Terminal Restriction Fragment Length Polymorphism Research Articles

Metal phytostabilization by mastic shrub (Pistacia lentiscus L.) and its root-associated bacteria in different habitats of Sardinian abandoned mining areas (Italy).

Pistacia lentiscus L. is an excluder metallophyte proposed for the revegetation and phytostabilization of metal-contaminated sites in the Mediterranean area. The present study aims at evaluating the linking between bacterial communities and plants spontaneously growing in ecosystems chronically impacted by mining activities. Environmental properties and metal accumulation into hypogeal and epigeal tissues were analyzed in wild plants of two contrasting habitats with extreme metal contamination (> 2300mg/kg for Zn, > 1100mg/kg for Pb, > 10mg/kg for Cd). The community structures of rhizospheric and root endophytic bacteria were fingerprinted by terminal restriction fragment length polymorphism of the 16S rRNA gene. The wild shrubs efficiently restrict the accumulation of the three major contaminants to the epigeal tissues in the two habitats under study (249 ± 68mg/kg dw for Zn, 43 ± 21mg/kg dw for Pb, and 1.4 ± 0.5mg/kg dw for Cd). Evidence was provided that the combined but not individual effect of environmental conditions (moisture, inorganic carbon, pH) and proportion between Zn and Cd in the mine substrate play a role in structuring rhizosphere bacterial communities. The observed changes in community structures of root endophytes were found to be strongly associated with Pb level in roots and substrate properties (inorganic carbon and Zn/Cd ratio). Overall, our study highlights the importance of the analysis of multifactorial interactions among mine substrate, plant, and microbes for understanding how the environmental context affects phytoremediation under real conditions.

Soil management effects on soil organic matter properties and carbon sequestration (SOMPACS)

SOMPACS is a project recommended by EJP SOIL for funding under the 1st External Call "Towards Healthy, Resilient and Sustainable Agricultural Soils". The goal of this project is to assess management practices that enrich organic matter pools that are resilient to rapid microbial decomposition. The project started in 2022 as a consortium of 12 research institutions from Poland, Germany, Ireland, Lithuania, UK, Italy and USA for a period of three years. Soil and vegetation samples from eight long-term experiments that differ in soil management practices (i.e., conventional vs. no-tillage; mineral vs. organic fertilization; with and without catch crop; and arable land vs. undisturbed grassland) are investigated. Study sites include: 22- and 54-year long experiments in Lithuania; 26-year long experiment in Italy; 30- and 40-year long experiments in Ireland; 30- and 46- and 100-year long experiments in Poland; and 178-year long Broadbalk experiment in Great Britain. Additional experimentation includes assessing the impact of root growth promoting amendments (commercially available humic substances, biochar and biogas digestate) on stable organic matter pools. In parallel with soil sampling, plant productivity are measured in all field experiments. This investigation is couples fields studies with small-scale experimental plots and laboratory incubations under controlled conditions. In addition to assessing basic soil properties, the following state-of-the-art analyses are conducted: SOM composition and stability by Py-GC-MS; fractionation of aggregate size classes and C pools of increasing physicochemical protection; isotopic analysis of δ13C and δ15N performed on different SOM pools; microbiological properties (community-level physiological profiling, selected functional genes involved in C and N cycles, microbiome and mycobiome analyses by next-generation sequencing, genetic diversity using terminal restriction fragment length polymorphism); enzymatic activity; soil water retention and soil water repellency; mineral composition of clay fraction; (8) soil structure stability. SOM composition and stability by Py-GC-MS; fractionation of aggregate size classes and C pools of increasing physicochemical protection; isotopic analysis of δ13C and δ15N performed on different SOM pools; microbiological properties (community-level physiological profiling, selected functional genes involved in C and N cycles, microbiome and mycobiome analyses by next-generation sequencing, genetic diversity using terminal restriction fragment length polymorphism); enzymatic activity; soil water retention and soil water repellency; mineral composition of clay fraction; (8) soil structure stability. The most resistant SOM pool (humin) are isolated by different methods (isolation vs. extraction) and examined for chemical composition and structure, using spectrometric and spectroscopic techniques (mass spectrometry, NMR, FTIR, EPR, UV-Vis-NIR, fluorescence). The carbon stocks in the soil profile will be evaluated and the carbon extractable in cold water will be determined to assess the potential carbon leaching and microbial availability. Additionally, in-field soil carbon dioxide (CO2) fluxes from selected experiments is monitored. Thus far, soil samples (0-100 cm depth) were collected and the humin fraction from surface A horizon was isolated for spectroscopic studies. Crop yield and vegetation productivity was also assessed. The research was financed by NCBR (EJPSOIL/I/78/SOMPACS/2022).

Probiotics as an alternative to antibiotics in modulating the intestinal microbiota and performance of broiler chickens.

Gut bacteria play an important role in poultry nutrition and the immune defense system. Changes in the intestinal microbiome affect the physiological state, metabolism and innate im-munity of poultry. The present study aimed to characterize age-related changes in the gastrointestinal tract microflora in broiler chickens, depending on supplementation of the diet with the in-feed antibiotic Stafac® 110 and a Bacillus subtilis strain-based probiotic. In this regard, a comprehensive analysis of the taxonomic structure of the microbial community in the gastrointestinal tract (GIT) of broiler chickens was carried out using a molecular genetic technique of the Terminal-Restriction Fragment Length Polymorphism analysis and taking into account age dynamics and feeding treatment. A beneficial effect on the microbiological composition and body weight of broilers was observed when using the antibiotic and probiotic in compound feeds. Different bacterial communities were revealed in the duodenum and cecum and their positive impact on broiler growth was established. The results obtained shed light on the formation of GIT microflora of broiler chickens during the growing period and its changes in response to the use of the antibiotic and the probiotic. We suggest that the implementation of the tested in-feed antibiotic and probiotic can be beneficial in regulating the intestinal microflora microbiological processes in the GIT and improving the feeding efficiency and productivity of broiler chickens.

Microbiome Structure Analysis of Oil Palm Pollinator Elaeidobius kamerunicus (Coleoptera; Curculionidae)

The pollination of oil palm by Elaeidobius kamerunicus leads to an increase of over 70% in countries such as India, Malaysia, and Indonesia. The impact of insect-microbiome interactions on the pollination activity and fitness of E. kamerunicus is unknown. Our study aimed to gain insight into the bacterial communities of E. kamerunicus from two different sites with high and low fruit set percentages, using culturable and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis. Our result revealed distinctive T-RFs profiles in E. kamerunicus from two different sites. Additionally, the culturable approach showed that some of this microbiome were found only in the weevil population from the high fruit set site. Our findings suggest that these bacteria could contribute to the fitness of E. kamerunicus, leading to a higher fruit set in oil palm plantations.

The effect of heat-killed Lactobacillus brevis SBL88 on improving selective hepatic insulin resistance in non-alcoholic fatty liver disease mice without altering the gut microbiota.

There have been several reports that some probiotics improve non-alcoholic fatty liver disease (NAFLD); however, many studies have involved cocktail therapies. We evaluated whether heat-killed Lactobacillus brevis SBL88 (L.brevis SBL88) monotherapy improves the clinical features of NAFLD. The NAFLD model was induced in mice fed a high-fat diet (HFD) (HFD mice) or HFD+1% heat-killed L.brevis SBL88 (SBL mice) for 16weeks. Histopathological liver findings were analyzed. To evaluate the gut microbiota, a modified terminal restriction fragment length polymorphism analysis of the feces was performed. RNA sequencing in the liver was performed with Ion Proton™. To investigate the direct effects of heat-killed L.brevis SBL88, an in vitro study was performed. Histopathological findings revealed that fat droplets in the liver were significantly reduced in SBL mice; however, terminal restriction fragment length polymorphism did not show alterations in the gut microbiota between HFD mice and SBL mice. RNA sequencing and pathway analysis revealed that the regulation of lipid and insulin metabolism was affected. The mRNA expression of insulin receptor substrate 2 (IRS-2) was significantly higher in SBL mice, whereas the expression of IRS-1 was not significantly different. Phospho-IRS-2 expression was also significantly increased in SBL mice. In addition, an in vitro study revealed significant alterations in IRS-2 and forkhead box protein O1 expression levels. SBL mice exhibited partially improved selective hepatic insulin resistance. Our data suggest that heat-killed L.brevis SBL88 could attenuate the clinical features of NAFLD that are not mediated by alterations in the gut microbiota.

Molecular Approaches in Soil Microbial Analysis: Forensic Perspective

The growing field of soil microbial forensics provides the legal answer to how microorganisms play a crucial role in criminal investigations. It is an advanced crossdisciplinary science capable of offering significant physical evidence with considerable interest in criminal investigations, environmental crime, and counterterrorism. Microbial forensics of soil consists of different techniques to identify and evaluate microbial abundance, their complexity, and their interaction with soil and surrounding objects. The present review highlights various microbial analysis techniques such as Terminal restriction fragment length polymorphism, Temperature/Denaturing Gradient Gel Electrophoresis, Amplified Ribosomal DNA Restriction Analysis, Length Heterogeneity PCR, Phospholipid-derived fatty acids, Fluorescence in situ hybridization, Stable-isotope probing and metagenomics using nextgeneration sequencing. This article also summarises the challenges faced in soil microbial forensics, various statistical approaches, reference databases commonly used in forensic soil studies, and different methodological approaches used in forensic laboratories. Literature was studied using various online bibliographic databases like Google Scholar, Web of Science, Pub Med, Scopus, and several other search engines. Conclusive evidence generated by this paper signifies the importance of soil microbes to assist forensic scientists and researchers in selecting adequate methods to differentiate soil samples. The present comparative study concluded that targeted molecular analysis of microbes as a forensic soil typing tool has a lot of potential and should be investigated further.

Comparative Assessment of Success Rate Between Silver Nanoparticle Coated and Uncoated Infra-zygomatic Bone Screws: A Split-Mouth Study

Introduction In orthodontic treatment, infra-zygomatic crest screws have been effectively used in the correction of borderline skeletal malocclusion. The success of bone screws depends on factors such as biomechanical factors, placement technique, and microbial factors. Therefore, this study aimed to evaluate the success rate as well as assess the microbiota of silver nanoparticle coated and uncoated infra zygomatic crest (IZC) screws on clinical application. Method A sample of 36 stainless steel IZC screws was divided into 2 groups. Group I- Silver nanoparticle (AgNP) coated (n = 18) and group II uncoated (n = 18). A total of 18 patients (4 male, 14 female; mean age 22.58±3.52) received IZC screws (coated or uncoated) via a double-blind, split-mouth design. The success rate was evaluated based on the stability of IZC screws. And also molecular analysis was carried out using terminal restriction fragment length polymorphism complemented with cloning and identification of species. Results The success rate was higher in group I (77.80%) than in group II (50.00%). The mean rank for terminal restriction fragments in AgNP coated group (14.92%) was significantly lower than the uncoated group (22.08%) (P<0.05). The cloning results revealed a major difference in the predominant species composition between AgNP-coated (Prevotella oris sp.) and uncoated IZC screw (Capnocytophaga leadbetteri). Conclusion The success rate for AgNP-coated IZC screws was higher compared to uncoated screws, while predominant bacteria present in coated and uncoated samples were found to be of different biological significance which needs to be explored subsequently.

Co-Selection of Bacterial Metal and Antibiotic Resistance in Soil Laboratory Microcosms.

Accumulation of heavy metals (HMs) in agricultural soil following the application of superphosphate fertilisers seems to induce resistance of soil bacteria to HMs and appears to co-select for resistance to antibiotics (Ab). This study aimed to investigate the selection of co-resistance of soil bacteria to HMs and Ab in uncontaminated soil incubated for 6 weeks at 25 °C in laboratory microcosms spiked with ranges of concentrations of cadmium (Cd), zinc (Zn) and mercury (Hg). Co-selection of HM and Ab resistance was assessed using plate culture on media with a range of HM and Ab concentrations, and pollution-induced community tolerance (PICT) assays. Bacterial diversity was profiled via terminal restriction fragment length polymorphism (TRFLP) assay and 16S rDNA sequencing of genomic DNA isolated from selected microcosms. Based on sequence data, the microbial communities exposed to HMs were found to differ significantly compared to control microcosms with no added HM across a range of taxonomic levels.

Altered microbiota caused by disordered gut motility leads to an overactivation of intestinal immune system in APC1638T mice.

Adenomatous polyposis coli (APC) is recognized as an antioncogene related to familial adenomatous polyposis and colorectal cancers. However, APC is a large protein with multiple binding partners, indicating APC has diverse roles besides as a tumor suppressor. We have ever studied the roles of APC by using APC1638T/1638T (APC1638T) mice. Through those studies, we have noticed stools of APC1638T mice were smaller than those of APC+/+ mice and hypothesized there be a disturbance in fecal formation processes in APC1638T mice. The gut motility was morphologically analyzed by immunohistochemical staining of the Auerbach's plexus. Gut microbiota was analyzed by terminal restriction fragment length polymorphism (T-RFLP). IgA concentration in stools was determined by enzyme-linked immunosorbent assay (ELISA). As results, macroscopic findings suggestive of large intestinal dysmotility and microscopic findings of disorganization and inflammation of the plexus were obtained in APC1638T mice. An alteration of microbiota composition, especially increased Bacteroidetes population was observed. Increases in IgA positive cells and dendritic cells in the ileumwith high fecalIgA concentration were also confirmed, suggesting over-activation of gut immunity. Our findings will contribute to our understanding of APC's functions in the gastrointestinal motility, and lead to a development of novel therapies for gut dysmotility-related diseases.

Pioneer colonizers: Bacteria that alter the chicken intestinal morphology and development of the microbiota.

Microbes commonly administered to chickens facilitate development of a beneficial microbiome that improves gut function, feed conversion and reduces pathogen colonization. Competitive exclusion products, derived from the cecal contents of hens and shown to reduce Salmonella colonization in chicks, possess important pioneer-colonizing bacteria needed for proper intestinal development and animal growth. We hypothesized that inoculation of these pioneer-colonizing bacteria to day of hatch chicks would enhance the development of their intestinal anatomy and microbiome. A competitive exclusion product was administered to broiler chickens, in their drinking water, at day of hatch, and its impact on intestinal morphometrics, intestinal microbiome, and production parameters, was assessed relative to a control, no treatment group. 16S rRNA gene, terminal restriction fragment length polymorphism (T-RFLP) was used to assess ileal community composition. The competitive exclusion product, administered on day of hatch, increased villus height, villus height/width ratio and goblet cell production ∼1.25-fold and expression of enterocyte sugar transporters 1.25 to 1.5-fold in chickens at 3days of age, compared to the control group. As a next step, chicks were inoculated with a defined formulation, containing Bacteroidia and Clostridia representing pioneer-colonizing bacteria of the two major bacterial phyla present in the competitive exclusion product. The defined formulation, containing both groups of bacteria, were shown, dependent on age, to improve villus height (jejunum: 1.14 to 1.46-fold; ileum: 1.17-fold), goblet cell numbers (ileum 1.32 to 2.51-fold), and feed efficiency (1.18-fold, day 1) while decreasing Lactobacillus ileal abundance by one-third to half in birds at 16 and 42days of age, respectively; compared to the phosphate buffered saline treatment group. Therefore, specific probiotic formulations containing pioneer colonizing species can provide benefits in intestinal development, feed efficiency and body weight gain.

Effects of Chemical Fertilizer Reduction Combined with Organic Fertilizer Application on Bacterial Community Structure in Rhizosphere/Non-Rhizosphere Soil of Lemon

The aim of this study was to examine the effects of different fertilization methods on the physicochemical properties and bacterial community structure of lemon rhizosphere/non-rhizosphere soil in order to provide theoretical basis for scientific and rational fertilization of orchards. A pot experiment was carried out, and six fertilization treatments were set up:control (CK), conventional fertilization (FM), organic fertilizer (P), fresh organic fertilizer (NP), 70% chemical fertilizer+30% organic fertilizer (70FP), and 50% chemical fertilizer+50% organic fertilizer (50FP). Chemical analysis, real-time fluorescence quantitative PCR, and terminal restriction fragment length polymorphism (T-RFLP) were used to study the effects of different fertilization treatments on the physicochemical properties of rhizosphere and non-rhizosphere soils, the abundance of the bacterial 16S rRNA gene, and bacterial community structure. Redundancy analysis (RDA) was used to explore the environmental factors affecting the bacterial community structure of lemon rhizosphere/non-rhizosphere soil. The results showed the following:① the pH and contents of organic matter, alkali-hydrolyzed nitrogen, available phosphorus, available potassium, and nitrate nitrogen in rhizosphere/non-rhizosphere soil were significantly increased by reducing the amount of chemical fertilizer and applying organic fertilizer (50FP and 70FP) (P<0.05). Compared with conventional fertilization (FM) and single application of organic fertilizer (P and NP), the soil available P content, available K content, and nitrate nitrogen content increased by 24.76%-97.98%, 6.87%-45.11%, and 18.42%-55.82%, respectively. ② Fertilizer reduction combined with organic fertilizer significantly increased the abundance of soil bacteria and soil respiration intensity (P<0.05), and the abundance of soil rhizosphere bacteria and soil respiration intensity under the 50FP treatment increased by 15.83%-232.98% and 8.0%-162.5% compared with that under conventional fertilization and organic fertilizer alone, respectively. The bacterial abundance of rhizosphere soil was positively correlated with the pH and contents of organic matter, total nitrogen, and total phosphorus. ③ The PCoA and RDA analysis results showed that the single organic fertilizer and organic fertilizer and chemical fertilizer de-weighting of rhizosphere bacterial community structure and not adding fertilizer had a bigger difference between processing, and the main environmental factors influencing the rhizosphere/non rhizosphere bacterial community structure were organic matter, total nitrogen, total phosphorus, total potassium, alkali solution nitrogen, nitrate nitrogen, and available potassium. Fertilizer reduction combined with organic fertilizer could significantly increase soil nutrient content, increase soil bacterial abundance, and change the bacterial community structure of rhizosphere soil, and the 50FP treatment yielded better results. Therefore, 50% Chemical fertilizer+50% organic fertilizer (50FP) was a better fertilization method to improve the physical and chemical properties of orchard soil, increase the abundance of soil bacteria, and improve the soil respiration intensity.

Profile of Gut Bacteria in Hypertensive Patients Based on Terminal Restriction Fragment Length Polymorphism Analysis

Hypertension is a severe public health problem due to its high prevalence worldwide. About 7.5 million deaths or 12.8% of all annual deaths worldwide occur due to high blood pressure. The hypertensive disease is also associated with the intestinal bacteria. To our knowledge, the diversity of the gut bacteria in hypertensive patients has not been reported yet in Indonesia. Thus, the aims of this study were to analyze profile of gut bacteria in hypertensive patients compared to non-hypertensive based on metagenomic analysis, Terminal Restriction Fragment Length Polymorphism (T-RFLP). The results of the affiliation analysis of the entire Terminal Restriction Fragments (TRF) contained 6 groups of bacteria from 26 TRFs in hypertensive and non-hypertensive respondents. Cutting the 16S rRNA gene with the BslI restriction enzyme successfully detected intestinal bacterial groups, namely Clostridium subcluster XIVa, Prevotella, Clostridium cluster IV, Clostridium cluster XI, Bacteroides and others. In hypertensive patients, a higher relative abundance of bacterial groups showed in Clostridium cluster XI, Clostridium cluster IV and Clostridium subcluster XIVa. The abundance of Bacteroides and Prevotella groups in hypertensive patients were lower than non-hypertensive. The abundance of enterotype I and enterotype II was lower in hypertensive patients than non-hypertensive. Contrarily to that enterotype III cluster. It is worth noting that the F/B ratio was higher in hypertensive patients than that in non-hypertensive. Our data suggest that the gut bacteria profile of hypertensive patients differs to that non-hypertensive.

The Endophytic Plant Growth Promoting Methylobacterium oryzae CBMB20 Integrates and Persists into the Seed-Borne Endophytic Bacterial Community of Rice

Endophytic persistence of inoculated plant growth promoting bacteria (PGPB) involves interaction with the host plant and the host’s indigenous endophytic bacterial communities. This study investigated the persistence of Methylobacterium oryzae CBMB20 into the rice endosphere together with the impact of inoculation on the diversity and community structure of the root and shoot bacterial endophytes in Oryza sativa L. spp. indica cv. IR29. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis of the root and shoot showed that M. oryzae CBMB20 was able to integrate and persist in the rice endosphere without causing drastic shifts in bacterial endophytic diversity and community composition. The bacterial communities in the root and shoot are very similar to the seeds of IR29, suggesting that most of them are seed-borne. The root endosphere bacterial communities of inoculated and uninoculated IR29 plants are more diverse compared to the shoots in terms of richness and diversity indices. The dominant bacterial T-RFs of the root endosphere of IR29 belong to Microbacterium, Delftia, Pseudomonas, Xanthomonas and Stenotrophomonas, Herbaspirillum, Enterobacter, and Sphingomonas, as observed in the three restriction enzyme T-RFLP profiles. Bacterial clades identified as Curtobacterium, Enterobacter, Stenotrophomonas, and Xanthomonas were distinctly observed in both the root and shoot communities, and these bacterial groups are also the dominant endophytes of the shoot endosphere. This study showed that Methylobacterium oryzae CBMB20 could persist and incorporate into the endophytic bacterial community of the endosphere without causing long-term antagonistic interactions with its host plant and with the native microbiota.

Influence of seasonality, wastewater treatment plant process, geographical location and environmental parameters on bacterial community selection in activated sludge wastewater treatment plants treating municipal sewage in South Africa

This is the first comprehensive study that focusses on the correlation between the bacterial community composition and a range of previously identified selective criteria in activated sludge wastewater treatment plants on the African continent. Multivariate statistical analyses were used to determine the relative significance of the geographical location (factor: site), wastewater treatment plant process (factor: configuration), seasonality (factor: season), and environmental parameters on the bacterial communities in nine wastewater treatments plants from two sites in South Africa using terminal restriction fragment length polymorphism as a screening tool to rationalize the number of samples (to 50 samples) for high throughput (Illumina MiSeq) sequencing.Site was the most significant factor (Global ANOSIM R value = 0.91, p = 0.001), and it was established that the inter-site differences were not climatic in origin but related to differences in the composition of the influent and activated sludge. Previous studies that have reported associations between microbial community structure and environmental parameters have measured influent chemistry, and this is the first time, to our knowledge, that the comprehensive chemical character of activated sludge itself has been included in this type of study. It was found using BEST analysis that the activated sludge ammonia, activated sludge total phosphate and influent chemical oxygen demand were the most significant (p < 0.001) drivers for inter-site bacterial community selection (ANOSIM Global R values of 0.862, 0.782 and 0.428, respectively). This link would not have been established with only influent chemical analyses as there was no significant difference (t-test, p > 0.05) in the average influent phosphate concentrations between the 2 sites, but there was a highly significant difference (p < 0.001, t (15.5)>t-crit (2.01)) in the activated sludge total phosphate concentrations (20.8 ± 17.0 and 127.8 ± 40.2 mg/L). This is notable for all future studies on a global level aimed at identifying factors for selection of microbial communities in activated sludge.

The T-RFLP research method in the study of rumen microbiota in dairy cows with subclinical ketosis

The article presents the results of researches related to the study of the possibility of using the modern molecular genetics method -T-RFLP-analysis (terminal restriction fragment length polymorphism) to identify the community of microorganisms in the contents of the rumen in clinically healthy highly productive cows and cows with subclinical ketosis. The research methodology is based on the analysis of the conservative regions in the microorganisms’ genome variability. The results showed the high efficiency of the method used for the identification of microorganisms in the rumen contents in the studied animals. A large community of bacteria, archaea, protozoa and anaerobic fungi is determined in the rumen contents from highly productive cows with subclinical ketosis. The data obtained allow us to significantly amplify the information on the pathogenesis of subclinical ketosis in cows with high milk productivity. The presence of conditionally pathogenic and pathogenic microflora in sick cows in the rumen contents indicates a violation of rumen digestion, which leads to the development of concomitant non-contagious diseases in animals.

​Identification of Lactic Acid Bactria in Dairy Products using Culture-independent Methods: A Review

The microbiota of dairy products is composed of starter and non-starter lactic acid bacteria, as well as other bacteria, yeasts and filamentous fungi that form secondary microbiota and play an important role in cheese ripening. The use of combined LAB strains based on compatibility and/or coexistence tests plays an important role in food production and preservation, while combinations of strains that exhibit mutually inhibitory properties must be avoided. For centuries, researchers have focused on the exploration of autochthonous LAB biodiversity from various ecological dairy niches in order to address its role in texture and flavour formation and to select strains for use as starter cultures. Recent advanced technological tools like next-generation sequencing machines have played a significant role in the fast and accurate characterization and isolation of microbial communities from dairy products. However, the characterization and isolation of LAB using culture-independent methods is also a reliable and cost-effective method for studying a bacterial community without prior cultivation. These methods include single-strand conformation polymorphism (SSCP-PCR), denaturing gradient gel electrophoresis (DGGE), thermal gradient gel electrophoresis (TGGE), real-time PCR (qPCR), terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), automated ribosomal intergenic spacer analysis (ARISA), and fluorescence in situ hybridization (FISH). Therefore the goal of this review is to compare and describe the most recent culture-independent molecular-based methods for the characterization and isolation of LAB in dairy products.

Long-term enclosure at heavy grazing grassland affects soil nitrification via ammonia-oxidizing bacteria in Inner Mongolia

Enclosure and grazing can significantly change the turnover of nitrogen in grassland soil. Changes of soil nitrogen mineralization and ammonium-oxidizing microorganisms caused by enclosure in different grazing intensities (about 30 years of grazing history) grassland, however, has rarely been reported. We selected the grassland sites with high and medium grazing intensity (HG and MG, 4 and 2 sheep ha−1, respectively) and had them enclosed (45 × 55 m) in 2005 while outside the enclosure was continuously grazed year-round. A two factorial study was designed: grazing intensity (MG and HG sites) and enclosure (fence and non-fence). Nitrogen mineralization was detected through a laboratory incubation experiment. The abundance and community structure of soil ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were analyzed using quantitative PCR (q-PCR), terminal-restriction fragment length polymorphism (T-RFLP), cloning, and sequencing. Results showed that compared with MG site, at HG site the AOB abundance and community structure of AOB changed significantly while the AOA abundance and community structure did not change obviously. Enclosure significantly decreased the cumulative mineralized N, N mineralization rate, the abundance of AOB and the AOB community structure at the HG site, while at MG site, enclosure did not change these parameters. Potential nitrification rate (PNR) was positively correlated with the abundance of AOA and AOB at the MG and HG sites, respectively. The abundance of AOA was significantly correlated with soil pH; however, AOB abundance was significantly correlated with soil available N, total N, C/N ratio, pH, etc. The phylogenetic analysis showed that Nitrososphaeraceae and Nitrosomonadaceae were the dominant AOA and AOB, respectively. Totally, the responses of AOB and AOA mainly were associated to changes in soil physicochemical properties caused by different intensity grazing; AOB and AOA may be the dominant functional players in ammonia oxidation processes at HG and MG site, respectively.

Microbial Community Profiling Using Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Denaturing Gradient Gel Electrophoresis (DGGE).

In their natural environments, microorganisms usually live in organized communities. Profiling analysis of microbial communities has recently assumed special relevance as it allows a thorough understanding of the diversity of the microbiota, its behavior over time, and the establishment of patterns associated with health and disease. The application of molecular biology approaches holds the advantage of including culture-difficult and as-yet-uncultivated phylotypes in the profiles, providing a more comprehensive picture of the microbial community. This chapter focuses on two particular techniques, namely terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE), both of which have been widely used in environmental studies and have been recently successfully used by the authors in the study of the oral microbial communities associated with conditions of health and disease.

Molecular genetic studies of microbiocenosis and microstructure of jejunum wall in young rams grown on biofortified feed additives

The research featured the effect of a diet fortified with essential microelements on the ruminal microbiota of young rams. Ruminal microbiota is largely responsible for feed digestibility and body functioning of cattle. The study involved the contents of the rumens and jejuna of seven-month-old rams of the Edilbaev breed, which were subjected to a biofortified diet. The diet included the Russian feed additives Yoddar-Zn and DAFS-25 represent a protein-carbohydrate complex with plant silicon. The microflora of the digestive tract was tested using the molecular genetic method of terminal restriction fragment length polymorphism (T-RFLP) sequestration. The microstructural studies of the jejunum samples exploited light microscopy. The feed additives increased the population of cellulolytic and lactate-fermenting bacteria, as well as the Prevotella sp. microbiome and bifidobacteria in the rumen samples. The data obtained revealed the effect of essential microelements on the taxonomic pattern of microorganisms and the microflora profile. The research revealed the ratio of normal, opportunistic, pathogenic, nonculturable, and transit microflora. The jejunum wall samples obtained from the experimental group that fed on Yoddar-Zn and DAFS-25 had a more distinct micropicture of mucous membrane. Their rumen microflora balance had fewer pathogenic and opportunistic microorganisms, which was also confirmed by the jejunum morphology. The feed additives DAFS-25 and Yoddar-Zn proved beneficial for ram diet and inhibited the negative effect of pathogenic treponemas on the rumen. The additives improved digestion, absorption, and assimilation of food nutrients, as well as increased the livestock yield.

Analysis of decomposing wood wastes in the Lagos Lagoon using terminal-restriction fragment length polymorphism and illumina sequencing platform

Indiscriminate disposal of wood shavings and sawdust into the Lagos lagoon usually constitute environmental hazard and varying degree of threats to marine biotic communities. In this study we applied terminal-restriction fragment length polymorphism (T-RFLP) and illumina sequencing of 16S ribosomal RNA gene, to describe the microbial ecology of decomposing wood wastes in the Lagos lagoon, Nigeria. The terminal restriction fragments (TRFs) generated corresponds to over 100 bacterial genera and numerous uncultured bacterial clones. Phylogenetic analysis of the TRFs of 16S rRNA gene sequences and cultured bacterial sequences established genetic relatedness and divergent relationships. T-RFLP in combination with illumina sequencing platform identified bacterial species (Acinetobacter sp., Clostridium sp., Planctomyces sp., Escherichia coli, Pantoea agglomerans, Dyella japonica, Ochrobactrum sp., Prevotella sp., Runella slithyformis, Enterobacter aerogenes, Gordonia amicalis, Klebsiella sp., Thalassospira sp. and Serratia sp.) not previously accounted for in culture-dependent analysis. Bacterial strains such as Ruminococcus flavefaciens, Fibrobacter succinogenes, Simiduia agarivorans, Microbulbifer sp. and Cellulomonas fimi further identified in illumina sequences were found to be involved in the biodegradation of lignin and lignin derivatives. Hence, understanding of the diverse autochthonous microbiota of decomposing wood wastes in the Lagos lagoon is essential in the development of effective biotechnology programme, to tackling the menace of sawdust pollution.