point source. The results show that PAHs with the same MW are sometimes characterized by substantially different CTDs and therefore their air concentrations and hence MDRs are predicted to change as the distance from the original source increases. From the assessed pair-PAHs, the biggest CTD difference is seen for Fluoranthene (107 km) vs. Pyrene (26 km). This study provides a strong indication that MDRs are of limited use as a source identification tool. -- Highlights: • Model-based evaluation of the PAHs molecular diagnostic ratios efficiency. • Individual PAHs are characterized by different characteristic travel distances. • MDRs are proven to be a limited tool for source identification. • Use of MDRs for other environmental media is likely unfeasible. -- PAHs molecular diagnostic ratios which change greatly as a function of distance from the emitting source are improper for source identification purposes

The molecular structure of the insoluble organic matter isolated from Murchison carbonaceous chondrite.

Science.gov (United States)

Robert, F.; Derenne, S.

2009-04-01

During these last 10 years, our group has characterized the various molecular moieties of the insoluble organic matter (IOM) isolated from carbonaceous meteorites with the aim of reconstructing its overall molecular structure. Indeed, a precise knowledge of the structure of an organic macromolecule contains irreplaceable information that traces its mechanisms of synthesis and its conditions of formation. Such a modelled structure will be presented. Carbonaceous chondrites contain up to 3 wt % of carbon that is under the form of soluble and insoluble fractions. The IOM, which constitutes more than 75 wt% of the bulk organic matter, was isolated from the bulk rock through successive acid dissolutions. The chemical structure of the isolated IOM has been studied by both (1) destructive and (2) non destructive methods. Methods include thermal and chemical degradations followed by GC/MS, spectroscopic techniques (nuclear magnetic resonance, Fourier transform infra red spectroscopy; X-ray absorption near-edge spectroscopy, electron paramagnetic resonance) along with high resolution transmission electron microscopy. Although each technique alone cannot provide definite information on the chemical structure of such a complex material, the combination of the results can be used to reconstruct the molecular structure of the IOM. The proposed structure accounts for all these measured parameters. The details of this structure reveal information of the conditions of its formation in space and allow to discuss the mechanisms of organo-synthesis in the cosmochemical context of the formation of the solar system.

Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Science.gov (United States)

Vidal-Acuña, M Reyes; Ruiz-Pérez de Pipaón, Maite; Torres-Sánchez, María José; Aznar, Javier

2017-12-08

An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS. One hundred ninety strains (179 clinical isolates previously identified by sequencing and the 11 reference strains), cultured on solid medium, were blindy analyzed by the MALDI-TOF MS technology to validate the generated in-house reference database. A 100% correlation was obtained with both identification methods, gene sequencing and MALDI-TOF MS, and no discordant identification was obtained. The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.84%) and for the remaining 25 (13.16%) a genus level identification (score between 1.7 and 2.0) was obtained. The routine MALDI-TOF MS analysis with the new database, was then challenged with 200 Aspergillus clinical isolates grown on solid medium in a prospective evaluation. A species identification was obtained in 191 strains (95.5%), and only nine strains (4.5%) could not be identified at the species level. Among the 200 strains, A. tubingensis was the only cryptic species identified. We demonstrated the feasibility and usefulness of the new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species, grown either on solid or liquid media. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For

Identification of Extended-Spectrum β-Lactamases Escherichia coli Strains Isolated from Market Garden Products and Irrigation Water in Benin

Directory of Open Access Journals (Sweden)

Wassiyath Moussé

2015-01-01

Full Text Available The present study aimed at biochemical and molecular characterization of Escherichia coli strains isolated from horticultural products and irrigation water of Cotonou. The samples were collected from 12 market gardeners of 4 different sites. Rapid’ E. coli medium was used for identification of E. coli strains and the antimicrobial susceptibility was performed by the agar disk diffusion method. The β-lactamases production was sought by the liquid acidimetric method. The genes coding for β-lactamases and toxins were identified by PCR method. The results revealed that about 34.95% of the analyzed samples were contaminated by E. coli. Cabbages were the most contaminated by E. coli (28.26% in dry season. All isolated strains were resistant to amoxicillin. The penicillinase producing E. coli carried blaTEM (67.50%, blaSHV (10%, and blaCTX-M (22.50% genes. The study revealed that the resistance genes such as SLTI (35.71%, SLTII (35.71%, ETEC (7.15%, and VTEC (21.43% were carried. Openly to the found results and considering the importance of horticultural products in Beninese food habits, it is important to put several strategies aiming at a sanitary security by surveillance and sensitization of all the actors on the risks of some practices.

Identification of Extended-Spectrum β-Lactamases Escherichia coli Strains Isolated from Market Garden Products and Irrigation Water in Benin

Science.gov (United States)

Moussé, Wassiyath; Sina, Haziz; Baba-Moussa, Farid; Noumavo, Pacôme A.; Agbodjato, Nadège A.; Adjanohoun, Adolphe; Baba-Moussa, Lamine

2015-01-01

The present study aimed at biochemical and molecular characterization of Escherichia coli strains isolated from horticultural products and irrigation water of Cotonou. The samples were collected from 12 market gardeners of 4 different sites. Rapid' E. coli medium was used for identification of E. coli strains and the antimicrobial susceptibility was performed by the agar disk diffusion method. The β-lactamases production was sought by the liquid acidimetric method. The genes coding for β-lactamases and toxins were identified by PCR method. The results revealed that about 34.95% of the analyzed samples were contaminated by E. coli. Cabbages were the most contaminated by E. coli (28.26%) in dry season. All isolated strains were resistant to amoxicillin. The penicillinase producing E. coli carried blaTEM (67.50%), blaSHV (10%), and blaCTX-M (22.50%) genes. The study revealed that the resistance genes such as SLTI (35.71%), SLTII (35.71%), ETEC (7.15%), and VTEC (21.43%) were carried. Openly to the found results and considering the importance of horticultural products in Beninese food habits, it is important to put several strategies aiming at a sanitary security by surveillance and sensitization of all the actors on the risks of some practices. PMID:26770972

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

Identification of Burkholderia spp. in the Clinical Microbiology Laboratory: Comparison of Conventional and Molecular Methods

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van Pelt, Cindy; Verduin, Cees M.; Goessens, Wil H. F.; Vos, Margreet C.; Tümmler, Burkhard; Segonds, Christine; Reubsaet, Frans; Verbrugh, Henri; van Belkum, Alex

1999-01-01

Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli (n = 14), Ralstonia pickettii (n = 6), B. multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and Pseudomonas aeruginosa (n = 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as Burkholderia spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of B. cepacia appeared to be the API 20NE test. None of the biochemical assays successfully grouped the B. gladioli strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp. With this method, B. gladioli was also reliably classified in a separate group. For the laboratory diagnosis of B. cepacia, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a Burkholderia phenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR

Isolation, Identification and Seasonal Distribution of Soilborne Fungi in Different Areas of Erbil Governorate

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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.

Biodiversity of Bacteria Isolated from Different Soils

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Fatma YAMAN

2017-01-01

Full Text Available The aim of this study was to determine the biodiversity of PHB producing bacteria isolated from soils where fruit and vegetable are cultivated (onion, grape, olive, mulberry and plum in Aydın providence. Morphological, cultural, biochemical, and molecular methods were used for bacteria identification. These isolated bacteria were identified by 16S rRNA sequencing and using BLAST. The following bacteria Bacillus thuringiensis (6, Bacillus cereus (8, Bacillus anthrachis (1, Bacillus circulans (1, Bacillus weihenstephanensis (1, Pseudomonas putida (1, Azotobacter chroococcum (1, Brevibacterium frigoritolerans (1, Burkholderia sp. (1, Staphylococcus epidermidis (1, Streptomyces exfoliatus (1, Variovorax paradoxus (1 were found. The Maximum Likelihood method was used to produce a molecular phylogenetic analysis and a phylogenetic tree was constructed. These bacteria can produce polyhydroxybutyrate (PHB which is an organic polymer with commercial potential as a biodegradable thermoplastic. PHB can be used instead of petrol derivated non-degradable plastics. For this reason, PHB producing microorganisms are substantial in industry.

Distribution of Malassezia species on the skin of patients with atopic dermatitis, psoriasis, and healthy volunteers assessed by conventional and molecular identification methods.

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Jagielski, Tomasz; Rup, Elżbieta; Ziółkowska, Aleksandra; Roeske, Katarzyna; Macura, Anna B; Bielecki, Jacek

2014-03-07

The Malassezia yeasts which belong to the physiological microflora of human skin have also been implicated in several dermatological disorders, including pityriasis versicolor (PV), atopic dermatitis (AD), and psoriasis (PS). The Malassezia genus has repeatedly been revised and it now accommodates 14 species, all but one being lipid-dependent species. The traditional, phenotype-based identification schemes of Malassezia species are fraught with interpretative ambiguities and inconsistencies, and are thus increasingly being supplemented or replaced by DNA typing methods. The aim of this study was to explore the species composition of Malassezia microflora on the skin of healthy volunteers and patients with AD and PS. Species characterization was performed by conventional, culture-based methods and subsequently molecular techniques: PCR-RFLP and sequencing of the internal transcribed spacer (ITS) 1/2 regions and the D1/D2 domains of the 26S rRNA gene. The Chi-square test and Fisher's exact test were used for statistical analysis. Malassezia sympodialis was the predominant species, having been cultured from 29 (82.9%) skin samples collected from 17 out of 18 subjects under the study. Whereas AD patients yielded exclusively M. sympodialis isolates, M. furfur isolates were observed only in PS patients. The isolation of M. sympodialis was statistically more frequent among AD patients and healthy volunteers than among PS patients (P < 0.03). Whether this mirrors any predilection of particular Malassezia species for certain clinical conditions needs to be further evaluated. The overall concordance between phenotypic and molecular methods was quite high (65%), with the discordant results being rather due to the presence of multiple species in a single culture (co-colonization) than true misidentification. All Malassezia isolates were susceptible to cyclopiroxolamine and azole drugs, with M. furfur isolates being somewhat more drug tolerant than other Malassezia species

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

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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.

Molecular identification of Clonorchis sinensis and discrimination with other opisthorchid liver fluke species using multiple Ligation-depended Probe Amplification (MLPA

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Liang Chi

2011-06-01

Full Text Available Abstract Background Infections with the opisthorchid liver flukes Clonorchis sinensis, Opisthorchis viverrini, and O. felineus cause severe health problems globally, particularly in Southeast Asia. Early identification of the infection is essential to provide timely and appropriate chemotherapy to patients. Results In this study we evaluate a PCR-based molecular identification method, Multiplex Ligation-dependent Probe Amplification (MLPA, which allows rapid and specific detection of single nucleotide acid differences between Clonorchis sinensis, Opisthorchis viverrini and O. felineus. Three probe pairs were derived from the Internally Transcribed Spacer 1 (ITS1 of three opisthorchid liver flukes using a systematic phylogenetic analysis. Specific loci were detected in all three species, yielding three amplicons with 198,172 and 152 bp, respectively, while no cross reactions were observed. A panel of 66 C. sinensis isolates was screened using MLPA. All species were positively identified, and no inhibition was observed. The detection limit was 103 copies of the ITS gene for the three liver flukes, or about 60 pg genomic DNA for Clonorchis sinensis. Amplification products can be detected by electrophoresis on agarose gel or in a capillary sequencer. In addition, genomic DNA of Clonorchis sinensis in fecal samples of infected rats was positively amplified by MLPA. Conclusion The flexibility and specificity make MLPA a potential tool for specific identification of infections by opisthorchid liver flukes in endemic areas.

Molecular characterization of dengue viruses isolated from patients in Central Java, Indonesia.

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Kusmintarsih, Endang S; Hayati, Rahma F; Turnip, Oktaviani N; Yohan, Benediktus; Suryaningsih, Suhestri; Pratiknyo, Hery; Denis, Dionisius; Sasmono, R Tedjo

2017-10-19

Dengue is hyper-endemic in Indonesia. Purwokerto city in Central Java province is routinely ravaged by the disease. Despite the endemicity of dengue in this city, there is still no data on the virological aspects of dengue in the city. We conducted a molecular surveillance study of the circulating dengue viruses (DENV) in Purwokerto city to gain information on the virus origin, serotype and genotype distribution, and phylogenetic characteristics of DENV. A cross-sectional dengue molecular surveillance study was conducted in Purwokerto. Sera were collected from dengue-suspected patients attending three hospitals in the city. Diagnosis was performed using dengue NS1 antigen and IgG/IgM antibodies detection. DENV serotyping was performed using Simplexa Dengue real-time RT-PCR. Sequencing was conducted to obtain full-length DENV Envelope (E) gene sequences, which were then used in phylogenetic and genotypic analyses. Patients' clinical and demographic data were collected and analyzed. A total of 105 dengue-suspected patients' sera were collected, in which 80 (76.2%) were positive for IgM and/or IgG, and 57 (54.2%) were confirmed as dengue by NS1 antigen and/or DENV RNA detection using RT-PCR. Serotyping was successful for 47 isolates. All four serotypes circulated in the area with DENV-3 as the predominant serotype. Phylogenetic analyses grouped the isolates into Genotype I for DENV-1, Cosmopolitan genotype for DENV-2, and Genotype I and II for DENV-3 and -4, respectively. The analyses also revealed the close relatedness of Purwokerto isolates to other DENV strains from Indonesia and neighboring countries. We reveal the molecular and virological characteristics of DENV in Purwokerto, Banyumas regency, Central Java. The genotype and phylogenetic analyses indicate the endemicity of the circulating DENV in the city. Our serotype and genotype data provide references for future dengue molecular epidemiology studies and disease management in the region. Copyright © 2017 The

Isolation of Candida Species from Gastroesophageal Lesions among Pediatrics in Isfahan, Iran: Identification and Antifungal Susceptibility Testing of Clinical Isolates by E-test

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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.

Application of MALDI-TOF MS for requalification of a Candida clinical isolates culture collection

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Reginaldo Lima-Neto

2014-06-01

Full Text Available Microbial culture collections underpin biotechnology applications and are important resources for clinical microbiology by supplying reference strains and/or performing microbial identifications as a service. Proteomic profiles by MALDI-TOF MS have been used for Candida spp. identification in clinical laboratories and demonstrated to be a fast and reliable technique for the routine identification of pathogenic yeasts. The main aim of this study was to apply MALDI-TOF MS combined with classical phenotypic and molecular approaches to identify Candida clinical isolates preserved from 1 up to 52 years in a Brazilian culture collection and assess its value for the identification of yeasts preserved in this type of collections. Forty Candida spp. clinical isolates were identified by morphological and biochemical analyses. Identifications were also performed by the new proteomic approach based on MALDI-TOF MS. Results demonstrated 15% discordance when compared with morphological and biochemical analyses. Discordant isolates were analysed by ITS sequencing, which confirmed the MALDI-TOF MS identifications and these strains were renamed in the culture collection catalogue. In conclusion, proteomic profiles by MALDI-TOF MS represents a rapid and reliable method for identifying clinical Candida species preserved in culture collections and may present clear benefits when compared with the performance of existing daily routine methods applied at health centres and hospitals.

Isolation, Identification And Screening Antibacterial Activity from Marine Sponge-Associated Fungi Against Multidrug-Resistant (MDR) Escherichia coli

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Triandala Sibero, Mada; Sabdaningsih, Aninditia; Cristianawati, Olvi; Nuryadi, Handung; Karna Radjasa, Ocky; Sabdono, Agus; Trianto, Agus

2017-02-01

Irrational used of antibiotic in several decades ago causing resistant in bacteria and decreasing the cure rate of infectious diseases. Multidrug-resistant (MDR) Escherichia coli is known to cause various of infectious diseases such as urinary tract infection, nosocomial bloodstream infection, meningitis, bacteraemia, and gastrointestinal disease. Marine sponge-associated fungi have potential as source of new compound to combat MDR E. coli. The aims of this research were to isolate marine sponge-assosiated fungi, to screen potential fungi against MDR E. coli, to identify the potential fungi and its host sponge. There were 29 marine sponge-associated fungi successfully isolated from 9 sponges. Among 29 sponge-associated fungi screened, there were 7 isolates showed antibacterial activity against MDR E. coli. The best inhibition zone produced by MPS 14.1/MT 02 and MPS 14.3/MT 04 from sponge PP.SP.16.14. According to fungi identification result fungus MPS 14.1/MT 02 was identified as Trichoderma asperellum while MPS 14.3/MT 04 was identified as Trichoderma reesei. Sponge identification leaded the PP.SP.16.14 as Cinachyrella sp.

Molecular Epidemiology Reveals Genetic Diversity amongst Isolates of the Cryptococcus neoformans/C. gattii Species Complex in Thailand

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Kaocharoen, Sirada; Ngamskulrungroj, Popchai; Firacative, Carolina; Trilles, Luciana; Piyabongkarn, Dumrongdej; Banlunara, Wijit; Poonwan, Natteewan; Chaiprasert, Angkana; Meyer, Wieland; Chindamporn, Ariya

2013-01-01

To gain a more detailed picture of cryptococcosis in Thailand, a retrospective study of 498 C. neoformans and C. gattii isolates has been conducted. Among these, 386, 83 and 29 strains were from clinical, environmental and veterinary sources, respectively. A total of 485 C. neoformans and 13 C. gattii strains were studied. The majority of the strains (68.9%) were isolated from males (mean age of 37.97 years), 88.5% of C. neoformans and only 37.5% of C. gattii strains were from HIV patients. URA5-RFLP and/or M13 PCR-fingerprinting analysis revealed that the majority of the isolates were C. neoformans molecular type VNI regardless of their sources (94.8%; 94.6% of the clinical, 98.8% of the environmental and 86.2% of the veterinary isolates). In addition, the molecular types VNII (2.4%; 66.7% of the clinical and 33.3% of the veterinary isolates), VNIV (0.2%; 100% environmental isolate), VGI (0.2%; 100% clinical isolate) and VGII (2.4%; 100% clinical isolates) were found less frequently. Multilocus Sequence Type (MLST) analysis using the ISHAM consensus MLST scheme for the C. neoformans/C. gattii species complex identified a total of 20 sequence types (ST) in Thailand combining current and previous data. The Thai isolates are an integrated part of the global cryptococcal population genetic structure, with ST30 for C. gattii and ST82, ST83, ST137, ST141, ST172 and ST173 for C. neoformans being unique to Thailand. Most of the C. gattii isolates were ST7 = VGIIb, which is identical to the less virulent minor Vancouver island outbreak genotype, indicating Thailand as a stepping stone in the global spread of this outbreak strain. The current study revealed a greater genetic diversity and a wider range of major molecular types being present amongst Thai cryptococcal isolates than previously reported. PMID:23861989

Isolation and molecular characterization of Bacillus thuringiensis found in soils of the Cerrado region of Brazil, and their toxicity to Aedes aegypti larvae

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Katiane dos Santos Lobo

Full Text Available ABSTRACT This study investigated the potential of Bacillus thuringiensis isolates obtained in the Cerrado region of the Brazilian state of Maranhão for the biological control of Aedes aegypti larvae. The isolates were obtained from soil samples and the identification of the B. thuringiensis colonies was based on morphological characteristics. Bioassays were run to assess the pathogenicity and toxicity of the different strains of the B. thuringiensis against third-instar larvae of A. aegypti. Protein profiles were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polymerase chain reaction assays were used to detect the toxin genes found in the bacterial isolates. Overall, 12 (4.0% of the 300 isolates obtained from 45 soil samples were found to present larvicidal activity, with the BtMA-104, BtMA-401 and BtMA-560 isolates causing 100% of mortality. The BtMA-401 isolate was the most virulent, with the lowest median lethal concentration (LC50 (0.004 × 107 spores/mL, followed by the Bacillus thuringiensis var. israelensis standard (0.32 × 107 spores/mL. The protein profiles of BtMA-25 and BtMA-401 isolates indicated the presence of molecular mass consistent with the presence of the proteins Cry4Aa, Cry11Aa and Cyt1, similar to the profile of Bacillus thuringiensis var. israelensis IPS-82. Surprisingly, however, none of the cry and cyt genes analyzed were amplified in the isolate BtMA-401. The results of the present study revealed the larvicidal potential of B. thuringiensis isolates found in the soils of the Cerrado region from Maranhão, although further research will be necessary to better elucidate and describe other genes associated with the production of insecticidal toxins in these isolates.

[Molecular techniques applied in species identification of Toxocara].

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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).

Identification of Paramecium bursaria syngens through molecular markers--comparative analysis of three loci in the nuclear and mitochondrial DNA.

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Greczek-Stachura, Magdalena; Potekhin, Alexey; Przyboś, Ewa; Rautian, Maria; Skoblo, Inna; Tarcz, Sebastian

2012-09-01

This is the first attempt to resolve the phylogenetic relationship between different syngens of Paramecium bursaria and to investigate at a molecular level the intraspecific differentiation of strains originating from very distant geographical locations. Herein we introduce a new collection of five P. bursaria syngens maintained at St Petersburg State University, as the international collection of syngens was lost in the 1960s. To analyze the degree of speciation within Paramecium bursaria, we examined 26 strains belonging to five different syngens from distant and geographically isolated localities using rDNA (ITS1-5.8S-ITS2-5'LSU) fragments, mitochondrial cytochrome c oxidase subunit I (COI), and H4 gene fragments. It was shown that P. bursaria strains of the same syngens cluster together in all three inferred molecular phylogenies. The genetic diversity among the studied P. bursaria strains based on rDNA sequences was rather low. The COI divergence of Paramecium bursaria was also definitely lower than that observed in the Paramecium aurelia complex. The nucleotide sequences of the H4 gene analyzed in the present study indicate the extent of genetic differences between the syngens of Paramecium bursaria. Our study demonstrates the diagnostic value of molecular markers, which are important tools in the identification of Paramecium bursaria syngens. Copyright © 2011 Elsevier GmbH. All rights reserved.

Clinical and Laboratory Features of the Nocardia spp. Based on Current Molecular Taxonomy

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Brown-Elliott, Barbara A.; Brown, June M.; Conville, Patricia S.; Wallace, Richard J.

2006-01-01

The recent explosion of newly described species of Nocardia results from the impact in the last decade of newer molecular technology, including PCR restriction enzyme analysis and 16S rRNA sequencing. These molecular techniques have revolutionized the identification of the nocardiae by providing rapid and accurate identification of recognized nocardiae and, at the same time, revealing new species and a number of yet-to-be-described species. There are currently more than 30 species of nocardiae of human clinical significance, with the majority of isolates being N. nova complex, N. abscessus, N. transvalensis complex, N. farcinica, N. asteroides type VI (N. cyriacigeorgica), and N. brasiliensis. These species cause a wide variety of diseases and have variable drug susceptibilities. Accurate identification often requires referral to a reference laboratory with molecular capabilities, as many newer species are genetically distinct from established species yet have few or no distinguishing phenotypic characteristics. Correct identification is important in deciding the clinical relevance of a species and in the clinical management and treatment of patients with nocardial disease. This review characterizes the currently known pathogenic species of Nocardia, including clinical disease, drug susceptibility, and methods of identification. PMID:16614249

Promising Biological Indicator of Heavy Metal Pollution: Bioluminescent Bacterial Strains Isolated and Characterized from Marine Niches of Goa, India.

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Thakre, Neha A; Shanware, Arti S

2015-09-01

In present study, several marine water samples collected from the North Goa Beaches, India for isolation of luminescent bacterial species. Isolates obtained labelled as DP1-5 and AB1-6. Molecular characterization including identification of a microbial culture using 16S rRNA gene based molecular technique and phylogenetic analysis confirmed that DP3 & AB1 isolates were Vibrio harveyi. All of the isolates demonstrated multiple metal resistances in terms of growth, with altered luminescence with variable metal concentration. Present investigations were an attempt towards exploring and reporting an updated diversity of bioluminescent bacterial species from various sites around the Goa, India which would be explored in future for constructing luminescence based biosensor for efficiently monitoring the level of hazardous metals in the environment.

Isolating strong-field dynamics in molecular systems

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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.

Identification of lead-resistant endophytic bacteria isolated from rice

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