rDNA PCR Research Articles

Occurrence of Diplostomum pseudospathaceum Niewiadomska, 1984 and D. mergi Dubois, 1932 (Digenea: Diplostomidae) in Danish freshwater snails: ecological and molecular data

Freshwater pulmonate snails from three locations in Lake Furesø north of Copenhagen were screened for infection with furcocercariae (by shedding in the laboratory) and recovered parasite larvae were diagnosed by molecular methods (by performing PCR of rDNA and sequencing the internal transcribed spacer [ITS] region). Overall prevalence of infection in snails was 2%. Recovered cercariae from Lymnaea stagnalis (Linnaeus) were diagnosed as Diplostomum pseudospathaceum Niewiadomska, 1984 (prevalence 4%) and cercariae from Radix balthica (Linnaeus) as D. mergi (Dubois, 1932) (prevalence 2%). Pathogen-free rainbow trout were then exposed to isolated cercariae and infection success and site location of metacercariae in these fish were determined. Infection experiments confirmed that both species could infect rainbow trout with the eye lens as infection site for the metacercarial stage although infection success differed. Combination of molecular and biological assays may contribute to improvement of our knowledge on diagnosis, distribution and biology of diplostomids in fish.

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.

Molecular characterization of Trypanosoma (Megatrypanum) spp. infecting cattle (Bos taurus), white-tailed deer (Odocoileus virginianus), and elk (Cervus elaphus canadensis) in the United States

In the United States, the generally non-pathogenic trypanosome of cattle is designated Trypanosoma (Megatrypanum) theileri and is distinguished morphologically from Trypanosoma (M.) cervi, a trypanosome originally described in mule deer and elk. Phylogenetic studies of the Megatrypanum trypanosomes using various molecular markers reveal two lineages, designated TthI and TthII, with several genotypes within each. However, to date there is very limited genetic data for T. theileri, and none for the Megatrypanum trypanosomes found in wild ungulates, in the U.S. In this study U.S. isolates from cattle (Bos taurus), white-tailed deer (Odocoileus virginianus) (WTD), and elk (Cervus elaphus canadensis) were compared by ribosomal DNA (rDNA) sequence analysis and their incidence in cattle and WTD in south Texas counties was investigated. Phylogenetic analyses showed clear separation of the bovine and cervine trypanosomes. Both lineages I and II were represented in the U.S. cattle and WTD parasites. Lineage I cattle isolates were of a previously described genotype, whereas WTD and elk isolates were of two new genotypes distinct from the cattle trypanosomes. The cattle isolate of lineage II was of a previously reported genotype and was divergent from the WTD isolate, which was of a new genotype. In La Salle, Starr, Webb, and Zapata counties in south Texas a total of 51.8% of white-tailed deer were positive for trypanosomes by 18S rDNA PCR. Of the cattle screened in Webb County, 35.4% were positive. Drought conditions prevailing in south Texas when the animals were screened suggest the possibility of a vector for Trypanosoma other than the ked (Lipoptena mazamae) and tabanid flies (Tabanus spp. and Haematopota spp.).

Molecular Characterization of camphor utilizing bacterial isolates from refinery sludge and detection of target loci-Cytochrome P-450 cam mono oxygenase (cam C gene) by PCR and gene probe

This study presents the isolation and molecular characterization of bacterial strains utilizing 1, 7, 7-Trimethylbicyclo (2.2.1) heptane-2-one (camphor) as a sole source of carbon, isolated from the biomass sludge sample collected from an effluent treatment plant of Mathura Refinery Limited (MRL), India. Initial screening was carried out where the 16S rDNA PCR was performed using reported eubacterial primer set followed by Amplified Ribosomal DNA Restriction Analysis (ARDRA). About 47% of the isolates have shown unique ARDRA pattern based on which, 15 distinct isolates were selected and tested for the presence of cam C gene that was successfully demonstrated by PCR using gene specific primers. A Dot-Blot experiment was designed to detect the cam C loci in the plasmid DNA of all camphor isolates based on non-radioactive “Biotin-Streptavidin” detection system. The bacterial identity with respect to partial 16S rDNA gene sequences of all camphor isolates placed them in 9 major genera viz., Pseudomonas sp., Staphylococcus sp., Alcaligenes sp., Agromyces sp., Stenotrophomonas sp., Reichenowia sp., Achromobacter sp., Brevibacterium sp. and Pseudaminobacter sp. A detailed phylogentic tree was also constructed to establish their evolutionary status from the gene sequence data.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-170) contains supplementary material, which is available to authorized users.

16S rDNA PCR in diagnosis of Lemierre's syndrome

In their Grand Round paper, Krutika Kuppalli and colleagues have omitted an important device for the diagnosis of Lemierre’s syndrome. Molecular methods, including broad-range 16S rDNA PCR, are increasingly being used 1 Amoroso A, Etienne-Mesubi M, Edozien A, Mesubi OO, Guberski T. HIV-1 drug resistance in antiretroviral-naive patients in sub-Saharan Africa. Lancet Infect Dis 2012; 12: 585–86. 2 Hamers RL, Wallis CL, Kityo C, et al. HIV-1 drug resistance in antiretroviral-naive individuals in sub-Saharan Africa after rollout of antiretroviral therapy: a multicentre observational study. Lancet Infect Dis 2011; 11: 750–59. 3 Bennett DE, Jordan MR, Bertagnolio S, et al. HIV drug resistance early warning indicators in cohorts of individuals starting antiretroviral therapy between 2004 and 2009: World Health Organization global report from 50 countries. Clin Infect Dis 2012; 54 (suppl 4): 280–89. 4 Gupta RK, Sultan BJ, Hill A, et al. Global trends in antiretroviral resistance in untreated HIV-1 infected individuals following ART roll-out in resource-limited settings: a global collaborative study and meta-regression analysis. Lancet 2012; 380: 1250–58. 5 WHO. WHO HIV drug resistance report 2012. http://www.who.int/hiv/pub/drugresistance/ report2012/en/index.html (accessed Sept 10, 2012) 6 Hamers RL, Kityo C, Lange JM, de Wit TF, Mugyenyi P. Global threat from drug resistant HIV in sub-Saharan Africa. BMJ 2012; 344: e4159. on culture-negative specimens from normally sterile sites in developed countries. The authors acknowledge that a high index of clinical suspicion is key to the diagnosis of Lemierre’s syndrome. However, in my experience, the diagnosis is almost always delayed, either due to atypical presentation (ie, many suppurative lesions in the absence of a history of upper respiratory tract illness) or because the causative organism(s) cannot be recovered as a result of their fastidious nature or previous antibiotic treatment. 16S rDNA PCR has two major advantages in the management of patients presenting with a febrile illness and suppurative lesions, especially those who have received previous antibiotic treatment. First, the method encompasses several genera of bacteria, enabling diagnosis of other bacterial infections presenting with similar clinical features, such as Staphylococcus aureus, group A streptococci, and Streptococcus pneumoniae. Second, because the test detects molecular material from bacteria, previous antibiotic treatment would not inter fere with the diagnosis, as is the case with conventional culture methods. Molecular methods can also be used on throat swab for the diagnosis of Lemierre’s syndrome, but results should be interpreted with caution. As is the case with any other laboratory tests, molecular methods do have limitations regarding their sensitivity and specifi city. Nevertheless, 16S rDNA PCR has been shown to increase diagnostic yield. Although not perfect, if available, molecular methods should be used alongside routine culture methods in cases with a high probability of the illness being Lemierre’s syndrome.

Denaturing Gradient Gel Electrophoresis for Detection of Bacterial Population and Diversity in Amniotic Fluids and Neonatal Gastric Fluids

Purpose: Intra-amniotic infection (IAI) is often polymicrobial, and the 16S rDNA PCR assay has a major limitation that its interpretation is difficult in the presence of multiple 16S rDNAs. Denaturing gradient gel electrophoresis (DGGE) can overcome this limitation by separating PCR products based on sequence. We performed the DGGE analysis to investigate bacterial preva-lence and diversity in amniotic fluids from pregnant women with preterm births and gastric fluids from their newborns. Methods: DNA was extracted from bacterial cells in amniotic fluid (AF) and gastric fluid (GF) and was amplified with universal 16S rDNA primers. For DGGE analysis, the PCR products were loaded onto polyacrylamide gels that were made with denaturing gradients. Results: Bacterial 16S rDNA was detected by PCR from all AF and GF samples. The bacterial species in AF samples were the following: Lactobacillus reuteri (87.0%), uncultured Enterococcus species (65.2%), Ureaplasma urealyticum (13.0%), and Enterococcus faecalis (4.3%). The bacterial species in GF samples were the following: Lactobacillus reuteri (95.2%), uncultured Enterococcus species (42.9%), and Ureaplasma urealyticum (4.8%). Two or more species were identified from 69.6% of AF and 47.6% of GF samples. Conclusion: We suggest that DGGE analysis allows improved understanding of microbial diversity and community in AF and GF.

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.

Identification and characterization of lactic acid bacteria from forest musk deer feces

The aims of this study were to identify and characterize lactic acid bacteria (LAB) isolated from healthy forest musk deer (FMD) feces which have good potential to develop orally delivered probiotics applied in FMD. Eight LAB isolates were obtained from feces samples and were identified as Lactobacillus acidipiscis, Lactobacillus plantarum, Enterococcus faecium, Enterococcus mundtii, Enterococcus durans and Leuconostoc fallax using morphology, physiology, biochemical and 16s rDNA PCR methods. Five strains were selected after safety and function tests which showed no hemolytic activity, no harmful indole and hydrogen sulphide produced, safety to KM mouse, growth inhibition toEscherichia coli (ATCC25922), Salmonella pullorum (CVCC527) and Staphylococcus aureus (ATCC29213), and no acquired resistance genes detected. The growth of five strains could not be inhibited by 0.9% pig bile. Two Lactobacillus strains could survive at as low as pH 2.0. One L. acidipiscis strain and one E. durans strain could survive at as high as 70°C. Five strains identified as L. acidipiscis, L. plantarum, Enterococcus faeciumand L. fallax could be well suitable for developing an orally delivered probiotic. To our knowledge, this is the first time to isolate and identify LAB from forest musk deer feces and the characterized strains have good potential for developing an orally delivered probiotic to improve health condition of forest musk deer. Key words: Lactic acid bacteria (LAB), probiotics, Lactobacillus, Enterococcus,Leuconostoc.

WS20.1 Target minority bacterial population in cystic fibrosis lung microbiota

Staphylococcus aureus is one of the most frequently encountered species in Cystic Fibrosis (CF) lung microbiota. However its frequent presence on agar plates from sputum samples of CF patients likely impaired the culture of other minority bacterial species that may be responsible for lung exacerbations. In order to discover this minority bacterial population, we examined the sputum sample of a CF patient using conventional culture dependent with or without inhibition of S. aureus growth with vancomycin and compare our results to culture independent molecular techniques. Using standard cultures as well as selective media at different physico-chemical conditions, we isolate Staphylococcus aureus in the majority of our culture conditions and media. Interestingly, after pre-incubation of the sample with Vancomycin to inhibit Staphylococcus aureus growth, we isolate new bacterial species (Granulicatella, Streptococcus, Rothia, Lactobacillus and Propionibacterium species), especially Actinomyces radicidentis, a rare bacterial species associated with endodontic infections never described in the context of CF. Using 16S rDNA PCR amplification and sequencing of subsequent clones, we identified Streptococcus, Granulicatella, Rothia, Abiotropha, Peptostreptococcus, Lachnospiraceae and Actinomyces along with S. aureus confirming the culture dependent findings. In conclusion, the use of various bacterial inhibitors to inhibit the growth of the dominant bacteria may help to discover the minority bacterial population that could be new or emerging species in the context of CF.

Lemierre's syndrome due to Fusobacterium necrophorum

We present a case of a patient with Lemierre's syndrome caused by Fusobacterium necrophorum who developed a right frontal lobe brain abscess. We summarise the epidemiology, microbiology, pathogenesis, clinical presentation, diagnosis, complications, therapy, and outcomes of Lemierre's syndrome. F necrophorum is most commonly associated with Lemierre's syndrome: a septic thrombophlebitis of the internal jugular vein. Patients usually present with an exudative tonsillitis, sore throat, dysphagia, and unilateral neck pain. Diagnosis of septic thrombophlebitis is best confirmed by obtaining a CT scan of the neck with contrast. Complications of the disease include bacteraemia with septic abscesses to the lungs, joints, liver, peritoneum, kidneys, and brain. Treatment should include a prolonged course of intravenous beta-lactam antibiotic plus metronidazole.

Geographical variation of foxtail millet, Setaria italica (L.) P. Beauv. based on rDNA PCR–RFLP

The rDNA PCR–RFLP of foxtail millet (Setaria italica) germ-plasm collected throughout Eurasia and from a part of Africa was investigated with five restriction enzymes according to our previous study. Foxtail millet germ-plasms were classified by length of the rDNA IGS and RFLP; clear geographical differentiation was observed between East Asia, the Nansei Islands of Japan-Taiwan-the Philippines area, South Asia and Afghanistan-Pakistan. We also found evidence of migration of foxtail millet landraces between the areas. We calculated diversity index (D) for each region and found that center of diversity of this millet is East Asia such as China, Korea and Japan.

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.

Absence of Helicobacter pylori high tetracycline resistant 16S rDNA AGA926-928TTC genotype in gastric biopsy specimens from dyspeptic patients of a city in the interior of São Paulo, Brazil

BackgroundTreatment effectiveness of Helicobacter pylori varies regionally and is decreasing worldwide, principally as a result of antibiotic resistant bacterium. Tetracycline is generally included in second line H. pylori eradication regimens. In Brazil, a high level of tetracycline resistance (TetR) is mainly associated with AGA926-928TTC 16 S rDNA nucleotide substitutions. As H. pylori culture is fastidious, we investigated the primary occurrence of H. pylori 16 S rDNA high level TetR genotype using a molecular approach directly on gastric biopsies of dyspeptic patients attending consecutively at Hospital das Clinicas of Marilia, São Paulo, Brazil.MethodsGastric biopsy specimens of 68 peptic ulcer disease (PUD) and 327 chronic gastritis (CG) patients with a positive histological diagnosis of H. pylori were investigated for TetR 16 S rDNA genotype through a molecular assay based on amplification of a 16 S rDNA 545 bp fragment by polymerase chain reaction and HinfI restriction fragment length polymorphism (PCR/RFLP). Through this assay, AGA926-928TTC 16 S rDNA TetR genotype resulted in a three DNA fragment restriction pattern (281, 227 and 37 bp) and its absence originated two DNA fragments (264 and 281 bp) due to a 16 S rDNA conserved Hinf I restriction site.ResultsThe 545 bp 16 S rDNA PCR fragment was amplified from 90% of gastric biopsies from histological H. pylori positive patients. HinfI RFLP revealed absence of the AGA926–928TTC H. pylori genotype and PCR products of two patients showed absence of the conserved 16 S rDNA HinfI restriction site. BLASTN sequence analysis of four amplicons (two conserved and two with an unpredicted HinfI restriction pattern) revealed a 99% homology to H. pylori 16 S rDNA from African, North and South American bacterial isolates. A nucleotide substitution abolished the conserved HinfI restriction site in the two PCR fragments with unpredicted HinfI RFLP, resulting in an EcoRI restriction site.ConclusionsH. pylori AGA926-928TTC 16 S rDNA gene substitutions were not found in our population. More research is required to investigate if H. pylori TetR has a different genetic background in our region and if the nucleotide substitutions of the uncultured H. pylori 16 S rRNA partial sequences have biological significance.

DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)(1).

Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species-specific, semi-quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10-fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.

Molecular and morphological diversity of fungi and the associated functions in three European nearby lakes

This study presents an original rDNA PCR and microscopic survey of pelagic freshwater fungal communities, and was designed to unveil the diversity of true Fungi (i.e. the kingdom Eumycota) in three contrasting lake ecosystems (Lakes Pavin, Aydat and Vassivière) located in the French Massif Central. Three clone libraries were constructed from samples collected in the euphotic layers of the lakes during spring 2007. Phylogenetic analysis of the combined data from the three lakes clustered our sequences into thee divisions: Chytridiomycota (50% of total sequences), Ascomycota (40%) and Basidiomycota (10% in Pavin and Aydat only). Several sequences were assigned to a novel Chytridiomycota clade first recovered in Lake Pavin in 2005. Most of the sequences retrieved in the investigated lakes were affiliated with known fungal species, most of which were apparently well adapted to thrive in the pelagic realm. Their main functions (i.e. parasitism and saprophytism), putatively inferred from the closest relatives of the retrieved molecular sequences, were confirmed by microscopic approaches and by enrichment experiments with pollen grains. The occurrence of three fungal forms (zoosporic, yeast and mycelial) was associated with different trophic modes, establishing fungi as strong potential competitors for various niches in pelagic ecosystems, primarily in relation to the processing of particulate organic matter and the production of propagule food sources for grazers. For the first time, this study provides insight into the diversity and the associated functions of all members of the Kingdom Eumycota investigated in the whole plankton fraction of aquatic ecosystems.

Eukaryotic life in biofilms formed in a uranium mine

The underground uranium mine Königstein (Saxony, Germany), currently in the process of remediation, represents an underground acid mine drainage (AMD) environment, that is, low pH conditions and high concentrations of heavy metals including uranium, in which eye-catching biofilm formations were observed. During active uranium mining from 1984 to 1990, technical leaching with sulphuric acid was applied underground on-site resulting in a change of the underground mine environment and initiated the formation of AMD and also the growth of AMD-related copious biofilms. Biofilms grow underground in the mine galleries in a depth of 250 m (50 m above sea level) either as stalactite-like slime communities or as acid streamers in the drainage channels. The eukaryotic diversity of these biofilms was analyzed by microscopic investigations and by molecular methods, that is, 18S rDNA PCR, cloning, and sequencing. The biofilm communities of the Königstein environment showed a low eukaryotic biodiversity and consisted of a variety of groups belonging to nine major taxa: ciliates, flagellates, amoebae, heterolobosea, fungi, apicomplexa, stramenopiles, rotifers and arthropoda, and a large number of uncultured eukaryotes, denoted as acidotolerant eukaryotic cluster (AEC). In Königstein, the flagellates Bodo saltans, the stramenopiles Diplophrys archeri, and the phylum of rotifers, class Bdelloidea, were detected for the first time in an AMD environment characterized by high concentrations of uranium. This study shows that not only bacteria and archaea may live in radioactive contaminated environments, but also species of eukaryotes, clearly indicating their potential influence on carbon cycling and metal immobilization within AMD-affected environment.

Spatial Heterogeneity of Bacterial Communities in Sediments from an Infiltration Basin Receiving Highway Runoff

The bacterial community diversity of highway runoff-contaminated sediment that had undergone 19 years of acetate-based de-icing agents addition followed by three years of acetate-free de-icing agents was investigated. Analysis of 26 sediment samples from two drilled soil cores by means of 16S rDNA PCR generated 3,402 clones, indicating an overall high bacterial diversity, with no prominent members within the communities. Sequence analyses provided evidences that each sediment sample displayed a specific structure bacterial community. Proteobacteria-affiliated clones (58% and 43% for the two boreholes) predominated in all samples, followed by Actinobacteria (12% and 16%), Firmicutes (7% and 12%) and Chloroflexi (7% and 11%). The subsurface geochemistry complemented the molecular methods to further distinguish ambient and contaminant plume zones. Principal component analysis revealed that the levels of Fe(II) and dissolved oxygen were strongly correlated with bacterial communities. At elevated Fe(II) levels, sequences associated with anaerobic bacteria were detected in high levels. As iron levels declined and oxygen levels increased below the plume bottom, there was a gradual shift in the community structure toward the increase of aerobic bacteria.

Bacteria associated with the rhizosphere of manganese-deficient date palms affected by brittle leaf disease.

Brittle leaf disease or “Maladie des feuilles cassantes” (MFC) is a disorder affecting date palm (Phoenix dactylifera) orchards in oases of southern Tunisia. No causal pathogen has yet been identified, despite search for nematodes, fungi, bacteria, viruses and viroids. Although the soils of the oases in which affected palms grow have similar contents of total and exchangeable manganese (Mn) compared with oases with no disease, the leaflets of affected palms are deficient in this element. Here, we characterized the bacterial flora in the rhizosphere of three MFC-affected date palms and three non-affected controls. Our results show a high bacterial diversity in the date palm rhizosphere. From 201 clones corresponding to a 16S rDNA PCR fragment, we identified 177 different bacterial phylotypes. Some of them corresponded potentially to Mn-oxidizing bacteria that may be involved in soil Mn oxidation and, consequently, deplete this micronutrient in MFC-affected date palms. More detailed studies are necessary to establish the exact role of these bacteria in the disease syndrome.

Simultaneous Detection of Multiple Fish Pathogens Using a Naked-Eye Readable DNA Microarray

We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 103 CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens.