Culture-independent Molecular Approach Research Articles

Microbial Epidemiology of the Cystic Fibrosis Airways: Past, Present, and Future.

Progressive obstructive lung disease secondary to chronic airway infection, coupled with impaired host immunity, is the leading cause of morbidity and mortality in cystic fibrosis (CF). Classical pathogens found in the airways of persons with CF (pwCF) include Pseudomonas aeruginosa, Staphylococcus aureus, the Burkholderia cepacia complex, Achromobacter species, and Haemophilus influenzae. While traditional respiratory-tract surveillance culturing has focused on this limited range of pathogens, the use of both comprehensive culture and culture-independent molecular approaches have demonstrated complex highly personalized microbial communities. Loss of bacterial community diversity and richness, counteracted with relative increases in dominant taxa by traditional CF pathogens such as Burkholderia or Pseudomonas, have long been considered the hallmark of disease progression. Acquisition of these classic pathogens is viewed as a harbinger of advanced disease and postulated to be driven in part by recurrent and frequent antibiotic exposure driven by frequent acute pulmonary exacerbations. Recently, CF transmembrane conductance regulator (CFTR) modulators, small molecules designed to potentiate or restore diminished protein levels/function, have been successfully developed and have profoundly influenced disease course. Despite the multitude of clinical benefits, structural lung damage and consequent chronic airway infection persist in pwCF. In this article, we review the microbial epidemiology of pwCF, focus on our evolving understanding of these infections in the era of modulators, and identify future challenges in infection surveillance and clinical management.

Construction of Small-Insert and Large-Insert Metagenomic Libraries.

The vast majority of the Earth's biological diversity are hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is one cultivation-independent molecular approach to assess this unexplored genetic reservoir. High numbers of novel biocatalysts have been identified by function-based or sequence-based screening of metagenomic libraries derived from various environments. Here, we describe detailed protocols for the construction of metagenomic small-insert and large-insert libraries in plasmids and fosmids, respectively, from environmental DNA.

Exploring the Cystic Fibrosis Lung Microbiome: Making the Most of a Sticky Situation.

Chronic lower respiratory tract infections are a leading contributor to morbidity and mortality in persons with cystic fibrosis (pwCF). Traditional respiratory tract surveillance culturing has focused on a limited range of classic pathogens; however, comprehensive culture and culture-independent molecular approaches have demonstrated complex communities highly unique to each individual. Microbial community structure evolves through the lifetime of pwCF and is associated with baseline disease state and rates of disease progression including occurrence of pulmonary exacerbations. While molecular analysis of the airway microbiome has provided insight into these dynamics, challenges remain including discerning not only “who is there” but “what they are doing” in relation to disease progression. Moreover, the microbiome can be leveraged as a multi-modal biomarker for both disease activity and prognostication. In this article, we review our evolving understanding of the role these communities play in pwCF and identify challenges in translating microbiome data to clinical practice.

Diversity and functional prediction of microbial communities involved in the first aerobic bioreactor of coking wastewater treatment system.

The pre-aerobic process of coking wastewater treatment has strong capacity of decarbonization and detoxification, which contribute to the subsequent dinitrogen of non-carbon source/heterotrophic denitrification. The COD removal rate can reach > 90% in the first aerobic bioreactor of the novel O/H/O coking wastewater treatment system during long-term operation. The physico-chemical characteristics of influent and effluent coking wastewater in the first aerobic bioreactor were analyzed to examine how they correlated with bacterial communities. The diversity of the activated sludge microbial community was investigated using a culture-independent molecular approach. The microbial community functional profiling and detailed pathways were predicted from the 16S rRNA gene-sequencing data by the PICRUSt software and the KEGG database. High-throughput MiSeq sequencing results revealed a distinct microbial composition in the activated sludge of the first aerobic bioreactor of the O/H/O system. Proteobacteria, Bacteroidetes, and Chlorobi were the decarbonization and detoxification dominant phyla with the relative abundance of 84.07 ± 5.45, 10.89 ± 6.31, and 2.96 ± 1.12%, respectively. Thiobacillus, Rhodoplanes, Lysobacter, and Leucobacter were the potential major genera involved in the crucial functional pathways related to the degradation of phenols, cyanide, benzoate, and naphthalene. These results indicated that the comprehensive understanding of the structure and function diversity of the microbial community in the bioreactor will be conducive to the optimal coking wastewater treatment.

Nitrogen Fixation in Cereals.

Cereals such as maize, rice, wheat and sorghum are the most important crops for human nutrition. Like other plants, cereals associate with diverse bacteria (including nitrogen-fixing bacteria called diazotrophs) and fungi. As large amounts of chemical fertilizers are used in cereals, it has always been desirable to promote biological nitrogen fixation in such crops. The quest for nitrogen fixation in cereals started long ago with the isolation of nitrogen-fixing bacteria from different plants. The sources of diazotrophs in cereals may be seeds, soils, and even irrigation water and diazotrophs have been found on roots or as endophytes. Recently, culture-independent molecular approaches have revealed that some rhizobia are found in cereal plants and that bacterial nitrogenase genes are expressed in plants. Since the levels of nitrogen-fixation attained with nitrogen-fixing bacteria in cereals are not high enough to support the plant’s needs and never as good as those obtained with chemical fertilizers or with rhizobium in symbiosis with legumes, it has been the aim of different studies to increase nitrogen-fixation in cereals. In many cases, these efforts have not been successful. However, new diazotroph mutants with enhanced capabilities to excrete ammonium are being successfully used to promote plant growth as commensal bacteria. In addition, there are ambitious projects supported by different funding agencies that are trying to genetically modify maize and other cereals to enhance diazotroph colonization or to fix nitrogen or to form nodules with nitrogen-fixing symbiotic rhizobia.

Direct comparison of culture-dependent and culture-independent molecular approaches reveal the diversity of fungal endophytic communities in stems of grapevine (Vitis vinifera)

Grapevines (Vitis vinifera) are colonized by ubiquitous microorganisms known as endophytes, which may have advantageous or neutral effects without causing disease symptoms. Certain endophytes are uncultivable, so culture-independent approaches such as next generation sequencing (NGS) can help for a better understanding of their ecology and distribution. To date, there are no studies which directly link NGS results with taxa derived from a culturing approach, integrating morphological and multi-gene phylogenetic analysis of endophytes. In this study, a culture-dependent and high-resolution culture-independent approach (next generation sequencing) were used to identify endophytes in grapevine stems. In the culture-dependent approach, a total of 94 isolates were recovered from 84 of 144 healthy grapevine stem fragments (colonization rate = 58.3%). The study is unique as we used subsets of combined multi-gene regions to identify the endophytes to species level. Based on each multi-gene phylogenetic analysis, 28 species belong to 19 genera (Acremonium, Alternaria, Arthrinium, Ascorhizoctonia, Aspergillus, Aureobasidium, Bipolaris, Botryosphaeria, Botrytis, Chaetomium, Cladosporium, Curvularia, Hypoxylon, Lasiodiplodia, Mycosphaerella, Nigrospora, Penicillium, Phoma, Scopulariopsis) were identified. A higher number of culturable fungi were obtained from 13 year-old vines, followed by eight and three year-old vines. In the culture-independent approach, a fungal richness of 59 operational taxonomic units (OTU) was detected, being highest in 13 year-old grapevines, followed by eight and three years. Even though the cultivation approach detected lower fungal richness, the results related to stem are consistent for fungal community composition and richness. Comparison of the fungal taxa identified by the two approaches resulted in an overlap of 53% of the fungal genera. Due to interspecific variability of the sequences from NGS, in many cases the OTUs (even with the highly abundant ones) were only assignable to order, family or genus level. Incorporating multi-gene phylogenies we successfully identified many of the NGS derived OTUs with poor taxonomic information in reference databases to the genus or species levels. Hence, this study signifies the importance of applying both culture-dependent and culture-independent approaches to study the fungal endophytic community composition in Vitis vinifera. This principle could also be applied to other host species and ecosystem level studies.

Microbial Communities Inhabiting a Rare Earth Element Enriched Birnessite-Type Manganese Deposit in the Ytterby Mine, Sweden

ABSTRACTThe dominant initial phase formed during microbially mediated manganese oxidation is a poorly crystalline birnessite-type phyllomanganate. The occurrence of manganese deposits containing this mineral is of interest for increased understanding of microbial involvement in the manganese cycle. A culture independent molecular approach is used as a first step to investigate the role of microorganisms in forming rare earth element enriched birnessite-type manganese oxides, associated with water bearing rock fractures in a tunnel of the Ytterby mine, Sweden. 16S rRNA gene results show that the chemotrophic bacterial communities are diverse and include a high percentage of uncultured unclassified bacteria while archaeal diversity is low with Thaumarchaeota almost exclusively dominating the population. Ytterby clones are frequently most similar to clones isolated from subsurface environments, low temperature milieus and/or settings rich in metals. Overall, bacteria are dominant compared to archaea. Both bacterial and archaeal abundances are up to four orders of magnitude higher in manganese samples than in fracture water. Potential players in the manganese cycling are mainly found within the ferromanganese genera Hyphomicrobium and Pedomicrobium, and a group of Bacteroidetes sequences that cluster within an uncultured novel genus most closely related to the Terrimonas. This study strongly suggest that the production of the YBS deposit is microbially mediated.

Detection and cell sorting of Pseudonocardia species by fluorescence in situ hybridization and flow cytometry using 16S rRNA-targeted oligonucleotide probes.

Pseudonocardia spp. are receiving increasing attention due to their ability to biodegrade recalcitrant cyclic ether pollutants (e.g., 1,4-dioxane and tetrahydrofuran), as well as for their distinctive ecological niches (e.g., symbiosis with ants/plants and production of antibiotics). Isolating and characterizing Pseudonocardia spp. is thus important to discern their metabolic and physiological idiosyncrasies and advance their potential applications. However, slow growth, low cell yield, and dissimilar colony morphology hinder efficient isolation of Pseudonocardia using conventional plating methods. Here, we develop the first fluorescent probe (Pse631) targeting the 16S rRNA of Pseudonocardia members. In combination with flow cytometry and cell sorting, in situ hybridization with this probe enables sensitive and specific detection of Pseudonocardia cells in mixed cultures and enriched environmental samples without significant false positives, using Escherichia coli, Bacillus subtilis, and Mycobacterium spp. as negative controls. Pseudonocardia dioxanivorans CB1190 cells labeled with Pse631 as a positive control were detected when their relative abundance in the total bacterial community was as low as 0.1%. Effective separation of Pseudonocardia cells from the mixed consortium was confirmed by quantitative PCR analysis of sorted cells. This study provides a culture-independent high-throughput molecular approach enabling effective separation of Pseudonocardia populations from complex microbial communities. This approach will not only facilitate subsequent molecular analyses including species identification and quantification, but also advance understanding of their catabolic capacities and functional molecular diversity.

Gut microbiota and gut morphology of gilthead sea bream (Sparus aurata) juveniles are not affected by chromic oxide as digestibility marker

The present work aimed to evaluate whether the use of chromic oxide (Cr2O3) as dietary inert marker in fish digestibility studies interferes with gut microbial community modulation and gut morphology. To assess the effects of Cr2O3 under potential diverse microbiota populations, dietary Cr2O3 was tested using challenging plant feedstuffs (PF)-based diets supplemented or not with prebiotics, as prebiotics are expected to modify gut microbiota populations. For that purpose, three diets were formulated to include circa 20:80 fish meal and PF as protein sources, without (CTR) or with prebiotic supplementation (10 g/kg XOS or GOS). These diets did not include Cr2O3 (−Cr2O3 diets). Three similar additional diets were formulated to include 5 g/kg Cr2O3 (+Cr2O3 diets). Cr2O3 effects on gut microbiota were assessed for the first time in the allochthonous (digesta) and autochthonous (mucosa) community by a culture-independent molecular approach, using denaturing gradient gel electrophoresis (DGGE). No differences in gut bacterial profiles (number of operational taxonomic units, microbiota richness, diversity and similarity indices) were observed between dietary treatments. No significant alterations in submucosa layer structure, enterocytes and eosinophilic granular cells structure, goblet cells and leucocytes quantity were detected in the distal intestine among diets. In conclusion, data indicate that dietary inclusion of 5 g/kg Cr2O3 does not interfere with gilthead sea bream (Sparus aurata) gut microbiota and gut morphology, suggesting that a dietary incorporation level of 5 g/kg Cr2O3 can safely be used as inert marker in digestibility studies.

The role of the surface smear microbiome in the development of defective smear on surface-ripened red-smear cheese

The complex smear microbiota colonizing the surface of red-smear cheese fundamentally impacts the ripening process, appearance and shelf life of cheese. To decipher the prokaryotic composition of the cheese smear microbiome, the surface of a semi-hard surface ripened cheese was studied post-ripening by culture-based and culture-independent molecular approaches. The aim was to detect potential bacterial alterations in the composition of the cheese smear microbiota resulting from cheese storage in vacuum film-prepackaging, which is often accompanied by the development of a surface smear defect. Next-generation sequencing of amplified 16S rRNA gene fragments revealed an unexpected high diversity of a total of 132 different genera from the domains Bacteria and Archaea on the cheese surface. Beside typical smear organisms, our study revealed the presence of several microorganisms so far not associated with cheese, but related to milk, farm and cheese dairy environments. A 16S ribosomal RNA based analysis from total RNA identified the major metabolically active populations in the cheese surface smear as Actinobacteria of the genera Corynebacterium, Brevibacterium, Brachybacterium and Agrococcus. Comparison of data on a higher phylogenetic level revealed distinct differences in the composition of the cheese smear microbiome from the different samples. While the proportions of Proteobacteria and Bacteroidetes were increased in the smear of prepacked samples and in particular in defective smear, staphylococci showed an opposite trend and turned out to be strongly decreased in defective smear. In conclusion, next-generation sequencing of amplified 16S rRNA genes and 16S rRNA from total RNA extracts provided a much deeper insight into the bacterial composition of the cheese smear microbiota. The observed shifts in the microbial composition of samples from defect surface smear suggest that certain members of the Proteobacteria contribute to the observed negative organoleptic properties of the surface smear of cheese after prepacking in plastic foil.

Assessing Bacterial and Fungal Diversity in the Plant Endosphere.

Plants are colonized various microorganisms including endophytes. These microbes can play an important role in agricultural production as they promote plant growth and/or enhance the resistance of their host plant against diseases and environmental stress conditions. Although culture-independent molecular approaches such as DNA barcoding have greatly enhanced our understanding of bacterial and fungal endophyte communities, there are some methodical problems when investigating endophyte diversity. One main issue are sequence contaminations such as plastid-derived rRNA gene sequences which are co-amplified due to their high homology to bacterial 16S rRNA genes. The same is true for plant and fungal ITS sequences. The application of highly specific-primers suppressing co-amplification of these sequence contaminations is a good solution for this issue. Here, we describe a detailed protocol for assessing bacterial and fungal endophyte diversity in plants using these primers in combination with next-generation sequencing.

Construction of Small-Insert and Large-Insert Metagenomic Libraries.

The vast majority of the Earth's biological diversity is hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is a cultivation-independent molecular approach to assess this unexplored genetic reservoir. High numbers of novel biocatalysts have been identified by function-based or sequence-based screening of metagenomic libraries derived from various environments. Here, we describe detailed protocols for the construction of metagenomic small-insert and large-insert libraries in plasmids and fosmids, respectively, from environmental DNA.

Can inoculation with living soil standardize microbial communities in soilless potting substrates?

Soilless plant cultivation substrates are commercially produced at large scales, but can show considerable variation in their performance in terms of sustaining plant growth and/or nutrition. This variation may be due to varying composition of microbial communities present in the substrates, mainly when composted organic materials are used as their components. Here we analyzed the portion of variability in composition of microbial (mainly the fungal) communities due to identity of substrate batches and compared it with variability due to the addition of a living soil (inoculation) or the presence of plant root system (i.e., the rhizosphere effect). The analysis was based on profiling total (DNA-based) and active (RNA-based) fungal and total (DNA-based) bacterial communities by using cultivation-independent molecular approaches. Contrary to expected effect of inoculation and rather limited variation across the substrate batches, identity of substrate batches in fact turned to explain the largest portion of biological variability, followed by the rhizosphere effect. The inoculation was completely ineffective as a factor affecting the indigenous microbial communities. These results indicate that the microbial communities in the soilless substrates are particularly resilient to plant- or inoculation-induced changes, but still highly variable between the individual production batches. Active fungal communities were dominated by yeasts recruiting either from Asco- or Basidiomycota. Due to phylogenetically and functionally similar but mutually exclusive dominants (Galactomyces and Candida) of the microbial communities in the different substrate batches without obvious correlation with their physico-chemical properties, we assume functional redundancy to play an important role in microbial community assembly within the substrates. Our results thus demonstrate as yet undescribed variation in microbial community composition with possible functional impact on plant performance in soilless substrates deserving further experimental attention.

Ruminal bacterial community changes during adaptation of goats to fresh alfalfa forage

Using culture-independent molecular approaches, we studied the bacterial population changes in the rumen of goats abruptly converted from alfalfa hay to fresh alfalfa diet. Administration of fresh forage with significantly increased soluble nitrogen and soluble protein nitrogen resulted in frothy bloat. Changes of the bacterial composition of rumen were monitored using DGGE analysis of 16S rDNA gene amplicons and quantitative PCR method. As the diet changed, the bacterial population of Bacteroidetes and γ-Proteobacteria decreased, even if animals have not shown signs of frothy bloat. The most severely bloated animals showed an increase of Bacteroidetes phylum. Lactobacillus/Streptococcus group belonging to Firmicutes phylum decreased in response to transferring the animals from hay to a fresh forage-based diet, and did not achieve the values observed at the beginning of the experiment. In summary, changes in the diet and subsequent frothy bloat occurrence produce long-lasting changes in the structure of the microbial community and may be associated with a specific bacterial population belonging to the Bacteroidetes phylum.

Estimating genetic structure and diversity of cyanobacterial communities in Atlantic forest phyllosphere.

Cyanobacterial communities on the phyllosphere of 4 plant species inhabiting the endangered Brazilian Atlantic Forest biome were evaluated using cultivation-independent molecular approaches. Total genomic DNA was extracted from cells detached from the surface of leaves of Euterpe edulis, Guapira opposita, Garcinia gardneriana, and Merostachys neesii sampled in 2 Brazilian Atlantic Forest locations along an elevational gradient, i.e., lowland and montane forest. The DNA fingerprinting method PCR-DGGE revealed that the cyanobacterial phyllosphere community structures were mainly influenced by the plant species; geographical location of the plant had little effect. The 16S rRNA gene sequences obtained by clone libraries showed a predominance of nitrogen-fixing cyanobacteria of the order Nostocales, even though the majority of retrieved operational taxonomic units (∼60% of the sequences) showed similarity only to uncultured cyanobacteria phylotypes. The leaf surface of Guapira opposita had the highest richness and diversity of cyanobacteria, whereas the M. neesii (bamboo) had the largest number of copies of cyanobacterial 16S rRNA gene per cm2 of leaf. This study investigated cyanobacteria diversity and its distribution pattern in Atlantic forest phyllosphere. The results indicated that plant species is the main driver of cyanobacteria community assemblage in the phyllosphere and that these communities are made up of a high diversity of cyanobacterial taxa that need to be discovered.

Biodiversity of Living, Non-marine, Thrombolites of Lake Clifton, Western Australia

ABSTRACTLake Clifton in Western Australia is recognized as a critically endangered ecosystem and the only thrombolite reef in the southern hemisphere. There have been concerns that increases in salinity and nutrient run-off have significantly impacted upon the thrombolite microbial community. Here we used cultivation-independent molecular approaches to characterize the microbial diversity of the thrombolites at Lake Clifton. The most dominant phyla currently represented are the Proteobacteria with significant populations of Bacteroidetes and Firmicutes. Cyanobacteria, previously invoked as the main drivers of thrombolite growth, represent only a small fraction (∼1–3% relative abundance) of the microbial community. We report an increase in salinity and nitrogen levels at Lake Clifton that may be contributing to a change in dominant microbial populations. This heightens concerns about the long-term health of the Lake Clifton thrombolites; future work is needed to determine if phyla now dominating this system are capable of the required mineral precipitation for continued thrombolite growth.

Microbiome of the Paranasal Sinuses: Update and Literature Review

Our understanding of the resident microbiome of the paranasal sinuses has changed considerably in recent years. Once presumed to be sterile, healthy sinus cavities are now known to harbor a diverse assemblage of microorganisms, and, it is hypothesized that alterations in the kinds and quantities of these microbes may play a role in the pathogenesis of chronic rhinosinusitis (CRS). To review the current literature regarding the sinus microbiome and collate research findings from relevant studies published to date. A systematic literature review was performed on all molecular studies that investigated the microbial communities of the paranasal sinuses. Methods of detection, microbiome composition, and comparative profiling between patients with and without CRS were explored. A complex consortium of microorganisms has been demonstrated in the sinuses of both patients with and without CRS. However, the latter generally have been characterized by reduced biodiversity compared with controls, with selective enrichment of particular microbes (e.g., Staphylococcus aureus). Such disruptions in the resident microbiome may contribute to disease pathogenesis by enhancing the virulence of potential pathogens and adversely modulating immune responses. The advent of culture-independent molecular approaches has led to a greater appreciation of the intricate microbial ecology of the paranasal sinuses. Microbiota composition, distribution, and abundance impact mucosal health and influence pathogen growth and function. A deeper understanding of the host-microbiome relationship and its constituents may encourage development of new treatment paradigms for CRS, which target restoration of microbiome homeostasis and cultivation of optimal microbial communities.

Impact of soil solarization on the ciliate community structure of a greenhouse soil

Soil solarization is an ecologically friendly method of controlling various plant pathogens and pests, but also affects non-pathogenic members of the soil biota. Here, we studied the impact of soil solarization on the community structure of soil ciliates using a culture-independent molecular approach, namely denaturing gradient gel electrophoresis (DGGE) of targeted 18S rRNA gene fragments. Greenhouse soil with added organic fertilizers was solarized for 33 days at an average temperature of 47–48°C. Solarization caused a drastic change in the ciliate community. The variation between replicates was large, which suggested that the distribution of ciliates was spatially heterogeneous in the soil, probably due to their decreased numbers. In contrast, non-solarized soil had a stable and homogeneous ciliate community during the experimental period. In solarized soil, most of the original ciliate community recovered 76 days after solarization. Sequence analysis of DGGE fragments indicated that both r-selected and K-selected species of ciliates were affected by solarization but recovered with time after solarization. Our results demonstrated both the vulnerability and resilience of the ciliate community to soil solarization and also the utility of using molecular-based analysis of ciliate communities as bioindicators of soil stress caused by solarization.

Three-phase succession of autochthonous lactic acid bacteria to reach a stable ecosystem within 7days of natural bamboo shoot fermentation as revealed by different molecular approaches.

Microbial community structure and population dynamics during spontaneous bamboo shoot fermentation for production of 'soidon' (indigenous fermented food) in North-east India were studied using cultivation-dependent and cultivation-independent molecular approaches. Cultivation-dependent analyses (PCR-amplified ribosomal DNA restriction analysis and rRNA gene sequencing) and cultivation-independent analyses (PCR-DGGE, qPCR and Illumina amplicon sequencing) were conducted on the time series samples collected from three independent indigenous soidon fermentation batches. The current findings revealed three-phase succession of autochthonous lactic acid bacteria to attain a stable ecosystem within 7days natural fermentation of bamboo shoots. Weissella spp. (Weissella cibaria, uncultured Weissella ghanensis) and Lactococcus lactis subsp. cremoris predominated the early phase (1-2days) which was joined by Leuconostoc citreum during the mid-phase (3days), while Lactobacillus brevis and Lactobacillus plantarum emerged and became dominant in the late phase (5-7days) with concurrent disappearance of W.cibaria and L.lactis subsp. cremoris. Lactococcus lactis subsp. lactis and uncultured Lactobacillus acetotolerans were predominantly present throughout the fermentation with no visible dynamics. The above identified dominant bacterial species along with their dynamics can be effectively utilized for designing a starter culture for industrialization of soidon production. Our results showed that a more realistic view on the microbial ecology of soidon fermentation could be obtained by cultivation-dependent studies complemented with cultivation-independent molecular approaches. Moreover, the critical issues to be considered for reducing methodological biases while studying the microbial ecology of traditional food fermentation were also highlighted with this soidon fermentation model.

Changing bacterial profile of Sundarbans, the world heritage mangrove: impact of anthropogenic interventions.

Mangrove microbial communities and their associated activities have profound impact on biogeochemical cycles. Although microbial composition and structure are known to be influenced by biotic and abiotic factors in the mangrove sediments, finding direct correlations between them remains a challenge. In this study we have explored sediment bacterial diversity of the Sundarbans, a world heritage site using a culture-independent molecular approach. Bacterial diversity was analyzed from three different locations with a history of exposure to differential anthropogenic activities. 16S rRNA gene libraries were constructed and partial sequencing of the clones was performed to identify the microbial strains. We identified bacterial strains known to be involved in a variety of biodegradation/biotransformation processes including hydrocarbon degradation, and heavy metal resistance. Canonical Correspondence Analysis of the environmental and exploratory datasets revealed correlations between the ecological indices associated with pollutant levels and bacterial diversity across the sites. Our results indicate that sites with similar exposure of anthropogenic intervention reflect similar patterns of microbial diversity besides spatial commonalities.