Denaturing Gradient Gel Electrophoresis Research Articles

Denaturing Gradient Gel Electrophoresis Approach for Microbial Shift Analysis in Thermophilic and Mesophilic Anaerobic Digestions.

To determine the evolution of microbial community and microbial shift under anaerobic processes, this study investigates the use of denaturing gradient gel electrophoresis (DGGE). In the DGGE, short- and medium-sized DNA fragments are separated based on their melting characteristics, and this technique is used in this study to understand the dominant bacterial community in mesophilic and thermophilic anaerobic digestion processes. Dairy manure is known for emitting greenhouse gases (GHGs) such as methane, and GHG emissions from manure is a biological process that is largely dependent on the manure conditions, microbial community presence in manure, and their functions. Additional efforts are needed to understand the GHG emissions from manure and develop control strategies to minimize the biological GHG emissions from manure. To study the microbial shift during anaerobic processes responsible for GHG emission, we conducted a series of manure anaerobic digestion experiments, and these experiments were conducted in lab-scale reactors operated under various temperature conditions (28 °C, 36 °C, 44 °C, and 52 °C). We examined the third variable region (V3) of the 16S rRNA gene fingerprints of bacterial presence in anaerobic environment by PCR amplification and DGGE separation. Results showed that bacterial community was affected by the temperature conditions and anaerobic incubation time of manure. The microbial community structure of the original manure changed over time during anaerobic processes, and the community composition changed substantially with the temperature of the anaerobic process. At Day 0, the sequence similarity confirmed that most of the bacteria were similar (>95%) to Acinetobacter sp. (strain: ATCC 31012), a Gram-negative bacteria, regardless of temperature conditions. At day 7, the sequence similarity of DNA fragments of reactors (28 °C) was similar to Acinetobacter sp.; however, the DNA fragments of effluent of reactors at 44 °C and 52 °C were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 97%) and Tepidimicrobium ferriphilum (strain: DSM 16624) (similarity: 100%), respectively. At day 60, the analysis showed that DNA fragments of effluent of 28 °C reactor were similar to Galbibacter mesophilus (strain: NBRC 10162) (similarity: 87%), and DNA fragments of effluent of 36 °C reactors were similar to Syntrophomonas curvata (strain: GB8-1) (similarity: 91%). In reactors with a relatively higher temperature, the DNA fragments of effluent of 44 °C reactor were similar to Dielma fastidiosa (strain: JC13) (similarity: 86%), and the DNA fragments of effluent of 52 °C reactor were similar to Coprothermobacter proteolyticus (strain: DSM 5265) (similarity: 99%). To authors' knowledge, this is one of the few studies where DGGE-based approach is utilized to study and compare microbial shifts under mesophilic and thermophilic anaerobic digestions of manure simultaneously. While there were challenges in identifying the bands during gradient gel electrophoresis, the joint use of DGGE and sequencing tool can be potentially useful for illustrating and comparing the change in microbial community structure under complex anaerobic processes and functionality of microbes for understanding the consequential GHG emissions from manure.

Impact of whole grain highland hull-less barley on the denaturing gradient gel electrophoresis profiles of gut microbial communities in rats fed high-fat diets.

While next-generation sequencing has overshadowed polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), the latter still holds promise for advancing gut microbiota analysis due to its unique advantages. In this study, we used optimized nested PCR-DGGE to investigate the gut microbiota profile of high-fat diet rats after administering whole grain highland hull-less barley. High-throughput sequencing was employed to validate the DGGE results. Our results proved the reliability of PCR-DGGE for analyzing the dominant taxonomic composition while also providing visual evidence of a notable relationship between the composition of cecal and fecal microbial communities, highlighting substantial differences in both richness and abundance.

Symbiont Diversity in Imidacloprid-Resistant and Imidacloprid-Susceptible Populations of Nilaparvata lugens (Hemiptera: Delphacidae)1

Abstract Imidacloprid, a neonicotinoid insecticide, is efficacious against hemipterans, including the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae). However, frequent use of this insecticide has resulted in the development of high levels of resistance among brown planthopper populations. Endosymbionts of insects have contributed to host physiology and evolution and play a role in resistance to chemical toxins. In this study, polymerase chain reaction–denaturing gradient gel electrophoresis was used to analyze the bacterial and yeast-like symbiont communities of imidacloprid-resistant and -susceptible brown planthopper populations. The Shannon-Weaver diversity index and the evenness index indicated no differences in the richness and the expression of overall species distribution of the symbiotic communities of resistant and susceptible populations. The similarity coefficients of 0.53 and 0.56 for bacterial and yeast-like symbionts, respectively, indicated the main types of differences among microorganisms in resistant and susceptible populations. Sequence comparison analysis indicated the bacterial species in the susceptible population were members of the Enterobacteriaceae and Moraxellaceae, and those in the resistant population were members of the Enterobacteriaceae, Oxalobacteriaceae, Rhodobacteriaceae, and Sphingomonadaceae. Differences also were found in the composition of yeast-like symbionts of the two populations; Cryptococcus luteolus, Pseudozyma aphidis, and Pseudozyma antarctica were detected in the susceptible population, and Cladosporium perangustum was detected in the resistant population.

Emissions of N2O AND CH4 gases and soil bacterial community under integrated systems in the tropical region

The use of integrated production systems is widespread, mainly in tropical areas. This practice could contribute to soil conservation, increasing biodiversity and environmental services while reducing greenhouse gases emissions. The present work assessed bacterial biodiversity and correlations with nitrous oxide (N2O) and methane (CH4) emissions in soils under integrated production systems. Analyses of bacterial diversity and community structure were carried out using DGGE (denaturing gradient gel electrophoresis). Samples for the evaluation of greenhouse gases were collected in the field and measured using a gas chromatograph. The assessed treatments in a rainy and dry season were: crop (L), pasture (P), planted eucalypt forests (F), pasture followed by crop (IPL), crop followed by pasture (ILP), crop with forest (ILF); pasture with forests (IPF); crop with forest followed by pasture with forests, pasture with forests followed by crop with forest and crop, grassland and forest. In the rainy season, most N2O emissions occurred in the ILP with an average flow of 42.33 µg N m−2 h−1, and the lowest flow was observed in the ILF, with an average flow of 5.53 µg N m−2 h−1. In the dry season, higher emission occurred in the IPL, with an average flow of 4.17 ug N m−2 h−1. In this period, we observed gas consumption in F, the highest N2O consumer with a flow of −4.14 µg N m−2 h−1. In both seasons, CH4 was consumed, with IPF as the most significant consumer with an average flow of −14.19 µg C m−2 h 1(dry); F was the most significant consumer with an average flow −12.32 µg of C m−2 h−1. Bacterial community structure was affected by the agricultural production system and the seasons. Methane emissions and pH showed a correlation with bacterial diversity in different treatments. In contrast, nitrous oxide was negatively correlated with bacterial diversity. Based on our results, we suggest that integrated production systems and plant coverage modulate soil bacterial community structure and biodiversity which, in turn, affect soil processes related to higher or lower methane and nitrous oxide emissions.

Analysis of the bacterial diversity in Moroccan Jben cheese using TTGE, DGGE, and 16S rRNA sequencing.

This research investigated the physicochemical, microbiological, and bacterial diversity of Jben cheese, a popular artisanal variety in Morocco. The bacterial diversity was explored using culture-independent methods, including temporal temperature gel electrophoresis (TTGE), denaturing gradient gel electrophoresis (DGGE), and high-throughput sequencing (HTS). Significant intra-sample differences were observed for most physicochemical parameters within each milk type, while inter-sample differences occurred between cow and goat cheeses for dry matter and ash. Jben cheese exhibited distinct characteristics, with low pH values of 3.96, 4.16, and 4.18 for cow, goat, and mixed cheeses, respectively. Goat cheeses had higher fat (49.23 g/100 g), ash (1.91 g/100 g), and dry matter (36.39 g/100 g) than cow cheeses. All cheeses displayed high microbial counts, with a notable prevalence of the lactic acid bacteria (LAB) group, averaging 8.80 ± 0.92 log CFU/g. Jben cheese also displayed high contamination levels with total coliforms, faecal coliforms, yeast, and molds. Fatty acid profiling revealed fraudulent practices in Jben cheese marketing, with cow or mixed cheeses sold as goat cheese, as proven by low capric acid concentration. HTS analysis of Jben cheese identified ten genera and twenty-four species, highlighting Lactococcus lactis as predominant. TTGE and DGGE confirmed the presence of L. lactis but failed to provide the detailed profile achieved through HTS analysis. HTS has been demonstrated to be more reliable, whereas TTGE/DGGE methods, though informative, were more time-consuming and less reliable. Despite limitations, the combined use of TTGE, DGGE, and HTS provided a comprehensive view of indigenous bacterial communities in Jben cheese, identifying L. lactis as the main species.

Turkish population-based screening for first identified changes of BRCA1 and BRCA2 genes in breast and/or ovarian cancer patients

IntroductionIt is known that BRCA1 and BRCA2 genes’ mutation carriers are predisposed to breast and ovarian cancers and other organ cancers such as prostate, colon and cervix. In the previous study performed at X University, all coding exons of both genes were screened by denaturing gradient gel electrophoresis (DGGE). In addition to various nonsense, missense mutations, polymorphisms and intronic region changes, seven novel missense mutations, including H513L, H816P and S1517Y in BRCA1 and S326R, G258P, E2903K and N2742S in BRCA2, had been identified.MethodsTo determine whether these unclassified variants are pathogenic, DNA samples of 150 healthy individuals without a known cancer history in the family were screened in this study for these seven novel missense mutations. These DNA samples were recruited from archives of previous polymorphism studies. PCR performed DNA amplifications, and denaturing high-performance liquid chromatography (DHPLC) techniques did mutation screenings.ResultsPeak patterns suggestive of a change in DNA fragments were considered for sequencing analyses. Analyses revealed that none of the 150 DNA samples had any change in the seven screened fragments. As a result, it is assumed that these seven mutations might be novel pathogenic mutations described in the Turkish population.ConclusionIn conclusion, these carriers must be informed about the mutation and given appropriate genetic counseling by their physicians. In addition, genetic testing must be offered to high-risk individuals (men/women) in the family so that it would be possible for other family members to have genetic counseling and contribute to disease prevention. On the other hand, these findings would contribute to current literature with novel results and shed light on future research.

Microbial Diversity and Functional Profiles of Three Commercial Biofertilizers and Impacts on the Bacterial Communities of Avocado’s Soil Rhizosphere

Chilean avocado (Persea americana Mill.) exports have accounted for 60 % of the total production and are recognized for their high quality worldwide. However, avocado production has significantly decreased in recent years, which is mainly attributed to abiotic and biotic factors, among which are high and low temperatures, intense and sudden rains, unavailability of water resources, and agricultural salinity. Secondary factors include pests and diseases. Applying plant growth promoting microorganisms (PGPM)-based commercial biofertilizers is a potential practice to increase avocado production and resistance to edaphoclimatic factors. In this study, to determine the functionality of microbial communities present in three commercial biofertilizers (Biofert A, Biofert B, and Biofert C) and thus offer a structure of associated microbial communities, the rhizospheric soil of avocado was analysed using the Biolog EcoPlate™ technique, providing information on the community level physiologic profiles (CLPP) and biodiversity indices by denaturing gradient gel electrophoresis (DGGE). The findings revealed that the microbial diversity in the three commercial biofertilizers is highly different, showing a SIMPER overall dissimilarity of 83.9 % in the catabolic capacity. Concerning the impact on avocados’ rhizosphere soil bacterial communities, the results demonstrated significant changes in their composition, particularly with Biofert A and C. In contrast, Biofert B did not show significant changes, especially on days 15 and 30. Long-term studies are recommended to develop sustainable agricultural practices for Chilean avocados.

Gut bacterial community of Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Crambidae) is driven by rice varieties

AbstractTo understand the effect of diet on gut bacteria of Cnaphalocrocis medinalis (rice leaf folder, RLF), we compared the composition and diversity of gut bacterial community in C. medinalis larvae collected from three different rice variety fields of Minghui 82 (RLF1), Youngyong 15 (RLF2) and Minghui 2155 (RLF3), using culture‐independent PCR (V3 variable region)‐denaturing gradient gel electrophoresis (PCR‐DGGE) methods and hypervariable region (V4) of 16S rRNA sequenced by Illumina HiSeq platform. The DGGE result showed that the bacteria genera Asaia, Bacillus, Stenotrophomonas, Achromobacter and Serratia coexisted in the guts of RLF1, RLF2 and RLF3, while Cedecea, Burkholderia and Acinetobacter coexisted in the guts of RLF2 and RLF3. However, only the genus Enterococcus existed in the guts of RLF1, and Pantoea, Wolbachia and Tumebacillus in RLF3 larvae. A total of 25 bacterial phyla, 48 classes, 127 orders, 223 families, and 406 genera were identified when using 16s RNA sequencing, with 35 genera coexisted in RLF1, RLF2 and RLF3 larval guts, while 83, 36, 141 unique genera existed in RLF1, RLF2 and RLF3, respectively. The indices of ACE and Shannon were not significantly different among RLF1, RLF2 and RLF3. The dominant bacterial taxa were Proteobacteria, Bacteroidetes and Firmicutes at the phylum level, and Acinetobacter and Wolbachia at the genus level. The relative genus abundance for the genera with relative abundance (≥0.01) was significantly different among RLF1, RLF2 and RLF3. The findings indicated that different rice varieties had significant effects on the relative abundance of gut bacteria in RLF, which could provide new insights into the relationship between insect gut bacteria and their associated host plants.

Detection of SARS-CoV-2 spike protein D614G mutation using μTGGE.

The accurate and expeditious detection of SARS-CoV-2 mutations is critical for monitoring viral evolution, assessing its impact on transmission, virulence, and vaccine efficacy, and formulating public health interventions. In this study, a detection system utilizing micro temperature gradient gel electrophoresis (μTGGE) was developed for the identification of the D614 and G614 variants of the SARS-CoV-2 spike protein. The in vitro synthesized D614 and G614 gene fragments of the SARS-CoV-2 spike protein were amplified via polymerase chain reaction and subjected to μTGGE analysis. The migration patterns exhibited by the D614 and G614 variants on the polyacrylamide gel were distinctly dissimilar and readily discernible by μTGGE. In particular, the mid-melting pattern of D614 was shorter than that of G614. Our results demonstrate the capability of μTGGE for the rapid, precise, and cost-effective detection of SARS-CoV-2 spike protein D614 and G614 variants without the need for sequencing. Therefore, this approach holds considerable potential for use in point-of-care mutation assays for SARS-CoV-2 and other pathogens.

Response of Bacterial Community Structure in the Bulk Soil and Rice Straw Residues under Different Crop Rotation Systems

This study investigated the effect of upland crop rotation on soil bacterial community in bulk soil and rice straw residues in the alluvial soils. Soil samples and rice straw residues in two crop rotation models including triple rice and rice-upland crops were collected for incubation under anaerobic and aerobic conditions. Data were analyzed from Denaturing Gradient Gel Electrophoresis band patterns. The results showed that the composition and diversity of communities colonizing the rice straw residues differed from those inhabiting the bulk soil. The bacterial community composition and diversity were only moderately affected by rice straw residues in the bulk soil. Especially, this study indicated that the composition of the bacterial community associated with the bulk soil and rice straw residues was dynamic in two incubation conditions (aerobic or anaerobic) and the different crop rotation models. The findings of this study demonstrated that the bacterial diversity was not affected by the difference in continuous paddy rice cultivation compared to the upland crop rotation system.

Use of PCR-DGGE-Based Molecular Methods to Analyze Nematode Community Diversity.

DGGE (denaturing gradient gel electrophoresis) is a nucleic acid separation technique applied to the evaluation of microbial biodiversity. This technique is quite rapid and cheap compared to other types of analysis. Here we describe the comparison of nematode communities inhabiting different ecosystems. After an ecologically representative sampling collection and the nematode extraction from soil, nematodes are centrifuged in Eppendorf tubes to facilitate DNA extraction. DNA from the whole community of each type of soil is extracted, amplified with primers for 18 S rDNA and used in DGGE analysis. The profiles of DGGE can be analyzed with appropriate software, and biodiversity indices can be estimated.

Broiler farming practices using new or re-used bedding, inclusive of free-range, have no impact on Campylobacter levels, species diversity, Campylobacter community profiles and Campylobacter bacteriophages.

A multi-stage option to address food-safety can be produced by a clearer understanding of Campylobacter's persistence through the broiler production chain, its environmental niche and its interaction with bacteriophages. This study addressed Campylobacter levels, species, genotype, bacteriophage composition/ levels in caeca, litter, soil and carcasses across commercial broiler farming practices to inform on-farm management, including interventions. Broilers were sequentially collected as per company slaughter schedules over two-years from 17 farms, which represented four commercially adopted farming practices, prior to the final bird removal (days 39-53). The practices were conventional full clean-out, conventional litter re-use, free-range-full cleanout and free-range-litter re-use. Caeca, litter and soil collected on-farm, and representative carcases collected at the processing plant, were tested for Campylobacter levels, species dominance and Campylobacter bacteriophages. General community profiling via denaturing gradient gel electrophoresis of the flaA gene was used to establish the population relationships between various farming practices on representative Campylobacter isolates. The farming practice choices did not influence the high caeca Campylobacter levels (log 7.5 to log 8.5 CFU/g), the carcass levels (log 2.5 to log 3.2 CFU/carcass), the C. jejuni/C. coli dominance and the on-farm bacteriophage presence/levels. A principal coordinate analysis of the flaA distribution for farm and litter practices showed strong separation but no obvious farming practice related grouping of Campylobacter. Bacteriophages originated from select farms, were not practice-dependent, and were detected in the environment (litter) only if present in the birds (caeca). This multifaceted study showed no influence of farming practices on on-farm Campylobacter dynamics. The significance of this study means that a unified on-farm risk-management could be adopted irrespective of commercial practice choices to collectively address caeca Campylobacter levels, as well as the potential to include Campylobacter bacteriophage biocontrol. The impact of this study means that there are no constraints in re-using bedding or adopting free-range farming, thus contributing to environmentally sustainable (re-use) and emerging (free-range) broiler farming choices.

Application of Molecular Biology Technologies in Environmental restoration

With the development of molecular biology, various new technologies have been developed and applied to the monitoring and management of the ecological environment. This includes the biodiversity of the oceans, soils, and plant and microbial ecosystems. This paper introduces the types of molecular biology techniques and discusses their applications in bioremediation and monitoring of contaminated environments. Conventional techniques include genetic engineering, DNA sequence analysis, electrophoresis separation and purification, nucleic acid probe detection, and polymerase chain reaction. Denaturing gradient gel electrophoresis (denaturing gradient gel electrophoresis) is a new electrophoresis technique. It is a gel system that separates DNA fragments according to their melting properties. Gene microarray technology (also known as gene chip) began to be widely used in the 1890s with biological monitoring technology. There are thousands of nucleic acid probes, which detect nucleic acids in living organisms using fluorescence or electricity, in gene microarray technology. These technologies can be used to regulate the diversity of microbial communities, solve the virus infection of plants, and explore the contaminant degradation genes and functional strains. The development and progress of modern molecular biology technology and biochemical technology have promoted the research and detection of ecological environments. These methods would become very effective tools for the study of environmental ecology.

Microbial Diversity of Traditional Livno Cheese from Bosnia and Herzegovina

Traditional dairy products, especially cheeses, represent part of the cultural food heritage of many countries. In addition, these cheeses constitute microbiological “reservoirs”, of which many have been lost due to the introduction of the pasteurization of milk in the dairy industry. Increased awareness of the importance of microorganisms that make up the biodiversity of traditional cheeses, as well as the development of molecular methods in recent decades, have enabled efforts to identify and preserve them. Traditional Livno cheese is a full-fat hard cheese, considered one of the most famous traditional cheeses of Bosnia and Herzegovina and is seasonally produced from a mixture of raw sheep’s milk supplemented with cow’s milk. Often, Livno cheese has variable quality, due to microbial contamination and poor milk quality. In this study, traditional Livno cheese was studied during the ripening of cheeses produced by different producers during two seasons. Culture-dependent analyses were made during ripening using microbiological plating on suitable media. Likewise, culture-independent methods Denaturing Gradient Gel Electrophoresis (DGGE) and Automated Ribosomal Intergenic Spacer Analysis (ARISA) were used to elucidate the cheese microbiota. Results of analysis showed Lactococcus spp., Streptococcus spp., Lactobacillus spp., Pediococcus spp. and Leuconostoc spp. to be dominant species in traditional Livno cheese. However, when comparing the use of culture-dependent and culture-independent methods in the evaluation of Livno cheese microbiota, Enterococcus was not detected by culture-independent DGGE methods. The microbial population of both the milk and the environment determines the fermentation processes during cheese production and ripening, and thereby defines the quality of this cheese. The numbers of bacteria in the cheese were shown to be dependent on the manufacturer, the degree of ripening and the production season.

Hydrodynamic behavior and start-up performance of a periodic anaerobic baffled reactor in an "every second" switching manner treating traditional Chinese medicine wastewater.

Most studies focus on the "clockwise sequential" switching manner for a four-compartment periodic anaerobic baffled reactor (PABR), while the exploration of the "every second" option on the feasibility for real industrial wastewater treatment is rarely reported. Hence, a PABR-treating traditional Chinese medicine wastewater was run continuously in "every second" switching manner with both switching period T and hydraulic residence time of 48 h. Satisfactory start-up performance was achieved during the operation of a climbing average organic load rate at approximately 1, 2, 4, and 6 kg chemical oxygen demand (COD) m-3 d-1 for 12, 24, 24, and 6 days, respectively. The average COD removal was 87.20% after the second lifting of OLR and 89.98% after the third one. Denaturing gradient gel electrophoresis and its cluster analysis showed that the microbial communities in each compartment adapted their structure in response to the periodically changing micro-ecology conditions. Moreover, the residence time distribution test with tap water in the clean PABR was carried out in experiments and computational fluid dynamics (CFD) simulation, both of which were in good agreement. The CFD model output visualized the flow velocity field and hydrodynamic-mass transport inside the PABR. Optimization of operation pattern in PABR including switching manner and frequency depended on both the type of waste being treated and the flexibility of biomass to periodically changing micro-ecology conditions.

Bacterial diversity and detection of resistance genes to broad-spectrum betalactams in dairy family farm soils

Bovine mastitis is a complex disease that brings great losses to the dairy producer. The microbial diversity of the soils, as well as the presence of resistance genes in the environment directly influence the maintenance of mastitis in the farm. The objective of this work was to analyze the bacterial diversity in pasture soils of a dairy family farm, detecting enterobacteria that may be involved in the etiology of bovine mastitis, and to detect genes that encode broad-spectrum betalactamases in these soils. Twelve soil samples, representative of different areas of the farm located in the municipality of Barra do Piraí, Rio de Janeiro, were collected at different times of the year. Total DNA was extracted from the samples, gene amplified by Nested-PCR and then the amplification products were separated by DGGE (Denaturing Gradient Gel Electrophoresis). With the DGGE it was possible to construct dendograms that effectively represented the bacterial diversity of these soils. Eight of the soil samples were used to amplify the genes encoding the betalactamase enzymes TEM (blaTEM gene), SHV (blaSHV gene) and CTX (blaCTXM gene). In three of the eight soil samples, the blaSHV gene was found to be present. The blaTEM and blaCTX-M genes were not detected in any of the samples. The detection of genes encoding broad-spectrum betalactamases in dairy cattle pasture soils is of concern, because the transfer of gene material between pathogenic and non-pathogenic bacteria in this environment is a reality.

Impact of Rhizobacterial Inoculants and Organic Amendment on Bacterial Communities of Jerusalem artichoke Tissue and Rhizosphere by using Denaturing Gradient Gel Electrophoresis

The application of organic amendments can improve soil quality and influence bacterial communities, contributing to enhanced soil health in agricultural systems. This study investigated the effect of using rain tree leaf, corn husk, and peat moss as soil amendments, supplemented with plant growth promoting rhizobacteria on communities of bacteria in the tissue and rhizosphere of Jerusalem artichoke. The plants were observed for 120 days after transplanting. Polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) technique was used to investigate the impact of both organic amendments and bacterial consortium with comparative profiles. Comparison DGGE profile by clustering analysis did not clearly separate each organic amendment and bacterial inoculation, but principal component analysis (PCA) revealed that plant age had an influence on tissue and rhizosphere bacterial communities. The results indicated that soil amendments and bacterial inoculation had no effect on bacterial community shifts. Interestingly, the genera of bacteria able to most colonize in the tissue and rhizosphere were the uncultured bacteria. Thus, we provide promising data for boosting Jerusalem artichoke growth and soil quality with potential organic amendments to improve soil with sustainable strategies.

Analysis on the pharyngeal microbiota in patients with laryngopharyngeal reflux disease

Objective(s)In this study, the laryngopharynx microbiome alterations were characterized after proton pump inhibitor treatment in patients with Laryngopharyngeal Reflux Disease (LPRD) and healthy people. The potential outcome-predictive biomarker was explored. MethodsPatients with LPRD and healthy controls were enrolled. The composition of their laryngopharynx microbiota was analyzed both by traditional plate count of the main bacterial groups and PCR amplification followed by denaturing gradient gel electrophoresis. Shannon-Wiener index and evenness index based on Dice index were used to assess the bacterial diversity. Droplet digital PCR was used to determine the total bacterial RNA and relative abundance of Klebsiella oxytoca. Receiver operating characteristic curve was plotted to explore the potential of Klebsiella oxytoca as an outcome-predictive biomarker. ResultsA total of 29 LPRD cases and 28 healthy subjects were enrolled. The composition of the laryngopharynx microbiota was almost similar, except Klebsiella oxytoca. The cluster analysis showed that the similarity between healthy and treatment-effective groups, as well as pretreatment and treatment-invalid groups, was close. Statistical analysis showed that there were differences in the diversity index and richness among the healthy, treatment-effective, pretreatment and treatment-invalid groups. The abundance of Klebsiella oxytoca in the treatment-effective LPRD group was lower than that of the treatment-invalid LPRD group. The abundance of Klebsiella oxytoca can distinguish treatment-effective and -invalid groups (AUC=0.859) with a sensitivity of 77.78% and specificity of 90.91%. ConclusionThere were differences in the diversity of cecal contents microbial community between treatment-invalid and treatment-effective LPRD groups. Klebsiella oxytoca has potential to distinguish treatment outcomes. Level of evidenceHow common is the problem? Level 1. Is this diagnostic or monitoring test accurate? (Diagnosis) Level 4. What will happen if we do not add a therapy? (Prognosis) Level 5. Does this intervention help? (Treatment Benefits) Level 4. What are the COMMON harms? (Treatment Harms) Level 4. What are the RARE harms? (Treatment Harms) Level 4. Is this (early detection) test worthwhile?(Screening) Level 4.

Research Note: Preliminary assessment of the impact of dietary yeast products on egg production and cecal microbial profiles of laying hens

The objective of the current study was to conduct an initial comparison of commercial yeast products in layer hen diets on egg production parameters and the corresponding impact on the cecal microbiota. A short-term feeding study was conducted with 35 laying hens receiving either a control, or 1 of 4 different yeast fermentation products, Immunowall, Hilyses (both from ICC, São Paulo, Brazil), Citristim (ADM, Decatur, IL), and Maxi-Gen Plus (CBS Bio Platforms, Calgary, Canada) with 7 hens per treatment from 40 to 46 wk of age. At the end of the trial, hens were euthanized, the ceca removed and prepared for denatured gradient gel electrophoresis (DGGE) microbial compositional analyses. Although initial shell weight and shell thickness were similar among the treatment groups, hens fed Hilyses had lower shell weight and thickness at the end of the experiment. The most predominant DGGE bands with the strongest intensity were identified as Lactobacillus species and excised double bands were identified as Bacillus, Clostridium, or Lachnospiraceae. In this short-term feeding trial, the commercial yeast products tested had little effect on egg production and shell quality, and only moderately impacted the composition of mature layer hen cecal microbiota.

Mangroves: A hotspot for novel bacterial and archaeal diversity

Mangrove ecosystems are highly productive ecosystems that harbor diverse microbial lineage playing multiple roles in nutrient cycling. Though several novel mangrove rhizospheres associated with bacterial genera and species have been reported from these unexplored wetland ecosystems, but yet to be studied for their richness and diversity including actinobacterial and archaeal communities. Therefore, this study was undertaken to decode the bacterial and archaeal communities associated with the rhizosphere regions of true mangrove species-Avicennia marina and Rhizophora mucronata and their intersecting regions; and associated halophytic species -Suaeda maritima, and Salicornia brachiata of Pichavaram mangroves, Tamil Nadu, India. Amplified ribosomal DNA restriction analysis (ARDRA) and PCR denaturing gradient gel electrophoresis (PCR-DGGE) analysis techniques were adopted. About 567 culturable bacterial isolates with distinct morphology were purified, and nearly 88 distinct Operational Taxonomic Units (OTUs) were identified by ARDRA analysis. Phylogenetic analysis of representative groups indicated that the majority of the isolates belonged to Firmicutes (10 genera), followed by Proteobacteria- Alphaproteobacteria (10 genera) and Gammaproteobacteria (8 genera), Actinobacteria (8 genera), Bacteroidetes (1genera), and Halomonas (9 genera) representing 38 genera with 105 species. In addition, a few phylogenetically distinct novel groups belonging to the genera Mangrovibacter, Vibrio, Demequina, and Catenococcus were also isolated. PCR-DGGE with the RDP-Classifier program identified the prominent DGGE bands up to genus level namely Bacillus, Pseudomonas, Vibrio, Halomonas, etc. In addition, this study for the first time describes the genera Bhargavaea, Exiguobacterium, Hoeflea, Catenococcus, Albirhodobacter, Dietzia, Brachybacterium, Aerococcus, and Myroides association with mangrove rhizospheres. Likewise, DGGE analysis of archaea represented 2 major groups such as Crenarchaeota and Euryarchaeota, which were further classified into 7 different genera.