Unidentified Isolates Research Articles

Optimizing Identification of Clinically Relevant Gram-Positive Organisms by Use of the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used as a method for the rapid identification of microorganisms. This study evaluated the Bruker Biotyper (MALDI-TOF MS) system for the identification of clinically relevant Gram-positive organisms. We tested 239 aerobic Gram-positive organisms isolated from clinical specimens. We evaluated 4 direct-smear methods, including "heavy" (H) and "light" (L) smears, with and without a 1-μl direct formic acid (FA) overlay. The quality measure assigned to a MALDI-TOF MS identification is a numerical value or "score." We found that a heavy smear with a formic acid overlay (H+FA) produced optimal MALDI-TOF MS identification scores and the highest percentage of correctly identified organisms. Using a score of ≥2.0, we identified 183 of the 239 isolates (76.6%) to the genus level, and of the 181 isolates resolved to the species level, 141 isolates (77.9%) were correctly identified. To maximize the number of correct identifications while minimizing misidentifications, the data were analyzed using a score of ≥1.7 for genus- and species-level identification. Using this score, 220 of the 239 isolates (92.1%) were identified to the genus level, and of the 181 isolates resolved to the species level, 167 isolates (92.2%) could be assigned an accurate species identification. We also evaluated a subset of isolates for preanalytic factors that might influence MALDI-TOF MS identification. Frequent subcultures increased the number of unidentified isolates. Incubation temperatures and subcultures of the media did not alter the rate of identification. These data define the ideal bacterial preparation, identification score, and medium conditions for optimal identification of Gram-positive bacteria by use of MALDI-TOF MS.

Lactobacillus oryzae sp. nov., isolated from fermented rice grain (Oryza sativa L. subsp. japonica)

The taxonomic position of three Lactobacillus-like micro-organisms (strains SG293(T), SG296 and SG310) isolated from fermented rice grain (Oryza sativa L. subsp. japonica) in Japan was investigated. These heterofermentative lactic acid bacteria were Gram-stain-positive, rod-shaped, facultatively anaerobic, non-motile, non-spore-forming and did not show catalase activity. 16S rRNA gene sequence analysis of strain SG293(T) revealed that the type strains of Lactobacillus malefermentans (98.3 %), Lactobacillus odoratitofui (96.2 %), Lactobacillus similis (96.1 %), Lactobacillus kimchicus (96.1 %), Lactobacillus paracollinoides (95.9 %) and Lactobacillus collinoides (95.7 %) were the closest neighbours. Additional phylogenetic analysis on the basis of pheS and rpoA gene sequences, as well as biochemical and physiological characteristics, indicated that these three strains were members of the genus Lactobacillus and that the novel isolates had a unique taxonomic position. The predominant cellular fatty acids were C18 : 1ω9c and C19 : 1 cyclo 9,10. Because low DNA-DNA hybridization values among the isolates and Lactobacillus malefermentans JCM 12497(T) were observed, it is proposed that these unidentified isolates be classified as a novel species of the genus Lactobacillus, Lactobacillus oryzae sp. nov. The type strain is SG293(T) (= JCM 18671(T) = DSM 26518(T)).

Sebaran Geografi Non Polio Enterovirus (NPEV) dari Kasus Layu Akut di Bagian Barat Indonesia 2007-2010

Acute Flaccid Paralysis (AFP) surveillance which comprises cases and laboratory investigation has been established by WHO in order to achieve the goal of global eradication of polio. NIHRD is one of the national reference polio laboratories in Indonesia that conduct diagnostic test for AFP case specimens from the Western part of Indonesia. AFP cases can be caused by poliovirus and non polio viruses, such as enteroviruses. This recent study investigate the serotype of Non Polio Entero Viruses (NPEV) from AFP cases in 2007-2010 and its geographical distribution. This recent study used biological archieve from AFP cases in 2007-2010. APF specimens was cultured in cell line (RD/L20B) in BSL-2 laboratory. Positive isolates from RD cell lin in negative in L20B cell line in were tested by neutralization test to determine serotype of NPEV. Demography data were also collected from case investigation form of the patients and geographical data was analyzed using GIS arc View 3.3. From a total 2525 AFP cases, 6,8% are NPEV. NPEV cases were mostly found in North Sumater province (20,4%), There is variation fo the number of NPEV cases found each year in 16 province that sen AFP specimens to NIHRD. NPEV cases is commonly found in age group 1-5 years old, (54,3% in 2007, 86,7% in 2008, 62,2% in 2009 and 66,3% in 2010). Serotyping using WHO standard antisera showed that Echovirus is the common serotype found in 2007-2010. However, there were unidentified or untypeable NPEV isolates that need further investigation. NPEV is one of the etiologies that cause Acute Flaccid Paralysis (AFP) in Western part of Indonesia. Further detection of NPEV serotype using molecular tehcnology is needed to reveal other etiologies of AFP. Key words : Acute Flaccid Paralysis, Non Polio Enterovirus, Serotype Abstrak Surveilans lumpuh layu akut atau Acute Flaccid Paralysis (AFP) yang terdiri dari investigasi kasus dan pemeriksaan laboratorium telah ditetapkan oleh WHO sebagai bagian program pemberantasan polio global. Laboratium Virologi Badan Litbangkes adalah salah satu laboratium rujukan Nasional penyakit polio di Indonesia yang melakukan tes diagnostik untuk spesimen kasus AFP dari Indonesia bagian Barat. Kasus AFP dapat disebabkan oleh virus polio dan virus non polio seperti enterovirus. Tujuan penelitian ini adalah untuk menyelidiki serotipee Non Polio Entero Virus (NPEV) dari kasus AFP pada tahun 2007-2010 dan distribusi geografisnya. Studi ini menggunakan spesimen tersimpan dari kasus AFP pada tahun 2007-2010. Spesimen AFP dikultur dalam lini sel (RD/L20B) di laboratorium BSL-2. Isolat positif dari lini sel RD dan negatif pada lini sel L20B diuji netralisasi untuk menentukan serotipe NPEV. Data denografi juga dikumpulkan dari bentuk kasus investigasi dari pasien sedangkan, data geografis dianalisis menggunakan GIS View 3.3. Dari total 2625 kasus AFP, ditemukan 6,8% kasus NPEV. Kasus NPEV terbanyak ditemukan di provinsi Sumatera Utara (20,4%). Terdapat variasi jumlah kasus NPEV yang ditemukan setiap tahunnya pada 16 provinsi yang mengirim spesimen AFP ke Laboratium Badan Litbangkes. Kasus NPEV umunya ditemukan dalam kelompok usia 1-5 tahun, (54,3% pada tahun 2007, 86,7% pada tahun 2008, 62,2% pada tahun 2009, dan 66,3% pada tahun 2010). Serotyping menggunakan antiserum standar WHO menunjukkan bahwa Echovirus adalah serotipe yang paling banyak ditemukan pada spesimen AFP yang dikumpulkan pada tahun 2007-2010. Namun, terdapat isolat NPEV tidak dikenal atau untypeable yang perlu penyelidikan lebih lanjut. Echovirus merupakan salah satu etiologi yang menyebabkan Acute Flaccid Paralysis (AFP) di Indonesia bagian Barat. Deteksi lebih lanjut serotipe NPEV menggunakan teknologi molekuler diperlukan untuk mengungkapkan etiologi lain dari AFP. Kata Kunci : Acute Flaccid Paralysis, Non Polio Enterovirus,Serotype

Effectiveness of some microorganisms in the limitation of grapevine cuttings infection by Phomopsis viticola Sacc.

The possibilities of using antagonistic fungi and bacteria in the limitation of grapevine stems infection by <i>Phomopsis viticola</i> Sacc. were studied. <i>Trichodema koningii</i> Oud., <i>T.viride</i> Persoon ex S.F.,<i>T.harzianum Rifai, Gliocladium catenulatum</i> Gilman and Abbott, <i>G.fimbriatum</i> Gilman and Abbott, Bacillus sp., <i>Pseudomonas fluorescens</i> and five unidentified isolates of bacteria i.e.: 22a, 35, 40, 45, 66 were estimated. It was appeared what <i>Trichoderma</i> spp. were the most effective in protection of grapevine stems against the infection by <i>P.viticola</i>. After these antagonistic fungi were used on protected grapevine canes not numerous necrosis were observed and few cultures of pathogen were reisolated from them. Moreover, <i>Trichoderma</i> spp. survived on the grapevine stems during the period of experiment. The abilities of other microorganisms tested to protect grapevine cuttings against <i>P.viticola</i> infection and to exist on the stems were less than <i>Trichoderma</i> spp.

Physiological characteristics of the trunk sap rot pathogen Fomitiporia sp. on the “Sanbu-sugi” cultivar of Cryptomeria japonica

An unidentified Fomitiporia sp. initially identified as Fomitiporia punctata, causes severe trunk sap rot on Cryptomeria japonica cultivar “Sanbu-sugi”. We investigated the physiological characteristics of the mycelia of the causal fungus (F2, F6, and F43), in comparison with F. punctata (Fp) as a reference, in eight different experiments. The three unidentified isolates showed similar tendencies in change in mycelial growth during incubation, optimal growth temperatures (25 °C), optimal pH range (pH 5–6), glucose to yeast extract ratio (45), utilizable carbon sources (amylose, CM-cellulose, and pectin), utilizable nitrogen sources (yeast extract and polypepton), and water potential (−1.7 Mpa).

Trichosporon vanderwaltii sp. nov., an asexual basidiomycetous yeast isolated from soil and beetles

During a survey of unidentified yeast isolates deposited in the UNESCO-MIRCEN Biotechnological Yeast Culture Collection housed at the Department of Microbial, Biochemical and Food Biotechnology of the University of the Free State, one isolate obtained from soil in South Africa showed 100 % identity in D1/D2 rDNA sequence with undescribed basidiomycetous yeasts isolated from the gut of beetles from the United States of America and forest soil from Taiwan in the NCBI sequence database. Phylogenetic analyses using sequences of the D1/D2 rDNA and ITS regions indicated that all these isolates form a well-supported sub-clade that is the sister clade to the Brassicae plus Porosum clades of Trichosporon in the order Trichosporonales. Subsequent phenotypic tests revealed that asexual reproduction by budding is rare but dominated by arthroconidia resulting from segmentation of hyphae and that fusiform giant cells are characterized by budding from a broad base. These findings further suggest that these isolates belong to a single tremellomycetous yeast species for which the name Trichosporon vanderwaltii CBS 12124(T) (=NRRL Y-48732(T), =UOFS Y-1920(T)) is proposed.

High level multiple antibiotic resistance among fish surface associated bacterial populations in non-aquaculture freshwater environment

Freshwater fish, Alburnus alburnus (bleak), were captured from Lake Mogan, situated in Ankara, during spring. The surface mucus of the fish was collected and associated bacteria were cultured and isolated. By sequencing PCR-amplified 16S RNA encoding genes, the isolates were identified as members of 12 different genera: Acinetobacter, Aeromonas, Bacillus, Brevundimonas, Gordonia, Kocuria, Microbacterium, Mycobacterium, Pseudomonas, Rhodococcus, and Staphylococcus, in addition to one strain that was unidentified. The mucus-dwelling bacterial isolates were tested for resistance against ampicillin, kanamycin, streptomycin and chloramphenicol. About 95% of the isolates were found to be resistant to ampicillin, 93% to chloramphenicol, and 88% to kanamycin and streptomycin. A Microbacterium oxydans and the unidentified environmental isolate were resistant to all four antibiotics tested at very high levels (>1600 μg/ml ampicillin and streptomycin; >1120 μg/ml kanamycin; >960 μg/ml chloramphenicol). Only a Kocuria sp. was sensitive to all four antibiotics at the lowest concentrations tested (3.10 μg/ml ampicillin and streptomycin; 2.15 μg/ml kanamycin; 1.85 μg/ml chloramphenicol). The rest of the isolates showed different resistance levels. Plasmid isolations were carried out to determine if the multiple antibiotic resistance could be attributed to the presence of plasmids. However, no plasmid was detected in any of the isolates. The resistance appeared to be mediated by chromosome-associated functions. This study indicated that multiple antibiotic resistance at moderate to high levels is common among the current phenotypes of the fish mucus-dwelling bacterial populations in this temperate, shallow lake which has not been subjected to any aquaculturing so far but under anthropogenic effect being in a recreational area.

New phomactin analogues from an unidentified fungicolous fungal isolate obtained from the surface of a polypore

Our ongoing search for new bioactive natural products from fungicolous fungi led to investigation of the ethyl acetate extract of cultures of an undetermined dematiaceous fungus (MYC-1907) obtained from the surface of a polypore found on a dead hardwood branch collected in Lava Tree State Park, Hawaii. The extract showed antifungal activity against Aspergillus flavus and antiinsectan activity against fall armyworm. Chemical investigation of this extract led to the isolation of six new phomactin analogues, in addition to four known compounds (phomactins F, I, and J and epi-phomactin I). Previously reported phomactins have shown potent activity as platelet activating factor (PAF) antagonists. The groß structures of the new analogues were established using various 1D and 2D NMR techniques. Relative configurations were established by interpretation of NOESY data, and absolute configurations were assigned using X-ray diffraction analysis and/or electronic circular dichroism (ECD) in conjunction with computational methods. Absolute configurations of three additional new phomactin analogues isolated in our lab from an extract of different isolate (Mycelia sterilia MYC-1969) were also proposed by analysis of their ECD spectra. One of the latter phomactins showed significant antifungal activity against A. flavus and antibacterial activity against Escherichia coli and Staphylococcus aureus.

Molecular data place the hyphomycetous lichenicolous genus Sclerococcum close to Dactylospora (Eurotiomycetes) and S. parmeliae in Cladophialophora (Chaetothyriales)

The lichenicolous anamorphic fungus Sclerococcum parmeliae was isolated in pure culture, and ITS, nuLSU and mtSSU sequences were obtained from these isolates. For comparison, sequences from S. sphaerale, the generic type, were obtained directly from freshly collected specimens. Phylogenetic analyses place S. sphaerale with species of Dactylospora and an unidentified lichen-inhabiting isolate in a strongly supported clade that is sister to a lineage comprising members of the Chaetothyriales and Pyrenulales. In contrast, S. parmeliae is inferred as a member of the Herpotrichiellaceae (Chaetothyriales) and belongs to a robustly supported clade that also includes species of Cladophialophora, Capronia semiimmersa, and Phialophora verrucosa. Within the Herpotrichiellaceae, S. parmeliae most closely resembles members of the anamorph genus Cladophialophora. Accordingly, we propose the transfer of S. parmeliae and the morphologically similar species S. cladoniae, S. hawksworthii and S. normandinae to Cladophialophora. A new lichenicolous species, Clad. megalosporae, collected twice on Megalospora in Florida and Papua New Guinea, is also described.

Bacterial Community Composition and Diversity in Methane Charged Sediments Revealed by Multitag Pyrosequencing

Bacterial diversity in sediments obtained along the Chilean margin from areas containing methane seeps, and a hydrate mound were explored by cloning and sequencing and multitag pyrosequencing (MTPS). These libraries were statistically compared to determine the robustness of taxonomic assignment derived from multiplexed pyrosequencing strategies targeting variable regions V1 and V2 of the small subunit rRNA gene for environmental studies. There was no statistical difference in the composition of the libraries, thus, MTPS was utilized to describe diversity in three geochemical zones in these environments. Unidentified Cyanobacteria isolates were abundant in the sulfate reduction zone (SRZ), Deltaproteobacteria were concentrated at the sulfate methane transition zone (SMTZ) and Chloroflexi/GNS dominated methanogenesis zone (MGZ). Although there was variation among specific groups, communities in the SRZ and MGZ did not differ significantly. However, the community dominated by Deltaproteobacteria differentiates the SMTZ from the other zones. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental file.

Environmental isolation and characterisation of Cryptococcus species from living trees in Havana city, Cuba

Cryptococcus isolates from Cuban patients were identified as C. neoformans var. grubii. Although this species has since long been associated with bird droppings, a recent genotyping study provided strong evidence for additional origins of exposure. We sampled different species of trees in Havana, Cuba to identify other potential sources of exposure to this fungus. A total of 662 samples were collected from 331 trees and cacti from Havana, Cuba. Initial selection of the isolates was carried out by conventional techniques. Isolates were further characterised using a combination of AFLP analysis and DNA sequence analysis. Identification by conventional methods yielded 121 C. neoformans and 61 C. gattii isolates. Molecular analyses showed that none of these isolates was C. gattii and only one isolate proved to be C. neoformans var. grubii. A total of 27 different other species were identified. The most prevalent species was C. heveanensis (33%). Sixty-five unidentifiable isolates segregated into ten potentially novel species. Conventional cultivation methods have a low specificity for C. neoformans complex and molecular analyses need to be applied to confirm identification of isolates from environmental sources. Environmental niches responsible for most of human cryptococcal infections in Cuba remain to be identified.

Screening and Phylogenetic Analysis of Deep-Sea Bacteria Capable of Metabolizing Lignin-Derived Aromatic Compounds

Lignin is one of the most abundant biomasses in nature. It is composed of aromatic moieties and has great potential for use in the production of chemical alternatives to petroleum products. Because of increasing interest in biocatalysis, the potential for industrial application of microbial metabolism of lignin-derived compounds has gained considerable recent attention. Functional screenings of culturable bacteria isolated from sediments and sunken wood collected from the deep sea revealed the existence of a number of previously unidentified bacteria capable of metabolizing lignin-related aromatic compounds. Of the 510 isolates obtained in the present study, 208 completely or partially metabolized these compounds. The 208 isolates were classified into diverse phyla, including Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria. Among the 208 isolates, 61 unique 16S rRNA gene sequences were detected including previously unidentified marine lineage isolates. The metabolites of the isolates were analysed using liquid chromatography/mass spectrometry (LC/MS) or gas chromatography/mass spectrometry (GC/MS). Most of the representative 61 isolates non-oxidatively decarboxylated the substrates to produce the corresponding aromatic vinyl monomers, which are used as feed stocks for bio-based plastics production. Oxidative metabolism of the lignin-related compounds for assimilation was frequently observed. Our study showed that the deep-sea environment contains an abundance of microorganisms capable of both non-oxidative and oxidative bioconversion of lignin-derived aromatic compounds. The ability for bio-conversion of aromatic compounds found in this study will facilitate the development of future biotechnological applications.

First Report of Boeremia Blight Caused by Boeremia exigua var. exigua on Pyrethrum in Australia.

Pyrethrum (Tanacetum cinerariifolium) is produced for extraction of insecticidal compounds from the flower achenes. In 2004 and 2006, isolations from necrotic lesions on stems and leaves in three fields in northern Tasmania, Australia yielded four unidentified fungal isolates. Leaf lesions were medium brown and circular (2 to 4 mm in diameter) or irregular in shape (2 to 5 mm long). Stem lesions were irregular, necrotic spots, 5 to 15 mm below the flower peduncle, medium brown, 2 to 4 mm long, and 1 to 2 mm wide. Isolations were conducted on water agar following surface sterilization. Isolates were identified by colony characteristics and the presence of metabolite 'E' (1). On oatmeal agar (OA), colonies had irregular margins, were greenish olivaceous-to-olivaceous gray with sparse, white, floccose, aerial mycelia. On malt extract agar (MEA), cultures were variable in color with olivaceous black centers with soft, dense, aerial mycelia. Conidia were hyaline, ellipsoidal to oblong, mainly aseptate, but occasionally 1-septate with dimensions ranging from 2.5 to 7.5 × 1.8 to 3.8 μm (length/width ratio = 1.7 to 2.1). All isolates had moderate reactions to the NaOH test for metabolite 'E'. DNA was extracted from all four isolates with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA). For identification, the internal transcribed spacer region (ITS1, 5.8s, and ITS2) and part of the translation elongation factor (TEF) region were amplified and sequenced. Primers ITS1 and ITS4 (2) were used for the ITS region and primers EFCF1 (5'-AGTGCGGTGGTATCGACAAG) and EFCF6 (3'-CATGTCACGGACGGCGAAAC) were used for the TEF. Amplicons were sequenced in both directions and consensus sequences assembled. The ITS sequence was 100% identical to Boeremia exigua var. exigua (GenBank Accession No. GU237715). Base pairs 413 to 1,214 of the TEF sequence from the pyrethrum isolates matched base pairs 1 to 802 (799 of 802 identities) of B. exigua var. exigua (GenBank Accession No. GU349080). All isolates were confirmed as B. exigua var. exigua using morphology and sequencing. Pathogenicity tests were conducted three times in separate glasshouse trials for two of the four isolates. For each isolate, conidial suspensions in water (3 ml/plant) from MEA, adjusted to 5 × 105/ml were applied with Tween 20 (1 drop per 100 ml of water) to 8-week-old pyrethrum plants (five pots per isolate with four plants per pot) using a hand-held spray bottle. Twenty plants were sprayed with water and Tween 20 as nontreated controls. Plants were covered with plastic bags for 48 h after inoculation and examined for symptoms after 15 days at 20°C. Disease incidence (number of symptomatic leaves affected per total number of leaves) of the inoculated plants varied from 7.5 to 9.4%. Noninoculated plants did not develop symptoms. Isolations resulted in cultures morphologically identical on MEA and OA to those inoculated. To our knowledge, this is the first report of B. exigua var. exigua causing disease in pyrethrum. Cultures were deposited in the New South Wales Department of Agriculture collection (DAR79101 to 79104) and TEF and ITS sequences for DAR79101 in GenBank (Accession Nos. JF925328 and JF925329, respectively). Boeremia blight is likely to contribute to the fungal disease complex causing reductions in green leaf area in Australian pyrethrum production.

Specific and Sensitive Detection of Enterobacter mori Using Reliable RT-PCR.

Enterobacter mori, the causal agent of bacterial wilt in mulberry, is becoming a serious disease in mulberry orchards in China. Because no effective control strategy has been devised for this disease, the reliable screening of mulberry material for latent infection became necessary. Hence, a fast polymerase chain reaction (PCR) assay for the detection of E. mori was developed in this study. The primers were designed within regions of the RNA polymerase β-subunit (rpoB) gene. The method is fast and simple and showed 100% sensitivity (no false negatives) and 100% specificity (no false positives), which was tested with 4 representative E. mori strains, 9 Enterobacter type strains, 2 strains of the other major mulberry bacterial pathogens (Ralstonia solanacearum and Pseudomonas syringae pv. mori) in China, 7 strains of other plant-associated pathogens, and 50 unidentified epiphytic bacterial isolates from mulberry plants. The real-time PCR assays reliably detected the DNA at at least 10 fg/μl and the bacterial cells at 102 CFU/ml from mulberry shoots and roots suspension. The strong positive reaction in testing of all symptomatic plants (with 100% positive) and parts of asymptomatic latent infected plant samples (with 36.4% positive) provided proof that this method is reliable and sensitive and suitable for screening plant material with latent infections of E. mori.

Evaluation of MALDI-TOF MS as a tool for high-throughput dereplication

The present study examined the suitability of matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid grouping of bacterial isolates, i.e. dereplication. Dereplication is important in large-scale isolation campaigns and screening programs since it can significantly reduce labor intensity, time and costs in further downstream analyses. Still, current dereplication techniques are time consuming and costly. MALDI-TOF MS is an attractive tool since it performs fast and cheap analyses with the potential of automation. However, its taxonomic resolution for a broad diversity of bacteria remains largely unknown. To verify the suitability of MALDI-TOF MS for dereplication, a total of 249 unidentified bacterial isolates retrieved from the rhizosphere of potato plants, were analyzed with both MALDI-TOF MS and repetitive element sequence based polymerase chain reaction (rep-PCR). The latter technique was used as a benchmark. Cluster analysis and inspection of the profiles showed that for 204 isolates (82%) the taxonomic resolution of both techniques was comparable, while for 45 isolates (18%) one of both techniques had a higher taxonomic resolution. Additionally, 16S rRNA gene sequence analysis was performed on all members of each delineated cluster to gain insight in the identity and sequence similarity between members in each cluster. MALDI-TOF MS proved to have higher reproducibility than rep-PCR and seemed to be more promising with respect to high-throughput analyses, automation, and time and cost efficiency. Its taxonomic resolution was situated at the species to strain level. The present study demonstrated that MALDI-TOF MS is a powerful tool for dereplication.

Control of chickpea wilt (Fusarium oxysporum f.sp. ciceris) using Trichoderma spp. in Ethiopia

Thirty-two Trichoderma isolates were collected from soils grown with chickpea in central highlands of Ethiopia. The eight isolates were identified by CAB-International as Trichoderma harzianum, T. koningii and T. pseudokoningii. In in vitro tests, all Trichoderma isolates showed significant (P < 0.05) differences in their colony growth and in inhibiting the colony growth of Fusarium oxysporum f.sp. ciceris, race 3. In potted experiment, four Trichoderma isolates were tested as seed treatment on three chickpea cultivars (JG-62 susceptible, Shasho moderately susceptible and JG-74 resistant) against F. oxysporum f.sp. ciceris, race 3. The result showed that T. harzianum and unidentified Trichoderma isolate T23 significantly reduced wilt severity and delayed disease onset. The degree of wilt severity and delay of disease onset varied with chickpea cultivars. Our study revealed that biological control agents such as Trichoderma can be a useful component of integrated chickpea Fusarium wilt management.

Re-analysis of 178 previously unidentifiable Mycobacterium isolates in the Netherlands in 1999–2007

Nontuberculous mycobacteria (NTM) that cannot be identified to the species level by reverse line blot hybridization assays and sequencing of the 16S rRNA gene comprise a challenge for reference laboratories. However, the number of 16S rRNA gene sequences added to online public databases is growing rapidly, as is the number of Mycobacterium species. Therefore, we re-analysed 178 Mycobacterium isolates with 53 previously unmatched 16S rRNA gene sequences, submitted to our national reference laboratory in 1999–2007. All sequences were again compared with the GenBank database sequences and the isolates were re-identified using two commercially available identification kits, targeting separate genetic loci. Ninety-three out of 178 isolates (52%) with 20 different 16S rRNA gene sequences could be assigned to validly published species. The two reverse line blot assays provided false identifications for three recently described species and 22 discrepancies were recorded in the identification results between the two reverse line blot assays. Identification by reverse line blot assays underestimates the genetic heterogeneity among NTM. This heterogeneity can be clinically relevant because particular sub-groupings of species can cause specific disease types. Therefore, sequence-based identification is preferable, at least at the reference laboratory level, although the exact targets needed for clinically useful results remain to be established. The number of NTM species in the environment is probably so high that unidentifiable clinical isolates should be given a separate species status only if this is clinically meaningful.

Isolasi dan Identifikasi Kapang Saprofitik pada Sampel Tanah di Sekitar Kawasan Gunung Gamalama, Ternate

Microorganisms, such as mould and bacteria, have important roles in organic matter decomposition; and this process is fundamental to maintain soil structure and fertility. Moulds have hypha with filament structures that can penetrate to the substrates. Their enzymatic capability is higher than that of bacteria, especially in decomposing organic compounds like lignin and cellulose. The objective of the study was to isolate and identify saprophytic mould inhabiting soil of Mount Gamalama, Ternate, North Moluccas. The mould isolation was based on sample dillution method with Rose Bengal Cloramphenicol Agar isolation media. The abundance of saprophytic mould counted by measuring the average Colony Forming Unit (CFU)/ml of all mould colonies which grown on isolated media by Total Plate Count (TPC) method. The diversity of isolated mould was identified based on phenotypic characters by observing both macroscopic and microscopic morphology. The result showed that the average of mould colonies were between 13,30 x10 4 and 78,15 x10 4 CFU/ml. Identification based on morphology from selected mould isolates showed that 25 fungal taxa and two groups of unidentified fungal isolates were identified.

Barcoding Tetrahymena: Discriminating Species and Identifying Unknowns Using the Cytochrome c Oxidase Subunit I (cox-1) Barcode

DNA barcoding using the mitochondrial cytochromecoxidase subunit I (cox-1) gene has recently gained popularity as a tool for species identification of a variety of taxa. The primary objective of our research was to explore the efficacy of using cox-1 barcoding for species identification within the genusTetrahymena. We first increased intraspecific sampling forTetrahymena canadensis, Tetrahymena hegewischi, Tetrahymena pyriformis, Tetrahymena rostrata, Tetrahymena thermophila, and Tetrahymena tropicalis. Increased sampling efforts show that intraspecific sequence divergence is typically less than 1%, though it may be more in some species. The barcoding also showed that some strains might be misidentified or mislabeled. We also used cox-1 barcodes to provide species identifications for 51 unidentified environmental isolates, with a success rate of 98%. Thus, cox-1 barcoding is an invaluable tool for protistologists, especially when used in conjunction with morphological studies.

Metagenomic sequencing for virus identification in a public-health setting

The use of metagenomics for virus discovery in clinical samples has opened new opportunities for understanding the aetiology of unexplained illness. This study explores the potential of this sequence-independent approach in a public-health setting, by systematic analysis of samples cultured from patients with unexplained illness through a combination of PCR-based assays and viral metagenomics. In total, 1834 cell-culture isolates were collected between 1994 and 2007 through the Enterovirus Surveillance programme in the Netherlands. During the 13 year period, seven samples that exhibited reproducible cytopathogenic effects in cell culture tested negative in standard PCR assays for a range of viruses. In order to fill the diagnostic gap, viral metagenomics was applied to these culture supernatants, resulting in the rapid identification of viruses in all of the samples. The unexplained samples contained BK polyomavirus, herpes simplex virus, Newcastle disease virus and the recently discovered Saffold viruses (SAFV) (which dominated the unexplained samples; n=4). The full genomic sequences of four SAFV genotype 3 (SAFV-3) viruses, which share 88-93 % nucleotide identity with known SAFV-3 viruses, are reported. Further screening for SAFV in additional cultured, unidentified clinical isolates from 2008 and 2009 resulted in identification of another SAFV-positive sample. Although the pathogenicity of the identified viruses has not been established, this study demonstrates that viral metagenomics is a powerful tool that can be integrated into public-health monitoring efforts to investigate unidentified viruses in cell cultures from clinical isolates where standard PCR assays fail to detect viruses.