Independent Molecular Techniques Research Articles

Interactions of Bacteriophages and Bacteria at the Airway Mucosa: New Insights Into the Pathophysiology of Asthma.

The airway epithelium is the primary site where inhaled and resident microbiota interacts between themselves and the host, potentially playing an important role on allergic asthma development and pathophysiology. With the advent of culture independent molecular techniques and high throughput technologies, the complex composition and diversity of bacterial communities of the airways has been well-documented and the notion of the lungs' sterility definitively rejected. Recent studies indicate that the microbial composition of the asthmatic airways across the spectrum of disease severity, differ significantly compared with healthy individuals. In parallel, a growing body of evidence suggests that bacterial viruses (bacteriophages or simply phages), regulating bacterial populations, are present in almost every niche of the human body and can also interact directly with the eukaryotic cells. The triptych of airway epithelial cells, bacterial symbionts and resident phages should be considered as a functional and interdependent unit with direct implications on the respiratory and overall homeostasis. While the role of epithelial cells in asthma pathophysiology is well-established, the tripartite interactions between epithelial cells, bacteria and phages should be scrutinized, both to better understand asthma as a system disorder and to explore potential interventions.

Microbial biofilm community structure and composition on the lithic substrates of Herculaneum Suburban Baths

In this work, we want to investigate the impact of different substrates and different environmental condition on the biofilm communities growing on plaster, marble, and mortar substrates inside the Herculaneum Suburban Baths. To do so, we measured environmental conditions and sampled biofilm communities along the walls of the baths and used culture-dependent and -independent molecular techniques (DGGE) to identify the species at each sampling sites. We used the species pool to infer structure and richness of communities within each site in each substrate, and confocal light scanning microscopy to assess the three-dimensional structure of the sampled biofilms. To gather further insights, we built a meta-community network and used its local realizations to analyze co-occurrence patterns of species. We found that light is a limiting factor in the baths environment, that moving along sites equals moving along an irradiation gradient, and that such gradient shapes the community structure, de facto separating a dark community, rich in Bacteria, Fungi and cyanobacteria, from two dim communities, rich in Chlorophyta. Almost all sites are dominated by photoautotrophs, with Fungi and Bacteria relegated to the role of rare species., and structural properties of biofilms are not consistent within the same substrate. We conclude that the Herculaneum suburban baths are an environment-shaped community, where one dark community (plaster) and one dim community (mortar) provides species to a “midway” community (marble).

Microbiota Associated with Dromedary Camel Milk from Algerian Sahara.

Despite the fact that camel milk represents a valuable food source, the fungal diversity of raw camel milk has been poorly studied so far. Here, we investigated the fungal and bacterial communities found in dromedary camel milk from Ghardaia, a representative region of Algerian Sahara. The application of both culture-dependent and independent molecular techniques, based on dHPLC analysis and metabarcoding of ITS region, provided a complementary biodiversity assessment of camel milk fungi which was composed of 15 different taxa. Yeast species belonged to Filobasidium, Naganishia, Malassezia, Mrakia, Rhodotorula, and Yarrowia genera; and mold species belonged to Fusarium, Cladosporium, and Penicillium genera. All three techniques revealed that the fungal community was dominated by species belonging to the former genus Cryptococcus (Filobasidium and Naganishia) although none of them was able to encompass the entire fungal diversity alone. In addition, massive parallel 16S rRNA tag sequencing was applied to gain an insight into the diversity of bacterial communities which were dominated by Pseudomonas spp. Our results provide an initial insight about fungal and bacterial population found in dromedary camel milk from Algerian Sahara.

Mn2+ complex-modified polydopamine- and dual emissive carbon dots based nanoparticles for in vitro and in vivo trimodality fluorescent, photothermal, and magnetic resonance imaging

Multimodality molecular imaging has attracted more and more attention recently, as it possesses novel multiple imaging patterns via combining information from several independent molecular imaging techniques. Here, we investigated the multifunctional Mn2+ complex-modified polydopamine (PDA) and dual emissive carbon dots-based nanoparticles (NPs) for trimodality fluorescent, photothermal and magnetic resonance (MR) imaging in vitro and in vivo. In our system, N-doped carbon dots (N-CDs), a kind of green and red emissive CDs, were loaded or embedded onto the PDA NPs for fluorescent imaging. PDA NPs acted as a photothermal agent which showed obvious near-infrared (NIR) absorbance and high photothermal conversion efficiency (28.2%). Moreover, due to the automatic chelation of Mn2+ ions, PDA@N-CDs(Mn) NPs could offer contrast for magnetic resonance (MR) imaging. The application of PDA@N-CDs(Mn) NPs for fluorescent, photothermal, and MR in vivo imaging could be successfully demonstrated. Besides, the cytotoxicity analysis, hemolytic test, histological analysis of viscera sections, as well as blood biochemical analysis have showed that the PDA@N-CDs(Mn) NPs had excellent biocompatibility and low biological toxicity.

Effectiveness of a full-scale horizontal slow sand filter for controlling phytopathogens in recirculating hydroponics: From microbial isolation to full microbiome assessment

The microbial disinfestation efficiency of an innovative horizontal-flow slow sand filter (HSSF) for treating nutrient solution spent from an experimental closed-loop nursery was evaluated by means of a combination of culture-dependent and independent molecular techniques. A dense inoculum of the fungal plant pathogen Fusarium oxysporum f.sp. lycopersici was applied in the fertigation system (106 cells per mL). Indigenous and introduced populations of eubacteria and fungi were assessed in the nutrient solution, the HSSF influent/effluent, and a sand bed transect by isolation on selective media, as well as by quantitative qPCR and next-generation sequencing (NGS) on target ribosomal genes. The HSSF effectively reduced viable Fusarium propagules and fungal gene content with an efficiency consistently above 99.9% (5 orders of magnitude down). On the other hand, Fusarium cells accumulated in the sand bed, indicating that physical entrapment was the main removal mechanism. The viability of retained Fusarium cells tended to decrease in time, so that treatment efficiency might be enhanced by antagonistic species from the genera Bacillus, Pseudomonas, and Trichoderma, also identified in the sand bed. Indigenous bacterial populations from the HSSF effluent were reduced by 87.2% and 99.9% in terms of colony forming units and gene counts, respectively, when compared to the influent. Furthermore, microbial populations from the HSSF effluent were different from those observed in the sand bed and the influent. In summary, the HSSF microbial disinfestation efficiency is comparable to that reported for other more intensive and costly methodologies, while allowing a significant recovery of water and nutrients.

Metatrascriptomic Study of Microbes in Environment: A Community Function Based Approach

Research on microbial ecology in recent years had shifted methodologically from traditional culture based techniques to culture independent molecular techniques. This is due to the fact that it has already been established that less than 1% of microbes in environmental samples are cultivable. In recent years, metagenomics research has developed into analyzing partial or complete genomes as well as other types of molecules in environmental samples, including RNA (transcripts), proteins (translated transcripts), and metabolites (metabolic intermediates and final products). While the analyses of DNA sequences in environmental samples can provide evidence about the diversity and function of specific groups of organisms in the analyzed ecological niches, the relative importance of those genes in environmental functions cannot be determined from DNA sequences alone. To understand which genes are expressed under a specific ecological condition, the analysis of transcripts from the communities is often needed.

Characterisation of the yeast and mould biota in traditional white pickled cheeses by culture-dependent and independent molecular techniques.

Artisanal white pickled cheese of Western Serbia is a product of complex microbial community which detection by culture-dependent method only is hampered by its limitations. Thus, in the present study, we used a culture-independent, semi-quantitative technique based on construction of an internal transcribed spacer (ITS)-clone library from metagenomic DNA. This approach, based on direct DNA extraction followed by amplification of fungal internal transcribed regions (ITS) cloned into plasmid and restricted by endonucleases, revealed greater species richness in analysed cheeses and their by-products (17 species in total) compared to the more commonly used techniques of the culture-dependent method (8 species) and LSU-DGGE (10 species). The most frequently occurring yeast species which are commonly associated with cheeses production were Debaryomyces hansenii, Kluyveromyces lactis and Candida zeylanoides. On the other hand, Yarrowia lipolytica and Galactomyces geotrichum were detected only in one cheese sample. Moreover, some species, mainly moulds (Filobasidium globisporum, Cladosporium sp., Aspergillus sp. or Alternaria sp.) were identified only by culture-independent methods. The discrepancies between the techniques were confirmed by low correlation factor and by different indices of general biodiversity and dominance of species. The ITS-clone library approach provides the opportunity to analyse complex fungal communities associated with food products.

Genetic diversity of enterococci from Iranian home-made artisanal dairy products

Enterococci represent a significant part of microbiota in home-made dairy products due to their remarkable intrinsic resistance to critical growth conditions and technological properties. In this study, the predominant coccal lactic acid bacteria (LAB) were isolated from traditional dairy products made and largely consumed in villages of Chahar-Mahalo Bakhtiyari province of Iran. Thirty-six samples of home-made dairy products were collected from scattered households, and a total of 61 gram-positive and catalase-negative cocci were analyzed. Genetic fingerprinting (repetitive extragenic palindromic PCR (REP-PCR) and 16S–23S intergenic spacer (ITS) amplification) and gene sequencing (16S rRNA gene and pheS gene) were carried out to obtain an unambiguous identification of the isolates at the species level and to highlight genetic biodiversity among wild enterococci. It was shown that 42 out of 61 isolates belonged to Enterococcus spp., and Enterococcus faecium was the most frequently isolated species. Intraspecific diversity of the E. faecium species was further investigated, targeting the genes hyl, esp, and msrC, related to virulence and antibiotic resistance, and the results obtained suggest that two strains could be further characterized for possible use as starter cultures. In conclusion, the present study confirms that application of diverse independent molecular techniques is required to clearly identify Enterococcus spp. isolates and to unveil wild strain biodiversity. Moreover, a preliminary molecular screening of selected genetic features represents a fast and feasible approach to select, among a wide collection of isolates, the most promising candidates for further exhaustive characterization of safety and technological traits.

Protein extraction method for the proteomic study of a Mexican traditional fermented starchy food.

Several methodologies have been employed to study pozol, with a specific focus toward the identification of the microbiota of this fermented maize dough, using both traditional cultivation techniques and culture independent molecular techniques. However to date, the dynamics of this complex fermentation is not well understood. With the purpose to gain further insight into the nature of the fermentation, we used proteomic technologies to identify the origin of proteins and enzymes that facilitate substrate utilization and ultimately the development of the microbiota and fermentation. In this paper we overcome the first general challenge for such studies, obtaining a protein sample with adequate quality capable of representing the system.

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.

Assessment of tetracycline and erythromycin resistance transfer during sausage fermentation by culture-dependent and -independent methods

The food chain is considered one of the main routes of antibiotic resistance diffusion between animal and human population. The resistance to antimicrobial agents among enterococci could be related to the efficient exchange of transferable genetic elements. In this study a sausage model was used to evaluate the persistence of antibiotic resistant enterococci during meat fermentation and to assess horizontal gene transfer among bacteria involved in meat fermentation. Enterococcus faecalis OG1rf harbouring either pCF10 or pAMβ1 plasmid was used as donor strain. The analysis of population dynamics during fermentation confirmed that the human isolate E. faecalis OG1rf was able to colonize the meat ecosystem with similar growth kinetics to that of food origin enterococci and to transfer the mobile genetic elements coding for tetracycline and erythromycin resistances. Transconjugant strains were detected after only two days of fermentation and increased their numbers during ripening even in the absence of selective antibiotic pressure. By means of culture-dependent and -independent molecular techniques, transconjugant strains carrying both tetracycline and erythromycin resistance genes were identified in enterococci, pediococci, lactobacilli and staphylococci groups. Our results suggest that the sausage model provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among food microbiota.

Design of PCR Primers for the <i>mcr</i>A Genes Detection of Methanogen Communities

Methanogens play an important role to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent green house gases, during organic matter degradation in anaerobic environments. Therefore, it is necessary to better understand microorganisms that produce natural gas. Indeed, methanogens are difficult to perform through culture based methods. In addition, the culture independent methods using the 16S rRNA gene also revealed some disadvantages. For these reasons, the culture independent molecular techniques using the specific catabolic genes such as methyl coenzyme M reductase (MCR) were studied. In this study, a primer set which can amplify specific fragments from a wide variety of mcrA gene was designed based on the homologous regions of 100 mcrA genes listed in the GenBank. PCR with the mcrA primers amplified DNA fragments of the expected size from all the six samples which obtained from biogas production reactors. In addition, denaturing gradient gel electrophoresis PCR analysis using our designed primers also revealed the diversity of mcrA gene in each sample. These results revealed that our primers were successfully to detect the mcrA genes and it is also helpful to know the diversity of mcrA genes in methanogen communities.

Nebulette is the second member of the nebulin family fused to the MLL gene in infant leukemia

Genetic aberrations involving the mixed lineage leukemia ( MLL) gene are frequently diagnosed in infant acute lymphoblastic and acute myeloid leukemia. More than 60 fusion partner genes have been described at the molecular level, 31 of which have been characterized solely in infant leukemia cases. Here we describe a new MLL fusion partner gene, NEBL, which was identified in a case of acute myeloid leukemia in an infant. The chromosomal breakpoints of the MLL–NEBL and NEBL–MLL fusion genes were cloned by long-distance inverse polymerase chain reaction. The chromosomal breakpoints were located at 10p12, approximately 570 kb telomic of the MLLT10 ( AF10) gene. AF10 and NEBL are localized in such close vicinity that they cannot be distinguished cytogenetically by G banding. Therefore, the combination of cytogenetic and independent molecular techniques such as long-distance inverse polymerase chain reaction are indispensable for the rapid identification and characterization of rare MLL rearrangements.

Osteoradionecrosis contains a wide variety of cultivable and non-cultivable bacteria

Background: Direct microscopy, 11The chief editor, Ingar Olsen, has had no part in the review and decision process of this paper.anaerobic culture and DNA–DNA hybridization have previously demonstrated an association between microorganisms and osteoradionecrosis (ORN). The purpose of our study was to use culture independent molecular techniques to detect bacteria in necrotic bone lesions of the mandible after radiation therapy.Design: Bacterial DNA was extracted from eight deep medullar specimens from resected mandibles (six cases), including one patient with relapse. 16S rRNA genes were PCR amplified, cloned, transformed into Escherichia coli and sequenced to determine species identity and closest relatives.Results: From the analysis of 438 clones, 59 predominant species were detected, 27% of which have not been cultivated. The predominant species detected from radionecrotic mandibles were Campylobacter gracilis, Streptococcus intermedius, Peptostreptococcus sp. oral clone FG014, uncultured bacterium clone RL178, Fusobacterium nucleatum, and Prevotella spp. The study demonstrated intersubject variability of the bacteria present in ORN. In contrast to the diverse bacterial profile detected in primary infection, only a few members of the oral indigenous flora were identified from the relapse case.Conclusions: Diverse bacterial profiles in specimens of ORN in marrow spaces of the mandible were detected by culture independent molecular techniques. To better understand the pathogenesis and to improve the therapy of the infection, detection of all members of the complex bacterial flora associated with ORN is necessary.

Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers

We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate-one where methanogenesis was allowed and another in which it was completely inhibited with 2-bromoethane sulfonate. We observed a three-way syntrophy among ethanol fermenters, acetate-oxidizing anode-respiring bacteria (ARB), and a H2 scavenger. When methanogenesis was allowed, H2-oxidizing methanogens were the H2 scavengers, but when methanogenesis was inhibited, homo-acetogens became a channel for electron flow from H2 to current through acetate. We established the presence of homo-acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo-acetogens. Both methods documented the presence of the homo-acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol-fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H(2)-scavenging syntrophic partners that were either H2-oxidizing methanogens or homo-acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron-flow distribution and H2-scavenging mechanism.

Microbial diversity of intestinal contents and mucus in rainbow trout (Oncorhynchus mykiss)

The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture-dependent conventional and independent molecular techniques. Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. The fish gut harbours a larger bacterial diversity than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.

HIV drug resistance acquired through superinfection

HIV interclade B superinfection has previously been described in individuals initially infected with drug resistant virus who then become superinfected by a drug susceptible strain. We report an individual initially infected with a drug-sensitive clade B strain of HIV who was superinfected with another clade B strain resistant to two classes of antiretroviral drugs. To differentiate superinfection from possible co-infection we applied three independent molecular techniques: dye-primer sequencing of a pol fragment, length polymorphism analysis of the V4-5 coding region of the env gene and clonal sequencing of the V3 coding region of the env gene. To assess viral fitness we performed replication capacity assays of the pol gene. These investigations supported the conclusion that this was a case of superinfection and not co-infection. Coincident with acquiring the new strain, the individual's viral load increased by about 10,000 copies/ml with a decrease of 150 x CD4 T cells/mul over the next 6 months. The greater in vivo fitness of the second virus was not supported by the replication capacity assay. Furthermore, superinfection negatively impacted this individual's treatment course. It was not known that he had acquired a drug resistant strain before entering a treatment study, and he had an incomplete response to therapy most likely because the superinfecting viral strain had a decreased susceptibility to two of the prescribed medications. HIV drug resistance acquired through superinfection significantly lowers the likelihood of successful antiretroviral therapy and undermines the clinical value of a patient's prior drug resistance testing and lack of prior antiretroviral use.

Characterization of microbial diversity in hypersaline environments by melting profiles and reassociation kinetics in combination with terminal restriction fragment length polymorphism (T-RFLP).

The diversity of prokaryotes inhabiting solar saltern ponds was determined by thermal melting and reassociation of community DNA. These measurements were compared with fingerprinting techniques such as terminal restriction fragment length polymorphisms (T-RFLP) analysis, denaturant gradient gel electrophoresis (DGGE), and cloning and sequencing approaches. Three ponds with salinities of 22, 32, and 37% (NaCl saturation) were studied. The combination of independent molecular techniques to estimate the total genetic diversity provided a realistic assessment to reveal the microbial diversity in these environments. The changes in the prokaryotic communities at different salinity (22, 32, and 37% salt) were significant and revealed that the total genetic diversity increased from 22% to 32% salinity. At 37% salinity the diversity was reduced again to nearly half that at 22% salinity. Our results revealed that the community "genome" had a DNA complexity that was 7 (in 22% salinity pond), 13 (in 32% salinity pond), and 4 (in 37% salinity pond) times the complexity of an Escherichia coli genome. The base composition profiles showed two abundant populations, which changed in relative amount between the three ponds. They indicated an uneven taxon distribution at 22% and 37% salinity and a more even distribution at 32% salinity. The results indicated a large predominating population at 37% salinity, which might correspond to the abundance of square archaea (SPhT) observed by transmission electron microscopy (TEM) and also indicated by the same T-RFLP fragment as the SPhT. The SPhT phylotype has also been reported to be the most frequently retrieved phylotype from this environment by culture independent techniques. In addition, two different operational taxonomic units (OTU) were detected at 37% salinity based on PCR with bacterial specific primers and T-RFLP. One of these predominant phylotypes is the extreme halophilic bacterium belonging to the bacteroidetes group, Salinibacter ruber.

M-cone opsin gene number does not correlate with variation in L/M-cone sensitivity

Molecular genetic studies demonstrate that the human cone opsin gene array on the q-arm of the X-chromosome typically consists of one long-wave-sensitive (L) cone opsin gene and from one to several middle-wave-sensitive (M) cone opsin genes. Although the presence of the single L-cone opsin gene and at least one M-cone opsin gene is essential for normal red–green colour discrimination, the function of the additional M-cone opsin genes is still unclear. To investigate whether any variations in phenotype correlate with differences in the number of M-cone opsin genes, we selected 13 normal trichromat males, for whom four independent molecular techniques have exactly determined their number of M-cone opsin genes, ranging from one to four. Their phenotype was characterized by estimating their foveal L- to M-cone ratio from heterochromatic flicker photometric (HFP) thresholds, by measuring the wavelength corresponding to their `unique yellow', and by determining their L- and M-cone modulation thresholds (CMTs). No correlation was found between these psychophysical measures and the number of M-cone opsin genes. Although, we found a reasonably good correlation between the L/M-cone ratios based on HFP and on CMT, we did not find any correlation between the estimated L/M-cone ratios and the settings of `unique yellow'. Our results accord with previous molecular genetic studies that suggest that only the first two genes in the X-linked opsin gene array are expressed.

Complementary independent molecular, radioisotopic and fluorogenic techniques to assess biofilm communities in two wastewater wetlands

Optimisation of nutrient removal processes requires a detailed understanding of the microbial physiologies actually occurring within the biomass of different treatment zones, and also knowledge of how these communities respond to environmental factors. This paper describes a suite of four independent and complementary, microbiological techniques, utilised to obtain detailed assessments of wastewater bacterial biomass. Examples of these techniques are shown as applied to biofilms from two surface-flow wetlands used for treating sewage effluent. Intact biofilms were prepared via freezing, followed by cryosectioning and direct microscopic observation. This approach was combined with prior, in situ incubation of biofilm with radiolabelled thymidine to assess in situ bacterial growth rates. Individual bacterial cells were assessed microscopically for in situ respiratory activity and phylogenetic identity, respectively through use of an inducibly-fluorescent redox stain (CTC), and fluorescent in situ hybridisation (FISH) of ribosomal RNA (rRNA). These complementary techniques enabled assessment of individual biofilm clusters according to their in situ status (growth, respiration, spatial location and phylogenetic affiliation). This approach detected significant spatial variations in biofilm; limited the bias inherent in data obtained using any one technique, and provided greater detail of subpopulations within the microbial community.