rDNA PCR Products Research Articles

Chromosomal mapping of 5S rDNA in two species of the genus Acanthocephalus (Echinorhynchida)

Chromosomal mapping of 5S rDNA in two Acanthocephala species was performed for the first time using fluorescence in situ hybridization (FISH) with a 5S rDNA probe. The 5S rDNA PCR products from the genomes of both species were sequenced and aligned and an identical 141 bp long coding region was determined. The same patterns of 5S rDNA gene cluster distribution were observed, with FISH signal restricted to a single autosomal chromosome pair. A preference for distal positioning on the chromosomes (subtelomeric position) was observed in both species. In addition, two-color FISH was performed to examine the mutual positions of 5S and 18S rDNA on the chromosomes. Our knowledge of the organization of the Acanthocephala genome is extremely limited and its chromosomes are poorly studied. Any new information about the location of chromosomal markers as important features of the respective karyotype may be useful in solving evolutionary questions.

Isolation and Identification of Green Microalgae from Northern Jordan

In light of the rapid and extreme changes in climate and the steady depletion of natural resources, there is an urgent need to find innovative and sustainable solutions to these problems. Microorganisms such as microalgae can offer viable solutions to these challenges. Proper investment in such organisms requires the identification of the algal species that inhabit the region. Therefore, this study aimed to isolate and molecularly characterize green microalgae that inhabit freshwater at different locations in the governorates of Irbid and Ajloun in the northern region of Jordan. Water samples collected from these regions were used to isolate single colonies, some of which exhibited different morphological characteristics. Genomic DNA was extracted from the isolates and used as a template for PCR amplification of the 18S ribosomal DNA gene (18S rDNA) and the internal transcribed spacer (ITS) region. Phylogenetic trees were constructed based on 18S rDNA and ITS PCR product sequences, which were used to identify the isolates at the genus level. The obtained isolates belonged to three genera; Coelastrella, Desmodesmus, and Monoraphidium. The latter species has not been previously reported in Jordan.

First Report of 'Candidatus Phytoplasma solani' (16SrXII-A) Associated with Fox Nut (Euryale ferox Salisb.) Stolbur Disease in India.

Fox nut also known as Gorgon nut, Makhana (Euryale ferox Salisb.) is a high value aquatic crop belonging to the family Nymphaeaceae. In India, it is generally grown in flood prone areas of North Bihar, lower Assam, parts of West Bengal, Odisha, Manipur and Tripura (Jana et al., 2018). India contributes nearly 70-80% of the global fox nut production. During September 2021, a phytoplasma like symptom was noticed on fox nut leaves at Basudeopur Farm of Research Centre for Makhana, Darbhanga, Bihar, India (23° 9' N and 65° 53' E). The characteristic symptom was that some portion of leaf lamina deformed along the veins with wrinkled and raised overgrowth or hypertrophy. The veins were thickened and reddened in the infected leaf area. The infection occurs in petiole as well as in flower stalk. The disease incidence was found as high as 30% which caused severe yield loss which was calculated to be 40% in that particular field. Total of 20 sampled fox nut plants, 10 symptomatic and 10 asymptomatic ones, were collected and tested for the presence of phytoplasma. A nested PCR assay using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (J. Jović et al. 2011) amplified the expected ~1.2-kb 16S rDNA fragment in all 10 symptomatic samples. No amplification was detected from asymptomatic samples. One of the ten 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced and was deposited in NCBI under the Accession no.OL873590. BLAST analysis showed that the sequence of the PCR 16S rDNA product was 100% identical to several GenBank sequences of Ca. P. solani (16SrXII Stolbur group) viz. KF907506. Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup. Identification of 'Ca. P. solani' was conducted following the STOL11 stolbur-specific protocol (Radonjić et al. 2009). Sequencing of tuf gene (Elongation factor Tu) was performed by using tuf marker genes (Cvrković et al. 2014) from 10 symptomatic and 10 asymptomatic samples. Sequence of the amplified gene (896 bp) was deposited in GenBank under Accession number OM174272. The presence of 'Ca. P. solani' was detected in all symptomatic samples, while all control plants tested negative. The RFLP analysis of tuf gene nested PCR products using HpaII endonuclease (Fermentas) revealed uniform tuf-b type in all positive samples. Nucleotide blast analyses showed that the tuf gene was 100% identical to STOL11 strain of C. P. solani subgroup 16SrXII-A (Accession No JQ797670). For developing a suitable management strategy, identification of the vector is essential. Leaf hoppers visiting the infected plants as well as nearby crop fields will be tested for presence of the phytoplasma. To the best of our knowledge, this is the first report of Candidatus Phytoplasma solani' (16SrXII-A) infecting Fox nut (Euryale ferox Salisb.) in India. References Cvrković et al. 2014. Plant Pathol. 63:42. https://doi.org/10.1111/ppa.12080 Jana, B. R., et al. 2018. Int. J. Curr. Microbiol. App. Sci. 7(12): 578-587. https://doi.org/10.20546/ijcmas.2018.712.072 Jović, J. et al. 2011. B. Insectol. 64:S83. ISI Radonjić, S. et al. 2009. J. Phytopathol. 157:682. https://doi.org/10.1111/j.1439- 0434.2009.01560.

Campylobacter jejuni subdural hygroma infection in a 2-year old boy: case report and a brief literature review

BackgroundCampylobacter jejuni is a common cause of acute gastroenteritis, but central nervous system infections are rare manifestations of Campylobacter infection. Therefore, C. jejuni trauma-related subdural hygroma infection in children is poorly described in the literature.Case presentationWe described a 2-year old boy with lobar holoprosencephaly presenting with subdural hygroma following head trauma. C. jejuni infection was confirmed from a subdural hygroma sample by culture as well as by DNA sequencing of a broad range 16S rDNA PCR product. Cerebrospinal fluid from the ventriculoperitoneal shunt remained sterile. Combined neurosurgical and antimicrobial treatment led to complete recovery. Review of the literature showed that the most common manifestation of Campylobacter central nervous system infection is meningitis, mostly in neonates, and subdural hygroma infection was described for only one case.ConclusionsSubdural hygroma infection caused by C. jejuni is a rare clinical condition in children. Molecular methods represent an important tool for the detection of rare or unexpected pathogens. No standard recommendations for antimicrobial treatment of C. jejuni subdural space infection in children are available, but meropenem treatment combined with surgery seems to be an effective approach.

Isolation of a cellulase producing Bacillus cereus from cow dung and determination of the kinetic properties of the crude enzyme

The objective of the study was to isolate a thermostable alkaline cellulase producing bacterial strain from diverse natural sources such as goat excreta, cow dung, tropical soil, and organic matter. Opened hot cellulose agar plates were transferred on to the selective carboxy methyl cellulose (CMC) agar media and incubated for 2 days at 37 oC. The samples were cultured on Nutrient Agar in order to isolate cellulolytic bacteria. The bacteria isolated were screened for cellulolytic activity using serial dilution and pour-plate method after which they were characterised. The bacterial isolate with the highest CMC hydrolytic capacity which was obtained from cow dung was selected for further studies. Based on the morphological, biochemical and cultural analysis, the strain from cow dung was been identified as Bacillus sp. Pure culture of this bacterial strain was grown overnight, DNA was extracted and 16S rDNA was amplified by thermocycler using universal primers. When the amplified 16S rDNA PCR product was sequenced using automated sequencer and sequence similarity search was done for the 16S rDNA sequence using BLAST, the unknown organism was identified as Bacillus cereus with the GenBank accession no AF290555. Cellulase from Bacillus cereus showed zero order kinetics for 20 minutes. The optimum pH for the crude cellulase from Bacillus cereus was 9.0 at 45oC and the temperature optimum was 45oC at pH 9.0. Michaelis constant for the cellulase from this isolate to soluble cellulose was 38.60 g-1L and Vmax was 3.32 µmol mL-1 min-1 at pH 9.0 and at 45oC. Since this cellulase enzyme was stable for more than an hour at 40oC and at alkaline pH values, it could be a good candidate for industrial applications.

Isolation ofRickettsia,Rickettsiella, andSpiroplasmafrom Questing Ticks in Japan Using Arthropod Cells

Ticks are blood-sucking ectoparasites that transmit zoonotic pathogens to humans and animals. Ticks harbor not only pathogenic microorganisms but also endosymbionts. Although some tick endosymbionts are known to be essential for the survival of ticks, their roles in ticks remain poorly understood. The main aim of this study was to isolate and characterize tick-borne microorganisms from field-collected ticks using two arthropod cell lines derived from Ixodes scapularis embryos (ISE6) and Aedes albopictus larvae (C6/36). A total of 170 tick homogenates originating from 15 different tick species collected in Japan were inoculated into each cell line. Bacterial growth was confirmed by PCR amplification of 16S ribosomal DNA (rDNA) of eubacteria. During the 8-week observation period, bacterial isolation was confirmed in 14 and 4 samples using ISE6 and C6/36 cells, respectively. The sequencing analysis of the 16S rDNA PCR products indicated that they were previously known tick-borne pathogens/endosymbionts in three different genera: Rickettsia, Rickettsiella, and Spiroplasma. These included four previously validated rickettsial species namely Rickettsia asiatica (n = 2), Rickettsia helvetica (n = 3), Rickettsia monacensis (n = 2), and Rickettsia tamurae (n = 3) and one uncharacterized genotype Rickettsia sp. LON (n = 2). Four isolates of Spiroplasma had the highest similarity with previously reported Spiroplasma isolates: Spiroplasma ixodetis obtained from ticks in North America and Spiroplasma sp. Bratislava 1 obtained from Ixodes ricinus in Europe, while two isolates of Rickettsiella showed 100% identity with Rickettsiella sp. detected from Ixodes uriae at Grimsey Island in Iceland. To the best of our knowledge, this is the first report on successful isolation of Rickettsiella from ticks. The isolates obtained in this study can be further analyzed to evaluate their pathogenic potential in animals and their roles as symbionts in ticks.

Comparison of ten different DNA extraction procedures with respect to their suitability for environmental samples

DNA extraction for molecular biological applications usually requires target optimized extraction procedures depending on the origin of the samples. For environmental samples, a range of different procedures has been developed. We compared the applicability and efficiency of ten selected DNA extraction methods published in recent literature using four different environmental samples namely: activated sludge from a domestic wastewater treatment plant, river sediment, anaerobic digestion sludge and nitrifying enrichment culture. We assessed the suitability of the extraction procedures based on both DNA yield and quality. DNA quantification was performed by both ultra violet (UV) spectrophotometry and fluorescence spectrophotometry after staining with PicoGreen.In our study, DNA yields based on UV measurement were overestimated in most cases while DNA yields from fluorescence measurements correlated well with the sample load on agarose gels of crude DNA. The quality of the DNA extracts was determined by gel electrophoresis of crude DNA and PCR products from 16S rDNA with the universal primer set 27f/1525r. It was observed that gel electrophoresis of crude DNA was not always suitable to evaluate DNA integrity and purity since interfering background substances (e.g. humic substances) were not visible. Therefore, we strongly recommend examining the DNA quality of both crude DNA and 16S rDNA PCR products by gel electrophoresis when a new extraction method is established.Summarizing, we found four out of ten extraction procedures being applicable to all tested samples without noticeable restrictions. The procedure G (according to the standard method 432_10401 of the Lower Saxony State Office for Consumer Protection and Food Safety) had the broadest application range over procedure J (published by Wilson, 2001). These were followed by procedures F (Singka et al., 2012) and A (Bourrain et al., 1999). All four extraction procedures delivered reliable and reproducible crude DNA and PCR products. From an economical point of view, all procedures tested during this study were cheaper compared to commercial DNA extraction kits.

Towards more sustainable surimi? PCR-cloning approach for DNA barcoding reveals the use of species of low trophic level and aquaculture in Asian surimi

In recent years, authentication of commercialized seafood products has become a market priority. In this study, 29 Asian surimi products produced in China, India and Singapore, and commercialized in two Mediterranean countries (Egypt and Spain), were analyzed in order to authenticate species contained in surimi products. Due to the processing treatments, classical identification methods are not effective. Therefore, we conducted two molecular tracing methodologies for species identification: direct sequencing of 16S rDNA PCR products, and DNA mini-barcoding-based PCR cloning for Cytochrome oxidase subunit I (COI), with subsequent plasmid sequencing. In total, 10 fish species corresponding to 7 families were found. The Singaporean and Chinese surimi contained principally species of low trophic levels, like fringe scale sardines Sardinella fimbriata, and other, likely farmed, species such as striped catfish Pangasianodon hypophthalmus. The presence of low trophic level and aquaculture species suggests that current trends in surimi production are moving towards sustainability. The exception was a vulnerable shovelnose ray (Rhinobatos jimbaranensis) found in one product, which encourages further studies to detect the use of endangered species in such morphologically indistinct food items. We suggest PCR-cloning methodology for efficient species authentication in seafood controls, especially for mixed products.

Prevalence and subtype analysis of Blastocystis in healthy Indian individuals

There is a growing interest in subtype (ST) analysis of the intestinal parasite Blastocystis due to its extensive genetic diversity that might reflect differences in pathogenicity. Although essential for reference, few studies are available on Blastocystis in healthy individuals. Moreover, molecular epidemiology data on Blastocystis in India still remain to emerge. In the present study we identified the prevalence and ST distribution of Blastocystis in healthy Indian individuals. A total of 220 stool samples were obtained; four of 100 samples from 100 adults were chosen randomly for construction of small subunit (SSU) rRNA gene clone libraries in order to elucidate micro-eukaryotic diversity in the human gut. From the SSU rDNA library, 64 sequences annotated to Blastocystis were used for ST analysis along with sequences obtained by direct sequencing of SSU rDNA PCR products amplified from the remaining samples and generated using primers targeting Blastocystis. Of 220 stool samples collected, 120 samples from 30 infants (aged 1week to 1year) were PCR-negative. Of the remaining 100 samples from 100 adults, 27 resulted in specific amplification. Out of these 27, four samples were suspected of mixed ST infection and so these samples were further analyzed by construction of clone libraries. Analysis of cloned sequences revealed that indeed 2 samples had mixed ST infection (ST1 and ST3) while the remaining two showed infection with two separate ST3 strains. ST3 was the most common ST present in our study group (100%) followed by ST1 (7.4%); ST1 was seen only in mixed infections. SSU rDNA clone library sequences generated by processing of pooled samples were identified as ST3. The majority of ST3 sequences exhibited allele 34 commonly found in the European population.

First Report of Candidatus Phytoplasma solani Associated with Potato Plants in Greece.

In August 2013, potato plants (Solanum tuberosum) cv. Banba displaying symptoms resembling those caused by Candidatus Phytoplasma solani (potato stolbur phytoplasma) were observed in a 2-ha field in the area of the Peripheral Unit of Drama (northern Greece). The plants were 10 weeks old and their symptoms included reddening and upward rolling of leaflets, reduced size of leaves, shortened internodes, and aerial tuber formation. Incidence of affected plants was estimated to be 40% in the field. Four symptomatic potato plants were collected for laboratory testing of possible phytoplasma infection. From each of these four plants, total DNA was extracted from mid veins of reddish leaflets from apical shoot parts and of leaflets emerging from aerial tubers, using a phytoplasma enrichment procedure (1). A nested PCR using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (3) amplified the expected ~1.2-kb 16S rDNA fragment in all four symptomatic potato plants. No amplification was observed with DNA similarly extracted from leaflets of asymptomatic potato plants of the same variety collected from an apparently healthy crop. One of the four 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced by Beckman Coulter Genomics (United Kingdom). At least twofold coverage per base position of the cloned PCR product was achieved. BLAST analysis showed that the obtained sequence of the PCR 16S rDNA product was: i) 100% identical to several GenBank sequences of Ca. P. solani strains, including strains detected previously in Greece infecting tomato (GenBank Accession No. JX311953) and Datura stramonium (HE598778 and HE598779), and ii) 99.7% similar to that of the Ca. P. solani reference strain STOL11 (AF248959). Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup (AF248959). The sequence of this PCR product was deposited in NCBI GenBank database under the accession no. KJ810575. The presence of the stolbur phytoplasma in all four symptomatic potato plants examined was further confirmed by nested PCR using the stolbur-specific STOL11 primers (3) targeting non-ribosomal DNA. Based on the observed symptoms in the field and laboratory molecular examinations, we concluded that the potato plants were infected by a Ca. P. solani related strain. The stolbur disease has been previously reported in Greece affecting tomato (2,5) and varieties of D. stramonium (4). To our knowledge, this is the first report of a Ca. P. solani related strain infecting a potato crop in Greece. As northern Greece is a center of potato production, the source of this pathogen is to be investigated.

Assessment of helminth biodiversity in wild rats using 18S rDNA based metagenomics.

Parasite diversity has important implications in several research fields including ecology, evolutionary biology and epidemiology. Wide-ranging analysis has been restricted because of the difficult, highly specialised and time-consuming processes involved in parasite identification. In this study, we assessed parasite diversity in wild rats using 18S rDNA-based metagenomics. 18S rDNA PCR products were sequenced using an Illumina MiSeq sequencer and the analysis of the sequences using the QIIME software successfully classified them into several parasite groups. The comparison of the results with those obtained using standard methods including microscopic observation of helminth parasites in the rat intestines and PCR amplification/sequencing of 18S rDNA from isolated single worms suggests that this new technique is reliable and useful to investigate parasite diversity.

Effects of inclusion of waste vinegar residue in the diet of laying hens on chyme characteristics and gut microflora

The objective of this study was to evaluate the effects of inclusion of waste vinegar residue (WVR) in the diet of laying hens on chyme characteristics and gut microflora. A total of 96 60-wk-old Hy-Line Brown laying hens were selected and fed three diets including a corn and soybean meal-based control diet and two other diets with 20g/kg and 40g/kg of WVR. After 27d feeding period, digesta was collected for protease activity, pH value and microflora determination. Bacterial genetic diversity was determined using DGGE analysis of the V3 region of 16S rDNA PCR products (230bp) obtained from primers specific for the domain Bacteria. Inclusion of 20g/kg and 40g/kg of WVR in the diet decreased the digesta pH value in the gizzard (P=0.003) and increased pepsin activities in the gizzard of laying hens (P=0.032). Species of Enterobacter and Peptostreptococcaceae were more predominant in the cecum and ileum of laying hens, respectively. It is concluded that inclusion of 40g/kg of WVR in the laying hen diet modifies intestinal pH value and pepsin activities in upper gastrointestinal tract and affects the lower gut microflora in laying hens.

First Report of Two Distinct Phytoplasma Species, 'Candidatus Phytoplasma cynodontis' and 'Candidatus Phytoplasma asteris,' Simultaneously Associated with Yellow Decline of Wodyetia bifurcata (Foxtail Palm) in Malaysia.

The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV 'Candidatus Phytoplasma cynodontis' group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the 'Candidatus Phytoplasma asteris' 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia.

Babesia lengau associated with cerebral and haemolytic babesiosis in two domestic cats

BackgroundAlthough reported sporadically from various countries, feline babesiosis appears to be a significant clinical entity only in South Africa, where Babesia felis is usually incriminated as the causative agent. Babesia lengau, recently described from asymptomatic cheetahs, has now possibly been incriminated as the causative agent in two severe clinical cases in domestic cats.FindingsBoth cats were euthanised in extremis. While typical feline babesiosis in South Africa is an afebrile disease with a chronic manifestation, there was acute onset of severe clinical signs in both cats and their body temperatures were above the normal range when they were presented for treatment. Haemolytic anaemia was confirmed in one case. To our knowledge, this is the first report of cerebral babesiosis in cats.On reverse line blot 18S rDNA PCR products obtained from both cats showed positive hybridization profiles with the B. lengau species-specific probe. The two partial parasite 18S rRNA gene sequences obtained, showed high sequence similarity (99.9%) to B. lengau. In a representative tree constructed by the neighbor-joining method using the two-parameter model of Kimura the two obtained partial 18S rDNA sequences and that of B. lengau formed a monophyletic group with B. conradae and sequences previously isolated from humans and wildlife in the western USA.ConclusionAll clinical cases of feline babesiosis in South Africa are not necessarily caused by B. felis. Other piroplasms, e.g. B. lengau, may be incriminated in clinical cases, especially those occurring outside the known endemic area.

Morphological and molecular characterization of Gordionus alpestris (Nematomorpha) from the North-West Caucasus, Russia

Abstract During the survey of Pachyiulus krivolutskyi Golovatch, 1977 (Diplopoda) near Nickel’ in Adygei Republic (Russia) in the summer 2009 the infestation of these millipeds with nematomorphs of the species Gordionus alpestris was revealed. The morphology of naturally emerging nematomorphs was studied in SEM. The partial sequences of 18S (883 bp) and 28S (393 bp) rDNA PCR-products were obtained and analyzed. An unexpectedly high level of differences between Gordionus alpestris found in diplopods of North-West Caucasus and other molecularly studied representatives of the family Chordodidae was observed.

16S rRNA gene-based identification of microbiota associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga (Diptera, Psychodidae) from central Amazon, Brazil

Bacteria associated with the parthenogenetic troglobiont sand fly Deanemyia maruaga were characterized by sequencing cloned 16S rDNA PCR products. Eleven novel partial 16S rDNA sequences, with varying degrees of similarity to Actinobacteria, were identified. None of the sequences identified had homology to those known from parthenogenesis-inducing bacteria.

Species Identification of Bacteria and Fungi from Solid and Liquid Culture Media by MALDI-TOF Mass Spectrometry

Within the past years MALDI-TOF mass spectrometry has become a powerful tool for the species identification of cultured bacteria and fungi. In the present study, the implementation of MALDI-TOF mass spectrometry in a commercial high throughput laboratory is described. The impact of different growth conditions on the identification results was evaluated. Although slight differences in MALDI-TOF spectra of E. coli and S. aureus strains cultured on blood agar for various periods (5, 18, 24 and 48 hours) were noticed, reliable species identification was obtained for all periods. The same was true when E. coli and S. aureus strains were cultured for 18 hours on various solid media. Reliable identification was also achieved when fungi were cultured on solid and in liquid media (Sabouraud bouillon). Moreover, growth of fungi in bouillon resulted in accelerated identification. MALDI-TOF mass spectrometry also allowed reliable identification of microorganisms from positive blood culture samples. In total, 2,900 specimens (234 different species) predominantly derived from clinical samples were examined. Microorganisms were cultured on solid media, in blood culture bottles and in liquid Sabouraud bouillon. 98.6% (n=2,860) of the MALDI-TOF identification results matched those of conventional methods (e.g. Gram staining, carbohydrate degradation ability, Phoenix system) and 16S rDNA PCR product sequencing. Mismatches were mainly based on missing reference spectra in the database of the analysis system. The study was performed in 2009. Due to continuous improvement of the database, even higher accuracy would be achieved when performing the study nowadays. In summary, the use of MALDI-TOF mass spectrometry in a clinical high throughput environment leads to reliable results, even when various culture conditions are used, for instance bouillons for culture of fungi. MALDI-TOF mass spectrometry is a fast and robust identification system, which is on its way to become a new standard for the identification of microorganisms in high throughput laboratories.

Correlation of microbiomic profiles with disease status and MDR1 methylation in head and neck squamous cell carcinoma (HNSCC).

5573 Background: Recent studies of the aerodigestive tract have uncovered the functional importance of the microbiome, the total collection of microorganisms that reside within the human host. Furthermore, specific bacterial species have been shown to be etiologic agents or show potential as a screening biomarker in cancers including gastric, colon and pancreas. Here, we hypothesize that specific microbial populations may contribute to HNSCC pathogenesis via epigenetic modifications in inflammatory- and HNSCC-associated genes. Methods: Matched tumor and adjacent normal fresh frozen tissue specimens were collected from 55 prospectively enrolled HNSCC patients. Microbiomic profiles were obtained using Sanger sequencing of 16S rDNA PCR products. Methylation status of four genes previously linked to HNSCC or inflammation (MDR1, IL8, RARB, TGFBR2) was assessed in 49 samples by combined bisulfite restriction analysis and by Illumina HumanMethylation27 chip. Principle component analysis and multivariate analysis of covariance (MANCOVA) were used to identify bacterial subpopulations significantly associated with HNSCC and relevant clinical variables. Results: In this cohort, median age is 62, male:female ratio is 3:2, 16% are HPV+. Specific bacterial subpopulations associate with HNSCC over normal tissue and correlate with larger tumor size and higher nodal stage (p<0.05). Furthermore, microbial populations can separate tumors by tobacco (p<0.005) but not alcohol (p>0.7) status. Bacterial profiles are independent of HPV status. MDR1 promoter methylation is seen in tumor samples but not in normal oral mucosa (22/49 vs 0/49), and associates with specific microbiomic subpopulations in the order Enterobacteriales and phylum Tenericutes (p<0.001). Additionally, MDR1 methylation correlates with regional nodal metastases. Conclusions: Specific bacterial subpopulations differ between normal and tumor tissue and show association to specific clinicopathologic features, suggesting a role for microbial profiling in HNSCC as a novel area of investigation for diagnosis, prognostication, and therapeutic targeting.

Identification and characterization of Burkholderia isolates obtained from bacterial rot of saffron (Crocus sativus L.) grown in Italy

Summary. Twenty five isolates of Burkholderia gladioli, the causal agent of a bacterial disease recently reported on saffron (Crocus sativus L.) grown in central Sardinia (Italy), were characterized using different approaches. The characteristic symptoms of the disease on saffron plants were rot of emerging shoots and leaves and spots on leaves and corms. In the field, the disease was destructive and reduced flowering by about 80%. Two types of colonies of bacteria cultured from affected plants were selected on the basis of their characteristic morphology and pigment production on nutrient-glucose-agar. One type was round, wrinkled, and producing yellowish pigment; while the second was round, smooth and without pigment. All 25 selected isolates were pathogenic on saffron leaves and corms. Ten were pathogenic on gladiolus and lily leaves. None of the tested isolates was pathogenic on onion plants. The isolates were characterized by conventional tests, Biolog, PCR and PCR-RFLP analysis. Conventional tests and PCR identified all isolates as B. gladioli. PCR-RFLP analysis of 16S rDNA products digested with the three restriction enzymes Alu I, Dde I and Bss KI, identified ten of the isolates as B. gladioli pv. gladioli. Sequencing and comparison of the 16S rDNA PCR products confirmed that ten of the isolates were B. gladioli and the remaining 15 were an unidentified Burkholderia species. Sequencing the gene encoding for β-subunit polypeptide of DNA gyrase (gyrB) did not assist identification of these isolates. This study suggests that other Burkholderia species are involved with bacterial softrot of saffron in Sardinia, and further studies are in progress to verify this hypothesis.

Physical Mapping of 5S rDNA in Two Species of Knifefishes: Gymnotus pantanal and Gymnotus paraguensis (Gymnotiformes)

Physical mapping of 5S rDNA in 2 species of knifefishes, Gymnotuspantanal and G. paraguensis (Gymnotiformes), was performed using fluorescence in situ hybridization with a 5S rDNA probe. The 5S rDNA PCR product from the genomes of both species was also sequenced and aligned to determine non-transcribed spacer sequences (NTS). Both species under study had different patterns of 5S rDNA gene cluster distribution. While in the karyotype of G. pantanal two 5S rDNA-bearing pairs were observed, the karyotype of G. paraguensis possessed as many as 19 such pairs. Such multiplication of 5S rDNA gene clusters might be caused by the involvement of transposable elements because the NTS of G. paraguensis was 400 bp long with high identity (90%) with a mobile transposable element called Tc1-like transposon, described from the cyprinid fish Labeo rohita.