Culture-independent Molecular Techniques Research Articles

Influences of microbial community structures and diversity changes by nutrients injection in Shengli oilfield, China

Abstract After dozens of years of crude oil recovery, oilfields are facing increasing depletion of oil resources. More than 60% of the Earth's oil resources are likely still in the subsurface oil deposits. Although related theories and technologies of enhanced oil recovery are not routinely used, microbial enhanced oil recovery (MEOR) has the potential to be one of the most valuable technologies in crude oil exploitation. The diverse microbial communities found in oil/water samples from the production well of the Shengli oilfield were analyzed by culture-independent molecular techniques, including a 16S rRNA gene clone library, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). In this study, nutrients (carbon source, nitrogen source, inorganic salts and trace elements) were injected into S12-4 wells, and the microbial diversity, composition, dynamics, quantity and structures were subsequently determined from samples extracted 0–17 months after the nutrient injection. The results show that the nutrient injection had a profound effect on the diversity, composition, and relative number of the petroleum microorganisms. The dominant species in the S12-4 well were mainly of the genus Pseudomonas , belonging to the order Pseudomonadales and Marinobacter , of the order Alteromonadales, as well as a number of methanogenic archaea. This study is the first time microbial communities in an oil reservoir have been examined with so many samples before and after nutrient injection. All of the data illustrate that in nutrient-rich conditions, indigenous microbes of different communities accompanied by anaerobic organisms play an important role in oil emulsion and MEOR.

Microbiota in allergy and asthma and the emerging relationship with the gut microbiome.

Asthma and atopy, classically associated with hyper-activation of the T helper 2 (Th2) arm of adaptive immunity, are among the most common chronic illnesses worldwide. Emerging evidence relates atopy and asthma to the composition and function of the human microbiome, the collection of microbes that reside in and on and interact with the human body. The ability to interrogate microbial ecology of the human host is due in large part to recent technological developments that permit identification of microbes and their products using culture-independent molecular detection techniques. In this review we explore the roles of respiratory, gut, and environmental microbiomes in asthma and allergic disease development, manifestation, and attenuation. Though still a relatively nascent field of research, evidence to date suggests that the airway and/or gut microbiome may represent fertile targets for prevention or management of allergic asthma and other diseases in which adaptive immune dysfunction is a prominent feature.

Molecular analysis of products of conception obtained by hysteroembryoscopy from infertile couples.

To analyze the molecular cytogenetic data obtained from products of conception (POC) obtained by selective biopsy of first trimester miscarriages and to estimate the rate of chromosomal anomalies in miscarriages from pregnancies achieved by natural conception (NC) or by assisted reproductive technology (ART) interventions. We used KaryoLite™ BoBs™ (PerkinElmer LAS, Wallac, Turku, Finland) technology to analyze 189 samples from ART or NC pregnancies. All POC were successfully evaluated. A higher incidence of chromosomal abnormalities was observed in POC after ART using the patient's own oocytes than from NC pregnancies (62.7% vs. 40.6%; p < 0.05). The lowest incidence of chromosomal abnormalities was observed in POCs ART using donor eggs from women younger than 35 years (12.8%). No statistical differences in the percentage of abnormal miscarriages were observed in correlation with sperm concentration: a sperm concentration less than 5 million/mL produced 75% abnormal results and a concentration higher than 5 million/mL produced 51%. POC analysis is essential to determine the cause of pregnancy loss. Using culture-independent molecular biology techniques to analyze POCs avoids limitations such as growth failure and reduces the time required for analysis. Selective biopsy of fetal tissue by hysteroembryoscopy avoids the risk of misdiagnosis due to maternal cell contamination. Our results show that maternal age, sperm quality, and ART-assisted pregnancies are risk factors for abnormal gestations.

Diet, microbiota, and colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer in the world causing nearly 500,000 deaths every year. In addition to genetic background, environmental factors including diet and lifestyle are accepted as major contributors to adenoma and CRC development. Lifestyle factors include high BMI, obesity, and reduced physical activity. Growing interest and accumulating data on human microbiota implicate that host-microbe interplay has an important role in the development of metabolic, neoplastic, and inflammatory diseases. Findings from recent studies suggest that colon cancer risk is determined by the interaction between diet and gut microbiota. Dietary changes affect gut microbiota and conversely microbiota mediates the generation of dietary factors triggering colon cancer. Identification of the microbial communities associated with carcinogenesis is of crucial importance. Nowadays, with the evolvement of culture-independent molecular techniques, it has become possible to identify main bacterial species in healthy individuals, inflammatory conditions, and CRC. Some recent studies have shown the differences in intestinal microbiota between colon cancer patients and healthy individuals. Animal studies have provided a better understanding of interaction between pathobionts and symbionts in the development of colon cancer. There is no single causative organism identified in CRC; however, there is strong evidence that reduction of protective bacteria, increase in some bacteria (ie, fusobacterium members; Bacteroides/Prevotella), and age-related changes in microbiota have an impact on adenoma or cancer development. Future studies will enable us to understand procarcinogenic and anticarcinogenic mechanisms and give insights to rational manipulation of the microbiota with prebiotics, probiotics, or dietary modifications.

Acetic acid bacteria from biofilm of strawberry vinegar visualized by microscopy and detected by complementing culture-dependent and culture-independent techniques

Acetic acid bacteria (AAB) usually develop biofilm on the air–liquid interface of the vinegar elaborated by traditional method. This is the first study in which the AAB microbiota present in a biofilm of vinegar obtained by traditional method was detected by pyrosequencing. Direct genomic DNA extraction from biofilm was set up to obtain suitable quality of DNA to apply in culture-independent molecular techniques. The set of primers and TaqMan – MGB probe designed in this study to enumerate the total AAB population by Real Time – PCR detected between 8 × 105 and 1.2 × 106 cells/g in the biofilm. Pyrosequencing approach reached up to 10 AAB genera identification. The combination of culture-dependent and culture-independent molecular techniques provided a broader view of AAB microbiota from the strawberry biofilm, which was dominated by Ameyamaea, Gluconacetobacter, and Komagataeibacter genera. Culture-dependent techniques allowed isolating only one genotype, which was assigned into the Ameyamaea genus and which required more analysis for a correct species identification. Furthermore, biofilm visualization by laser confocal microscope and scanning electronic microscope showed different dispositions and cell morphologies in the strawberry vinegar biofilm compared with a grape vinegar biofilm.

Depth-related coupling relation between methane-oxidizing bacteria (MOBs) and sulfate-reducing bacteria (SRBs) in a marine sediment core from the Dongsha region, the South China Sea.

The vertical distributions of methane-oxidizing bacteria (MOBs) and sulfate-reducing bacteria (SRBs) in the marine sediment core of DH-CL14 from the Dongsha region, the South China Sea, were investigated. To enumerate MOBs and SRBs, their specific genes of pmoA and apsA were quantified by a culture-independent molecular biological technique, real-time polymerase chain reaction (RT-PCR). The result shows that the pmoA gene copies per gram of sediments reached the maximum of 1,118,679 at the depth of 140-160 cm. Overall considering the detection precision, sample amount, measurement cost, and sensitivity to the seepage of methane from the oil/gas reservoirs or gas hydrates, we suggest that the depth of 140-160 cm may be the optimal sampling position for the marine microbial exploration of oils, gases, and gas hydrates in the Dongsha region. The data of the pmoA and apsA gene copies exhibit an evident coupling relation between MOBs and SRBs as illustrated in their vertical distributions in this sediment core, which may well be interpreted by a high sulfate concentration inhibiting methane production and further leading to the reduction of MOBs. In comparison with the numbers of the pmoA and apsA copies at the same sediment depth, we find out that there were two methane-oxidizing mechanisms of aerobic and anaerobic oxidation in this sediment core, i.e., the aerobic oxidation with free oxygen dominantly occurred above the depth of 210-230 cm, while the anaerobic oxidation with the other electron acceptors such as sulfates and manganese-iron oxides happened below the depth of 210-230 cm.

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.

Cuticles of European and American lobsters harbor diverse bacterial species and differ in disease susceptibility.

Diseases of lobster shells have a significant impact on fishing industries but the risk of disease transmission between different lobster species has yet to be properly investigated. This study compared bacterial biofilm communities from American (Homarus americanus) and European lobsters (H. gammarus), to assess both healthy cuticle and diseased cuticle during lesion formation. Culture-independent molecular techniques revealed diversity in the bacterial communities of cuticle biofilms both within and between the two lobster species, and identified three bacterial genera associated with shell lesions plus two putative beneficial bacterial species (detected exclusively in healthy cuticle or healing damaged cuticle). In an experimental aquarium shared between American and European lobsters, heterospecific transmission of potentially pathogenic bacteria appeared to be very limited; however, the claws of European lobsters were more likely to develop lesions when reared in the presence of American lobsters. Aquarium biofilms were also examined but revealed no candidate pathogens for environmental transmission. Aquimarina sp. ‘homaria’ (a potential pathogen associated with a severe epizootic form of shell disease) was detected at a much higher prevalence among American than European lobsters, but its presence correlated more with exacerbation of existing lesions rather than with lesion initiation.

The relationships between environmental bacterial exposure, airway bacterial colonization, and asthma

Recent application of advanced culture-independent molecular techniques for the identification of microorganisms has contributed to our knowledge on the role of early-life microbial exposure and colonization in health and disease. The purpose of this review is to present the current perspectives regarding the role of microbial exposure and airway bacterial colonization on the development and the activity of asthma. Recent findings continue to support the protective role of early-life diverse microbial exposure against the development of atopic diseases. However, airway bacterial colonization early in life serves as a risk factor for the development of asthma. Culture-independent molecular techniques for the identification of microorganisms have challenged the traditional paradigm that the lower airway is a sterile compartment. Asthmatics, compared with nonasthmatics, appear to have a different lung microbiome composition and some of these differences might contribute to asthma activity, severity, and corticosteroid response. Bacterial presence in the airway appears to influence the inception and may affect the activity of asthma. Complex interactions between different types and routes of bacterial exposures, the airway, and the immune system early in life may determine whether these exposures augment or reduce the risk of asthma development.

The fungal microbiome in chronic rhinosinusitis: richness, diversity, postoperative changes and patient outcomes

Our understanding of fungi in chronic rhinosinusitis (CRS) has been limited by previously employed detection techniques. This study examines the fungal component of the microbiome in CRS patients and controls using a highly sensitive culture-independent molecular technique. The aims of this study include the characterization of fungal richness, prevalence, abundance, temporal changes, and their relationship with patient outcomes. Swabs were collected from the sinuses of 23 CRS patients and 11 controls. Collection occurred intraoperatively, and at 6 and 12 weeks postoperatively. DNA was extracted from the swabs and fungal outcomes were determined through 18S ribosomal DNA (rDNA) fungal tag-encoded FLX amplicon pyrosequencing. Fungi were ubiquitous to all patients. A total of 207 fungal genera were detected, with a mean sample richness of 8.18 and 12.14 in the control and CRS groups, respectively. Malassezia was detected in all patients at surgery and was also the most abundant. Postoperatively, fungal richness decreased (p < 0.05) and was associated with declines in the prevalence of Fusarium and Neocosmospora (p < 0.05). Neocosmospora was also less abundant postoperatively (p < 0.05). No correlations were found with quality of life. This is the first study to use a highly sensitive pyrosequencing technique to reveal the true diversity of fungi in the sinuses of CRS patients and postoperative changes in richness. The presence of Malassezia, a genus not previously described in the sinuses, is of great interest, and its potential as a disease modifier should see further investigation given its association with atopic disease.

Comparing fungal richness and community composition in coarse woody debris in Central European beech forests under three types of management

Managing forests by selection cutting is a promising silvicultural technique for maintaining forest biodiversity. Despite the importance of fungi in decomposition and nutrient cycling in forest ecosystems, no study to date has investigated the effects of selection cutting on fungal communities, especially using a culture-independent molecular technique to assess more than just the species that are fruiting at the time of sampling. Based on operational taxonomic units (OTUs) found in coarse woody debris, we compared the richness and community composition of wood-inhabiting fungi from selection cutting, age-class, and unmanaged European beech-dominated forests. We found that fungal OTU richness in selection cutting and unmanaged forests was not significantly different (P > 0.05), but it was higher, in both cases, than that in the age-class forest (P = 0.0002). Fungal community composition was not significantly different among the three forest types (P > 0.05). Abundances of common, wood-inhabiting fungal OTUs in different forest types were significantly correlated: the highest and lowest correlations were found between unmanaged forests and selection cutting (ρ = 0.52, P < 0.0001, n = 94), and between unmanaged and age-class forests (ρ = 0.30, P = 0.0080, n = 79), respectively.

Microbiota and Pelvic Inflammatory Disease

Female genital tract microbiota play a crucial role in maintaining health. Disequilibrium of the microbiota has been associated with increased risk of pelvic infections. In recent years, culture-independent molecular techniques have expanded understanding of the composition of genital microbiota and the dynamic nature of the microbiota. There is evidence that upper genital tract may not be sterile and may harbor microflora in the physiologic state. The isolation of bacterial vaginosis-associated organisms in women with genital infections establishes a link between pelvic infections and abnormal vaginal flora. With the understanding of the composition of the microbiota in healthy and diseased states, the next logical step is to identify the function of the newly identified microbes. This knowledge will further expand our understanding of the causation of pelvic infections, which may lead to more effective prevention and treatment strategies.

Overgrowth of the indigenous gut microbiome and irritable bowel syndrome

Culture-independent molecular techniques have demonstrated that the majority of the gut microbiota is uncultivable. Application of these molecular techniques to more accurately identify the indigenous gut microbiome has moved with great pace over recent years, leading to a substantial increase in understanding of gut microbial communities in both health and a number of disorders, including irritable bowel syndrome (IBS). Use of culture-independent molecular techniques already employed to characterise faecal and, to a lesser extent, colonic mucosal microbial populations in IBS, without reliance on insensitive, traditional microbiological culture techniques, has the potential to more accurately determine microbial composition in the small intestine of patients with this disorder, at least that occurring proximally and within reach of sampling. Current data concerning culture-based and culture-independent analyses of the small intestinal microbiome in IBS are considered here.

Bacterial Communities Associated with Porites White Patch Syndrome (PWPS) on Three Western Indian Ocean (WIO) Coral Reefs

The scleractinian coral Porites lutea, an important reef-building coral on western Indian Ocean reefs (WIO), is affected by a newly-reported white syndrome (WS) the Porites white patch syndrome (PWPS). Histopathology and culture-independent molecular techniques were used to characterise the microbial communities associated with this emerging disease. Microscopy showed extensive tissue fragmentation generally associated with ovoid basophilic bodies resembling bacterial aggregates. Results of 16S rRNA sequence analysis revealed a high variability between bacterial communities associated with PWPS-infected and healthy tissues in P. lutea, a pattern previously reported in other coral diseases such as black band disease (BBD), white band disease (WBD) and white plague diseases (WPD). Furthermore, substantial variations in bacterial communities were observed at the different sampling locations, suggesting that there is no strong bacterial association in Porites lutea on WIO reefs. Several sequences affiliated with potential pathogens belonging to the Vibrionaceae and Rhodobacteraceae were identified, mainly in PWPS-infected coral tissues. Among them, only two ribotypes affiliated to Shimia marina (NR043300.1) and Vibrio hepatarius (NR025575.1) were consistently found in diseased tissues from the three geographically distant sampling localities. The role of these bacterial species in PWPS needs to be tested experimentally.

Intestinal microbiota and metabolites—Implications for broiler chicken health and performance

SUMMARY Microbiota in the intestine of an animal species has evolved together with the host. This coevolution has produced specific host-microbe combinations, called superorganisms, with the best possible fitness in a given environment. Intestinal microbiota has an enormous metabolic potential and it affects both the nutrition and health of the host. The importance of intestinal microbiota for the performance of broiler chickens has been studied for decades. The microbial community structure of the cecum is significantly more complex and less well characterized than that of the crop and small intestine. Culture-independent molecular techniques have recently been introduced to bring fresh insights into the intestinal system of the broiler chicken. As a consequence, there is growing evidence of connection between the apparent metabolizable energy of the diet and microbiota composition in the hindgut of the host. This connection can be due to direct conversion of some dietary components into high-energy metabolites by specialized bacteria. The utilization of such fermentation end products would provide more energy for the host, which could be measured as improved feed conversion efficiency. However, it is also possible that cecal microbial profile is a reflection of feed digestion and nutrient absorption efficiency in the proximal intestine. Disorders in these functions cause excess nutrient bypass to the lower intestine, providing new, easily available substrates for bacteria that could otherwise not compete in that habitat. The 2 causal links described are likely to coexist in real life situations, but their relative dominance warrants further well-designed studies.

Metabolomics approaches for characterizing metabolic interactions between host and its commensal microbes

It is increasingly evident that the gut microbiota is involved in the regulation of multiple mammalian metabolic pathways through a series of interactive host-microbiota metabolic, signaling, and immune-inflammatory axes that physiologically connect the gut, liver, brain, and other organs. Correlation of the metabotypes with the gut microbial profiles derived from culture-independent molecular techniques is increasingly useful for deciphering inherent and intimate host-microbe relationships. Real-time analysis of the small molecule metabolites derived from gut microbial-host co-metabolism is essential for understanding the metabolic functions of the gut microbiome and has tremendous implications for personalized healthcare strategies. Metabolomics, an array of analytical techniques that includes high resolution NMR spectroscopy and chromatography-MS in conjunction with chemometrics and bioinformatics tools, enables characterization of the metabolic footprints of mammalian hosts that correlate with the microbial community in the intestinal tract. The metabolomics approach provides important information of a complete spectrum of metabolites produced from the gut microbial-mammalian co-metabolism and is improving our understanding of the molecular mechanisms underlying multilevel host-microbe interactions. In this review, the interactions of gut microbiota with their host are discussed and some examples of NMR- or MS-based metabolomics applications for characterizing the metabolic footprints of gut microbial-host co-metabolism are described. Advances in the metabolomic analysis of bile acids, short-chain fatty acids, and choline metabolism are also summarized.

Diagnostic tools using DNA bar coding for the identification of pathogen races and related species: a review

In the past, disease diagnostic has been an art which was based on visual scoring of disease symptoms. However, visual scoring was cumbersome and often confusing for the pathologists due to intermingling of the visual symptoms with other diseases, delayed appearance of symptoms or symptoms matching with nutrient deficiencies. As an alternative of visual scoring, several other methods were proposed such as microscopy to visualise the pathogen directly or to quantify the pathogen through enzyme linked immunosorbent assay. These techniques were associated with slow rate of analysis per sample and often were culture based. This led to the development of culture-independent DNA-based molecular techniques. These DNA-based molecular techniques have the potential for rapid, sensitive detection and accurate quantification of pathogens. Since a plant may be infected by the multiple pathogens, these techniques could also identify the number of pathogens by multiplex assay technique. In this manuscript, four different DNA-based techniques are reviewed which show that these are now routinely being used in diverse crop species for diversity, detection and diagnostic analyses of pathogens.

Fungal communities of young and mature hypersaline microbial mats

Microbial mats are a laminated organic-sedimentary ecosystem, found in a wide range of habitats. Fluctuating diel and seasonal physicochemical gradients characterize these ecosystems, resulting in both strata and microenvironments that harbor specific microbial communities. This study was undertaken to compare two types of microbial mats across seasons to further understand the structure of fungal communities in hypersaline microbial mats and their seasonal dynamics. The structure and diversity of fungal communities was documented in young transient and mature hypersaline microbial mats from a tropical region (Puerto Rico) using one culture-dependent and three culture-independent molecular techniques based on the internal transcribed spacer (ITS) region of ribosomal DNA: terminal restriction fragment length polymorphism (TRFLP), denaturing gradient gel electrophoresis (DGGE) and clone libraries. Two microbial mats (one young and transient, one mature) were sampled in Nov 2007 (wet season), Jan 2008 (intermediate season) and Mar 2008 (dry season) in the Cabo Rojo Solar Salterns on the southwestern coast of Puerto Rico. Traditional and molecular techniques revealed strong spatial and temporal heterogeneities in both microbial mats. Higher abundance of isolates and phylotypes were observed during the wet season, and diversity decreased from the top (oxic) to the bottom (anoxic) layers in both seasons. Some of the species isolated belong to the genera Aspergillus, Cladosporium, Hortaea, Pichia and Wallemia, which often are isolated from hypersaline environments. The most abundant clones belong to Acremonium strictum and Cladosporium halotolerans, which were not isolated in pure culture. The differences observed using culture-based and molecular techniques demonstrates the need of combining methods to study the diversity of fungi in a given substrate.

Microbial population dynamics in the faeces of wood-eating loricariid catfishes

Catfishes of the genus Panaque are known for their ability to feed on wood and hence to process cellulose fibres in their digestive systems. The paper industry uses cellulose fibres and thus has an interest in exploiting this property biomimetically: it could be employed as a pretreatment to lessen the energy required by the mechanical production stage of manufacturing nanocellulose fibres. Here, we characterize the diet-associated in situ microbial diversity and population dynamic in the faeces of catfish (Panaque sp.) exposed to consecutive diets of pellet food and then wood. Fish faeces samples were collected and investigated by parallel DNA deep amplicon sequencing of the bacterial 16SrRNA SSU for both diet conditions. The most frequently occurring bacterium in the faeces was Cetobacterium sp. The dominant cellulolytic bacterial genera found in ascending relative abundance were as follows: Aeromonas sp., Flavobacterium sp., Bacteroides sp., Pseudomonas sp. and Cellvibrio sp. Diet-associated changes in the faeces microbiome were noted for Flavobacterium sp. Extensive microbial diversity was found in catfish faeces, evidenced using culture-independent molecular techniques. No significant diet-associated effects on the microbiome in terms of biodiversity were observed in the catfish faeces, but diet-associated changes in the microbial population structure were observed. Although catfishes are not classified as true xylivores, inhabiting their faeces are bacteria that may provide a novel source of cellulolytic enzyme. Based on this first microbiology study, the faeces and thus the gastrointestinal microbiome of Panaque catfishes are an unexplored reservoir of microbial extracts with enhanced polysaccharide transforming enzyme activity. The biomimetical exploitation of this cellulolytic activity in the form of novel enzymes or by applying a mixture of cellulolytic micro-organisms could accomplish a pretreatment to the mechanical production process of nanocellulose fibres, thus could reduce the energy consumption costs significantly.

DNA extraction method selection for agricultural soil using TOPSIS multiple criteria decision-making model

There is an increased interest in the extraction of nucleic acids from various environmental samples since culture-independent molecular techniques contribute to deepen and broaden the understanding of a greater portion of uncultivable microorganisms. Due to difficulties to select the optimum DNA extraction method in view of downstream molecular analyses, this article presents a straightforward mathematical framework for comparing some of the most commonly used methods. Four commercial DNA extraction kits and two physical-chemical methods (bead-beating and freeze-thaw) were compared for the extraction of DNA under several quantitative DNA analysis criteria: yield of extraction, purity of extracted DNA (A260/280 and A260/230 ratios), degradation degree of DNA, easiness of PCR amplification, duration of extraction, and cost per extraction. From a practical point of view, it is unlikely that a single DNA extraction strategy can be optimum for all selected criteria. Hence, a systematic Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) was employed to compare the methods. The PowerSoil? DNA Isolation Kit was systematically defined as the best performing method for extracting DNA from soil samples. More specifically, for soil:manure and soil:manure:biochar mixtures, the PowerSoil?DNA Isolation Kit method performed best, while for neat soil samples its alternative version gained the first rank.