Temporal assessment of microbial communities in soils of two contrasting mangroves

ABSTRACT Variations in microbial communities promoted by alterations in environmental conditions are reflected in similarities/differences both at taxonomic and functional levels. Here we used a natural gradient within mangroves from seashore to upland, to contrast the natural variability in bacteria, cyanobacteria and diazotroph assemblages in a pristine area compared to an oil polluted area along a timespan of three years, based on ARISA (bacteria and cyanobacteria) and nifH T-RFLP (diazotrophs) fingerprinting. The data presented herein indicated that changes in all the communities evaluated were mainly driven by the temporal effect in the contaminated area, while local effects were dominant on the pristine mangrove. A positive correlation of community structure between diazotrophs and cyanobacteria was observed, suggesting the functional importance of this phylum as nitrogen fixers in mangroves soils. Different ecological patterns explained the microbial behavior in the pristine and polluted mangroves. Stochastic models in the pristine mangrove indicate that there is not a specific environmental factor that determines the bacterial distribution, while cyanobacteria and diazotrophs better fitted in deterministic model in the same area. For the contaminated mangrove site, deterministic models better represented the variations in the communities, suggesting that the presence of oil might change the microbial ecological structures over time. Mangroves represent a unique environment threatened by global change, and this study contributed to the knowledge of the microbial distribution in such areas and its response on persistent contamination historic events.

Molecular diversity pattern of intestinal lactic acid bacteria in Cemani chicken, Indonesian native chicken, as revealed by terminal restriction fragment length polymorphisms

Aims: An ecological study was conducted to investigate the diversity pattern of the lactic acid bacteria in the gastrointestinal tract of Cemani chicken, a native Indonesian chicken, using a molecular approach based on 16S rRNA genes. Methodology and results: Digesta samples of seven chickens were collected for terminal restriction fragment length polymorphism (T-RFLP) analysis. The molecular diversity of lactic acid bacteria in crop, ventriculus, ileum and cecum were determined. The results showed that microbial composition of lactic acid bacteria in cecum was relatively different with other upper gastrointestinal tract. Lactic acid bacteria phylotypes and diversity in ileum were higher than those in the crop, ventriculus, and cecum. Conclusion, significance and impact of study: We confirmed that cecum of native chicken has a different environment as compared to other gastrointestinal regions showing the lowest value of the Sorensen’s index. This first report of LAB diversity pattern in Cemani chicken contributes a more comprehensive understanding of the microbial ecology in the chicken.

Differential analysis of the bacterial community composition in mouse feces and intestinal contents / 中国实验动物学报

Objective To explore the objectivity and scientificity of fecal sampling , and to provide reference for investigating the relationship between intestinal microbes and diseases . Methods Terminal restriction fragment length polymorphism, degeneration gradient gel electrophoresis and real time fluorescent quantitative PCR techniques were applied to differentially analyze the bacterial community composition and abundance of intestinal contents and feces taken from dif -ferent sites of BALB/c mouse intestine .Results The predominant T-RFLP fragments ( T-RFs) in feces in the rectum and colon were 244 bp, 255 bp and 449 bp, however , those in feces of the small intestine including duodenum , jejunum and il-eum were 60 bp, 73 bp, 261 bp, 268 bp and 272 bp, and with a larger variation of the bacterial community composition in various parts of the small intestine .The bacterial abundance in the contents of duodenum and jejunum were 6.9 log ( cop-ies)/g and 8.3 log (copies)/g, fewer than in the other parts of the intestine , while the bacterial abundance in the feces was as high as 11.8 log (copies)/g, being about 2 times higher than that in the duodenum and jejunum (P0.05).Conclusions The inter-mouse variations of bacterial communi-ty composition in the large intestine contents are small .The bacterial composition and abundance are similar suggest that studies on the relationship between large intestine especially colorectal microbiota and diseases may be conducted via fecal sampling.

UV-B Resistant Yeast Inhabit the Phyllosphere of Strawberry.

The increase on the worldwide influx of solar ultraviolet radiation (UV-B) has inflicted a considerable challenge, due to its deleterious effects to live beings and pose a special threat to phyllosphere communities. However, UV-B influence on epiphytic yeasts associated with agricultural crops remains limited. Main aim of the present study was to determine the effect of ultraviolet-B radiation on the epiphytic yeast populations associated with strawberry under field conditions. Thus, strawberries (Fragaria x ananassa Duchesne cv. Oso Grande) were grown under three different treatments: a) environmental UV-B, b) enhanced UV-B and c) decreased UV-B; thereafter, their yeast epiphytic populations were analyzed by T-RFLP prior to yeast isolation, identification and in vitro test for the sensitivity against UV-B. Our results demonstrated that UV-B radiation did not significantly affect the strawberry epiphytic yeast populations. However, isolates directly exposed to radiation, generally revealed morphological abnormalities and a diminishing value in the percentage of survival, although they remained constant after 240 min of exposure. The increase in UV-B radiation was not sufficient to affect the dynamics and composition of epiphytic yeast communities from strawberry, there was a clear morphotype shift towards the selection of pigmented isolates.

An Improved Protocol for Extraction of Metagenomic DNA from High Humus, Alkaline and Saline Soil of Chinampa for T-RFLP Fingerprinting Analysis.

Aims: The principle aim of this study was to obtain high quality metagenomic DNA from the high humus-containing, alkaline soils of the chinampas, an artificial sustainable agroecosystem. Study Design: Different protocols reported previously were tested and were modified to extract the metagenomic DNA. Quality of the DNA samples was evaluated by amplification of 16S rRNA gene with PCR and T-RFLP (Terminal Restriction Fragment Length Polymorphism) analysis. Place and Duration of Study: This study was performed in Department of Microbiology, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional during 2011-2012. Methodology: Four soil samples were collected from two chinampas at the depth of 0-30 cm and 30-60cm. A protocol started with repeated prewashing before the direct cell lysis with lysozyme followed by SDS treatments, frozen and melting cycling was developed which combined the DNA isolation and purification procedures. The 16SrRNA genes were amplified from the extracted metagenomic DNAs and were used for T-RFLP fingerprinting analysis. Results: The 16SrRNA genes were amplified from all the DNA extracts corresponding to the four soil samples and were successfully used in the T-RFLP analysis, which generated 25 to 109T-RFs in the four soil samples digested separately with the restriction endonucleases HaeIII, HhaI and MspI. Conclusion: The protocol developed in the present study could generate high molecular weight and high quality metagenomic DNA from soils with high content of humic materials, for which the other reported protocols were not functioned. This soil harboured very diverse and unique bacterial communities belonging to at least nine phyla that might contribute to the high soil fertility.

Terminal restriction fragment length polymorphism analysis of microbial community in the bile of 117 patients with obstructive jaundice / 中华消化杂志

Objective To analyze the structure of microbial community in the bile of patients with obstructive jaundice.Methods From October 2010 to October 2013,117 patients with obstructive jaundice were selected.The results of bile microbial regular culture and anaerobic bacteria culture were both negative.A total of 10 mL bile of each case was aspired and DNA of bile microbial community was isolated.16S rDNA of bile microbial was amplified and underwent terminal restriction fragment length polymorphism (T-RFLP) analysis.Clone libraries were constructed and conducted sequencing and system analysis.Chi-square test was performed for data analysis.Results Among the 117 patients,16S rDNA of 50 cases was positive,and the total positive rate was 42.7 ％.The positive rate of bile bacterial 16S rDNA in stone and tumor cased obstruction was 97.3％ (36/37) and 17.5％ (14/80),and the difference was statistically significant (x2=65.828,P＜0.01).There was no statistically significant difference in the positive rate of bile bacterial 16S rDNA among hilar obstruction,above hilar obstruction and below hilar obstruction,which ware 43.3％ (13/30) and 42.5％(37/87),respectively (P＞0.05).Bile microbial community of obstruction coused by stone was mainly Enterobacteriaceae (Escherichia,Salmonella,Klebsiella,Proteus),Streptococcus (Streptococcus),digestive coccaceae (digestive bacteria genus,peptostreptococcus),Micrococcus branch (Staphylococcus),Propionibacterium (Propionibacterium) and Neisseriabacteria (Acinetobacter).Bile microbial community of obstruction caused by tumor was mainly Enterobacteriaceae (Clay Beth spp,Escherichia,Salnonellatyphi) and Micrococcus branch (Staphylococcus).Conclusion The condition and variety of bile microbial community of patients with obstructive jaundice could be effectively evaluated by 16S rDNA fragment through T RFLP analysis.

Bacterial community analysis of the water surface layer from a rice-planted and an unplanted flooded field

The bacterial communities in floodwater, from a rice-planted and an unplanted field were characterized at the beginning (flooding stage) and at the end (harvest stage) of the rice cropping cycle. Most probable number estimations and plate counts of aerobic and anaerobic heterotrophic bacteria and of several metabolic bacterial groups (methanogens, sulfate-reducers, anaerobic sulfur and nonsulfur phototrophs, denitrifiers and ammonifiers) were similar in rice and unplanted floodwater at both sampling times. The analysis of denitrifiers and methanogens by fluorescent in situ hybridization revealed a shift in the phylogenetic affiliation only of the former group in the rice-planted floodwater. Terminal restriction fragment length polymorphism of 16S rRNA gene amplicons indicated that the bacterial communities of the rice-planted and unplanted soils were consistently diverse and strongly influenced by the season.

Quantitative analysis of Terminal Restriction Fragment Length Polymorphism (T-RFLP) microbial community profiles: peak height data showed to be more reproducible than peak area / Análise quantitativa de perfis de T-RFLP de comunidades microbianas: dados de altura de picos mostraram-se mais reprodutíveis do que os de área

Terminal Restriction Fragment Length Polymorphism (T-RFLP) is a culture-independent fingerprinting method for microbial community analysis. Profiles generated by an automated electrophoresis system can be analysed quantitatively using either peak height or peak area data. Statistical testing demontrated that peak height data showed to be more reproducible than peak area data.

Terminal Restriction Fragment Length Polymorphism (T-RFLP) é um método molecular, independente de cultivo, para análise de comunidades microbianas. Perfis gerados por um sistema automatizado de eletroforese podem ser analisados quantitativamente usando dados de altura ou área dos picos. Os dados de altura mostraram-se mais reprodutíveis do que os de área.

An Emerging Method for Characterizing Microbial Ecology-T-Rflp Technique / 微生物学通报

Terminal restriction fragment length polymorphism(T-RFLP) is a resent molecular approach that can assess subtle genetic differences between strains as well as provide insight into the structure and function of microbial communities. This method overcomes the confinement of conventional culture-dependent methods and has both high sensitivity and throughput making it ideal for comparative analyses. Though there is still no application in our country, more and more investigators are highlighting it. In this article, the fundamental principle of this technique is introduced. The recent application and the development of this technique are also summed up .

A New Method of Soil Bacterial DNA Extraction and T-RFLP Analysis for Diversity / 微生物学通报

Obtaining soil bacterial DNA of good quality is a key step in soil bacterial ecology study.A quick, efficient,sensitive and stably method of DNA extraction from soil were established by combining strongpoints of two kits(Soilmaster kit and DNA IQ~(TM)kit).In addition,the 16S rDNA gene and T-RFLP(Terminal restriction fragment length polymorphism)were used in the analysis of soil bacterial community diversity and the result show that T-RFLP is a powerful tool for bacterial community study.

T_RFLP Technique and Its Application on Community Analysis of Nitrifying Bacteria / 微生物学通报

Terminal restriction fragment length polymorphism (T_RFLP) analysis is a culture- independent approach for analyzing microbial community in environment. It bases on PCR technology, and its process includes DNA extraction of environmental samples, amplification of genes encoding the 16S rRNA, 18S rRNA or enzymes with fluorescently labeled primers, the restriction enzyme digestion of PCR products, cap- illary electrophoresis and the analysis of T_RFLP profile. It has been proved to be powerful applied on mi- crobial community in environment since developed in 1997. Currently, T_RFLP rarely applied in China, and it has no applications on microbial community analysis of nitrifying bacteria. In this article, the fundamental principle of this technique and the recent applications of T_RFLP on microbial community are summarized; in addition, it illustrates the confinements of conventional culture-dependent of nitrifying bacteria and the foreground of T_RFLP applying on microbial community structure analysis of nitrifying bacteria.

The application exploring of bacterial community diversity profiling in forensic comparison of soil / 中国法医学杂志

Objective To explore the application of bacterial community diversity profiling in forensic comparison of soil.Methods The DNA profilings of bacterial community from 5 soil samples of different source and 4 samples of same source were obtained through the specific amplification of 16S rDNA segments followed by terminal restriction fragment length polymorphism(T-RFLP)analysis,and the similarity indexes between different soil samples were calculated.Result The ranges of similarity indexes between different sources of soil samples were 0.3～0.44,and the ranges between same sources of soil samples were 0.76～0.87.Conclusion There is a great relativity between the bacterial community diversity profiling and the source of soil sample.