Culture independent analysis of greyback canegrub-associated microflora and microbial community comparison using subtractive hybridisation

Greyback canegrubs cost the Australian sugarcane industry around $13 million per annum in damage and control. A novel and cost effective biocontrol bacterium could play an important role in the integrated pest management program currently in place to reduce damage and control associated costs. During the course of this project, terminal restriction fragment length polymorphism (TRFLP), 16-S rDNA cloning, suppressive subtractive hybridisation (SSH) and entomopathogen-specific PCR screening were used to investigate the little studied canegrub-associated microflora in an attempt to discover novel pathogens from putatively-diseased specimens. Microflora associated with these soil-dwelling insects was found to be both highly diverse and divergent between individual specimens. Dominant members detected in live specimens were predominantly from taxa of known insect symbionts while dominant sequences amplified from dead grubs were homologous to putativelysaprophytic bacteria and bacteria able to grow during refrigeration. A number of entomopathogenic bacteria were identified such as Photorhabdus luminescens and Pseudomonas fluorescens. Dead canegrubs prior to decomposition need to be analysed if these bacteria are to be isolated. Novel strategies to enrich putative pathogen-associated sequences (SSH and PCR screening) were shown to be promising approaches for pathogen discovery and the investigation of canegrubsassociated microflora. However, due to inter- and intra-grub-associated community diversity, dead grub decomposition and PCR-specific methodological limitations (PCR bias, primer specificity, BLAST database restrictions, 16-S gene copy number and heterogeneity), recommendations have been made to improve the efficiency of such techniques. Improved specimen collection procedures and utilisation of emerging high-throughput sequencing technologies may be required to examine these complex communities in more detail. This is the first study to perform a whole-grub analysis and comparison of greyback canegrub-associated microbial communities. This work also describes the development of a novel V3-PCR based SSH technique. This was the first SSH technique to use V3-PCR products as a starting material and specifically compare bacterial species present in a complex community.

The gut microbial community of midas cichlid fish in repeatedly evolved limnetic-benthic species pairs

Gut bacterial communities are now known to influence a range of fitness related aspects of organisms. But how different the microbial community is in closely related species, and if these differences can be interpreted as adaptive is still unclear. In this study we compared microbial communities in two sets of closely related sympatric crater lake cichlid fish species pairs that show similar adaptations along the limnetic-benthic axis. The gut microbial community composition differs in the species pair inhabiting the older of two crater lakes. One major difference, relative to other fish, is that in these cichlids that live in hypersaline crater lakes, the microbial community is largely made up of Oceanospirillales (52.28%) which are halotolerant or halophilic bacteria. This analysis opens up further avenues to identify candidate symbiotic or co-evolved bacteria playing a role in adaptation to similar diets and life-styles or even have a role in speciation. Future functional and phylosymbiotic analyses might help to address these issues.

Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

Microbial indicators related to yield and disease and changes in soil microbial community structure with ginger farm management practices

TRFLP (terminal restriction fragment length polymorphism) was used to assess whether management practices that improved disease suppression and/or yield in a 4-year ginger field trial were related to changes in soil microbial community structure. Bacterial and fungal community profiles were defined by presence and abundance of terminal restriction fragments (TRFs), where each TRF represents one or more species. Results indicated inclusion of an organic amendment and minimum tillage increased the relative diversity of dominant fungal populations in a system dependant way. Inclusion of an organic amendment increased bacterial species richness in the pasture treatment. Redundancy analysis showed shifts in microbial community structure associated with different management practices and treatments grouped according to TRF abundance in relation to yield and disease incidence. ANOVA also indicated the abundance of certain TRFs was significantly affected by farming system management practices, and a number of these TRFs were also correlated with yield or disease suppression. Further analyses are required to determine whether identified TRFs can be used as general or soil-type specific bio-indicators of productivity (increased and decreased) and Pythium myriotylum suppressiveness.

Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range

Ruminant livestock are important sources of human food and global greenhouse gas emissions. Feed degradation and methane formation by ruminants rely on metabolic interactions between rumen microbes and affect ruminant productivity. Rumen and camelid foregut microbial community composition was determined in 742 samples from 32 animal species and 35 countries, to estimate if this was influenced by diet, host species, or geography. Similar bacteria and archaea dominated in nearly all samples, while protozoal communities were more variable. The dominant bacteria are poorly characterised, but the methanogenic archaea are better known and highly conserved across the world. This universality and limited diversity could make it possible to mitigate methane emissions by developing strategies that target the few dominant methanogens. Differences in microbial community compositions were predominantly attributable to diet, with the host being less influential. There were few strong co-occurrence patterns between microbes, suggesting that major metabolic interactions are non-selective rather than specific. © 2015 Macmillan Publishers Limited.

Microbial community profiling shows dysbiosis in the lesional skin of Vitiligo subjects

Healthy human skin harbours a diverse array of microbes that comprise the skin microbiome. Commensal bacteria constitute an important component of resident microbiome and are intricately linked to skin health. Recent studies describe an association between altered skin microbial community and epidemiology of diseases, like psoriasis, atopic dermatitis etc. In this study, we compare the differences in bacterial community of lesional and non-lesional skin of vitiligo subjects. Our study reveals dysbiosis in the diversity of microbial community structure in lesional skin of vitiligo subjects. Although individual specific signature is dominant over the vitiligo-specific microbiota, a clear decrease in taxonomic richness and evenness can be noted in lesional patches. Investigation of community specific correlation networks reveals distinctive pattern of interactions between resident bacterial populations of the two sites (lesional and non-lesional). While Actinobacterial species constitute the central regulatory nodes (w.r.t. degree of interaction) in non-lesional skin, species belonging to Firmicutes dominate on lesional sites. We propose that the changes in taxonomic characteristics of vitiligo lesions, as revealed by our study, could play a crucial role in altering the maintenance and severity of disease. Future studies would elucidate mechanistic relevance of these microbial dynamics that can provide new avenues for therapeutic interventions.

Amplicon metagenomics for elucidating microbial community changes associated to conventional and organic farming of Beta vulgaris and Solanum lycopersicum

Soil microbial community changes associated to conventional and organic farming of two relevant crops (Beta vulgaris and Solanum lycopersicum) were analysed through 16s rRNA amplicon sequencing. This study revealed microbial communities in the agricultural soils studied to be similar to other reported nutrient-rich microbiomes, and some significant differences between the microbial communities associated to the two farming practices were found. Some phyla (Chloroflexi and Thermi) were found to be present in different abundances according to soil treatment. As chloroplast interference can be a stumbling block in plant-associated 16s rRNA amplicon metagenomics analysis of aerial plant tissues, two protocols for bacterial cell detachment (orbital shaking and ultrasound treatment) and two protocols for microbial biomass recovery (centrifugation and filtration) were tested regarding their efficiency at excluding plant-DNA. An alternative method to the one proposed by Rastogi et al (2010) for evaluating the chloroplast-amplicon content in post-PCR samples was tested, and the method revealed that filtration was the most efficient protocol in minimising chloroplast interference.

Microbial community analysis of Acropora palamata mucus swabs, water and sediment samples from Hawksnest Bay, St. John, U.S. Virgin Islands

Colonies of the scleractinian coral Acropora palmata, listed as threatened under the US Endangered Species Act in 2006, have been monitored in Hawksnest Bay, within Virgin Islands National Park, St. John, from 2004 through 2010 by scientists with the US Geological Survey, National Park Service, and the University of the Virgin Islands. The focus has been on documenting the prevalence of disease, including white band, white pox (also called patchy necrosis and white patches), and unidentified diseases (Rogers et al., 2008; Muller et al., 2008). In an effort to learn more about the pathologies that might be involved with the diseases that were observed, samples were collected from apparently healthy and diseased colonies in July 2009 for analysis. Two different microbial assays were performed on Epicentre Biotechnologies DNA swabs containing A. palmata coral mucus, and on water and sediment samples collected in Hawksnest Bay. Both assays are based on polymerase chain reaction (PCR) amplification of portions of the small rRNA gene (16S). The objectives were to determine 1) if known coral bacterial pathogens Serratia marcescens (Acroporid Serratiosis), Vibrio coralliilyticus (temperature-dependent bleaching, White Syndrome), Vibrio shiloi (bleaching, necrosis), and Aurantimonas coralicida (White Plague Type II) were present in any samples, and 2) if there were any differences in microbial community profiles of each healthy, unaffected or diseased coral mucus swab. In addition to coral mucus, water and sediment samples were included to show ambient microbial populations. In the first test, PCR was used to separately amplify the unique and diagnostic region of the 16S rRNA gene for each of the coral pathogens being screened. Each pathogen test was designed so that an amplified DNA fragment could be seen only if the specific pathogen was present in a sample. A positive result was indicated by bands of DNA of the appropriate size on an agarose gel, which separates DNA fragments based on the size of the molecule. DNA from pure cultures of each of the pathogens was used as a positive control for each assay.

Aerobic biodegradation of hexachlorobenzene by an acclimated microbial community

The aerobic degradation of hexachlorobenzene (HCB) by an acclimated microbial community which isolated from a contaminated site and acclimated in our laboratory was investigated. The enriched microbial community was capable of biodegrading HCB when cultivated in minimal salts medium and supplied HCB as the sole carbon source. The efficiencies of microbial community in the degradation of HCB under different pH and temperatures were examined. The phylogenetic analysis for the nearly complete sequences of 16S rDNA demonstrated that the bacteria assemblage in the microbial community was dominated by Azospirillum and Alcaligenes groups.

Use of microbial community to evaluate performance of a wetland system in treating Pb/Zn mine drainage

The performance of a wetland system in treating lead (Pb)/zinc (Zn) mine drainage was evaluated by using the polyurethane foam unit (PFU) microbial community (method), which has been adopted by China as a standardized procedure for monitoring water quality. The wetland system consisted of four cells with three dominant plants: Typha latifolia, Phragmites australis and Paspalum distichum. Physicochemical characteristics [pH, EC, content of total suspended solid (TSS) and metals (Pb, Zn, Cd, and Cu)] and PFU microbial community in water samples had been investigated from seven sampling sites. The results indicated that the concentrations of Pb, Zn, Cd, Cu, and TSS in the mine drainage were gradually reduced from the inlet to the outlet of the wetland system and 99%, 98%, 75%, 83%, and 68% of these metals and TSS respectively, had been reduced in concentration after the drainage passed through the wetland system. A total of 105 protozoan species were identified, the number of protozoa species and the diversity index (DI) gradually increased, while the heterotrophic index (HI) gradually decreased from the inlet to the outlet of the wetland system. The results indicated that DI, HI, and total number species of protozoa could be used as biological indicators indicating the improvement of water quality.

Soil microbial community analysis using denaturing gradient gel electrophoresis

The most biological diversity on this planet is probably harbored in soils. Understanding the diversity and function of the microbiological component of soil poses great challenges that are being overcome by the application of molecular biological approaches. This review covers one of many approaches being used: separation of polymerase chain reaction (PCR) amplicons using denaturing gradient gel electrophoresis (DGGE). Extraction of nucleic acids directly from soils allows the examination of a community without the limitation posed by cultivation. Polymerase chain reaction provides a means to increase the numbers of a target for its detection on gels. Using the rRNA genes as a target for PCR provides phylogenetic information on populations comprising communities. Fingerprints produced by this method have allowed spatial and temporal comparisons of soil communities within and between locations or among treatments. Numerous samples can be compared because of the rapid high throughput nature of this method. Scientists now have the means to begin addressing complex ecological questions about the spatial, temporal, and nutritional interactions faced by microbes in the soil environment.

Soil microbial community analysis using terminal restriction fragment length polymorphisms

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-fingerprinting method that is commonly used for comparative microbial community analysis. The method can be used to analyze communities of bacteria, archaea, fungi, other phylogenetic groups or subgroups, as well as functional genes. The method is rapid, highly reproducible, and often yields a higher number of operational taxonomic units than other, commonly used PCR-fingerprinting methods. Sizing of terminal restriction fragments (T-RFs) can now be done using capillary sequencing technology allowing samples contained in 96- or 384-well plates to be sized in an overnight run. Many multivariate statistical approaches have been used to interpret and compare T-RFLP fingerprints derived from different communities. Detrended correspondence analysis and the additive main effects with multiplicative interaction model are particularly useful for revealing trends in T-RFLP data. Due to biases inherent in the method, linking the size of T-RFs derived from complex communities to existing sequence databases to infer their taxonomic position is not very robust. This approach has been used successfully, however, to identify and follow the dynamics of members within very simple or model communities. The T-RFLP approach has been used successfully to analyze the composition of microbial communities in soil, water, marine, and lacustrine sediments, biofilms, feces, in and on plant tissues, and in the digestive tracts of insects and mammals. The T-RFLP method is a user-friendly molecular approach to microbial community analysis that is adding significant information to studies of microbial populations in many environments.