rRNA-targeted Oligonucleotide Probes Research Articles

The Improvement of Fluorescence In Situ Hybridization Technique Based on Explorations of Symbionts in Cicadas.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is widely used for the identification of microbes in complex samples, but it suffers from some limitations resulting in the weak or even absence of fluorescence signals of microbe(s), which may lead to the underestimation or misunderstanding of a microbial community. Herein, we explored symbionts in the bacteriomes and fat bodies of cicadas using modified FISH, aiming to improve this technique. We initially revealed that the probes of Candidatus Sulcia muelleri (Sulcia) and the yeast-like fungal symbiont (YLS) are suitable for detection of these symbionts in all cicadas and some other species of Auchenorrhyncha, whereas the probe of Candidatus Hodgkinia cicadicola (Hodgkinia) is only suitable for detection of Hodgkinia in a few cicada species. The fluorescence signal of Sulcia, Hodgkinia and YLS exhibited weak intensity without the addition of unlabeled oligonucleotides (helpers) and heat shock in some cicadas; however, it can be significantly improved by the addition of both helpers and heat shock. Results of this study suggest that heat shock denaturing rRNA and proteins of related microbe(s) together with helpers binding to the adjacent region of the probe's target sites prevent the re-establishment of the native secondary structure of rRNA; therefore, suitable probe(s) can more easily access to the probe's target sites of rRNA. Our results provide new information for the significant improvement of hybridization signal intensities of microbes in the FISH experiment, making it possible to achieve a more precise understanding of the microbial distribution, community and density in complex samples.

DETERMINATION OF SPECIFIC ENTEROPATHOGEN PRESENCE IN CAPTIVE CHEETAHS (ACINONYX JUBATUS) FED VARIOUS DIETS USING FLUORESCENCE IN SITU HYBRIDIZATION.

Chronic enteropathies pose an important difficulty in the captive management of cheetahs (Acinonyx jubatus) because of suspected multifactorial pathogenesis and the complex nature of enteric microbiota dynamics. Enterobacteriaceae, Campylobacter spp., Clostridium perfringens, Helicobacter spp., and Salmonella spp. are enteropathogens of interest because of their zoonotic potential and suspected contribution to enteropathies. This study aimed to determine the presence of these enteropathogens of interest in fecal samples from cheetahs (N = 48) fed different diets from three different institutions and to investigate the associations between diet, fecal score, and specific enteropathogen presence. Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes were used to visualize and quantify putative enteropathogens in each sample concurrent with selective culturing for Salmonella and Clostridium perfringens. From FISH counts, carcass-fed animals had greater numbers of Enterobacteriaceae compared with animals fed low-fat dog food, although this trend was not statistically significant (P = 0.088). Furthermore, no significant associations were found between fecal score and bacterial load. Abundance of Campylobacter spp., Clostridium perfringens, or Helicobacter spp. as measured by FISH were not correlated with diet or fecal score. On the basis of these data, in agreement with published literature, it is concluded that these microbes may be commensals in the cheetah gastrointestinal tract and do not appear to be a primary cause of abnormal fecal scores.

Linking Microbes to Their Genes at Single Cell Level with Direct-geneFISH.

Direct-geneFISH is a Fluorescence In Situ Hybridization (FISH) method that directly links gene presence, and thus potential metabolic capabilities, to cell identity. The method uses rRNA-targeting oligonucleotide probes to identify cells and dsDNA polynucleotide probes carrying multiple molecules of the same fluorochrome to detect genes. In addition, direct-geneFISH allows quantification of the cell fraction carrying the targeted gene and the number of target genes per cell. It can be applied to laboratory cultures, for example, enrichments and phage infections, and to environmental samples. This book chapter describes the main steps of the direct-geneFISH protocol: probe design and synthesis, the "core" direct-geneFISH protocol and lastly, microscopy and data analysis.

Detection of two pathogenic marine ciliates Ancistrum haliotis and A. crassum (Ciliophora: Scuticociliatia) by fluorescence in situ hybridization

The scuticociliatid ciliates Ancistrum haliotis and A. crassum are parasites that may cause high mortality in the cultured abalone Haliotis spp. and the bivalve Ruditapes philippinarum. Traditional identification with silver staining methods is hampered by their morphological similarities to closely related species and the complicated procedures of morphological analysis. We designed two SSU rRNA-targeted oligonucleotide probes labeled with a fluorochrome, and optimized the fluorescence in situ hybridization (FISH) protocols for identification of A. halioti and A. crassum, respectively. The assays resulted in a clear identification by strong fluorescence signals from the oligonucleotide probes. The method can be used for quick and accurate quantitative analysis of A. haliotis and A. crassum infections on host molluscs.

A refined set of rRNA-targeted oligonucleotide probes for in situ detection and quantification of ammonia-oxidizing bacteria

Ammonia-oxidizing bacteria (AOB) of the betaproteobacterial genera Nitrosomonas and Nitrosospira are key nitrifying microorganisms in many natural and engineered ecosystems. Since many AOB remain uncultured, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been one of the most widely used approaches to study the community composition, abundance, and other features of AOB directly in environmental samples. However, the established and widely used AOB-specific 16S rRNA-targeted FISH probes were designed up to two decades ago, based on much smaller rRNA gene sequence datasets than available today. Several of these probes cover their target AOB lineages incompletely and suffer from a weak target specificity, which causes cross-hybridization of probes that should detect different AOB lineages. Here, a set of new highly specific 16S rRNA-targeted oligonucleotide probes was developed and experimentally evaluated that complements the existing probes and enables the specific detection and differentiation of the known, major phylogenetic clusters of betaproteobacterial AOB. The new probes were successfully applied to visualize and quantify AOB in activated sludge and biofilm samples from seven pilot- and full-scale wastewater treatment systems. Based on its improved target group coverage and specificity, the refined probe set will facilitate future in situ analyses of AOB.

A Multicolor Fluorescence in situ Hybridization Approach Using an Extended Set of Fluorophores to Visualize Microorganisms.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a key method for the detection of (uncultured) microorganisms in environmental and medical samples. A major limitation of standard FISH protocols, however, is the small number of phylogenetically distinct target organisms that can be detected simultaneously. In this study, we introduce a multicolor FISH approach that uses eight fluorophores with distinct spectral properties, which can unambiguously be distinguished by confocal laser scanning microscopy combined with white light laser technology. Hybridization of rRNA-targeted DNA oligonucleotide probes, which were mono-labeled with these fluorophores, to Escherichia coli cultures confirmed that the fluorophores did not affect probe melting behavior. Application of the new multicolor FISH method enabled the differentiation of seven (potentially up to eight) phylogenetically distinct microbial populations in an artificial community of mixed pure cultures (five bacteria, one archaeon, and one yeast strain) and in activated sludge from a full-scale wastewater treatment plant. In contrast to previously published multicolor FISH approaches, this method does not rely on combinatorial labeling of the same microorganisms with different fluorophores, which is prone to biases. Furthermore, images acquired by this method do not require elaborate post-processing prior to analysis. We also demonstrate that the newly developed multicolor FISH method is compatible with an improved cell fixation protocol for FISH targeting Gram-negative bacterial populations. This fixation approach uses agarose embedding during formaldehyde fixation to better preserve the three-dimensional structure of spatially complex samples such as biofilms and activated sludge flocs. The new multicolor FISH approach should be highly suitable for studying structural and functional aspects of microbial communities in virtually all types of samples that can be analyzed by conventional FISH methods.

Combination of a flow cytometric bead system with 16S rRNA-targeted oligonucleotide probes for bacteria detection.

Here we report a bacteria detection method based on a flow cytometric bead system and 16S rRNA-targeted oligonucleotide probes. Polymerase chain reaction (PCR) was first used to acquire bacterial DNA including bacteria-specific sequences. Half of the resulting target DNA was then captured by a capture probe immobilized on a magnetic microbead (MB) surface. The other half of the target DNA was hybridized with a fluorescence-labeled signal probe. In this manner, a sandwich DNA hybridization involving a MB-based capture probe, the target DNA, and a signal probe was realized. The MB carriers modified with reporter dye were analyzed one by one by flow cytometry through a capillary. Using PCR amplicons and this flow cytometric bead system, a detection limit of 180cfumL-1 was achieved, along with high selectivity that permitted the discrimination of different targets when challenged with control bacteria targets and multiplexing capabilities that enabled the simultaneous detection of two kinds of bacteria. Given these advantages, the developed method can be used for the highly sensitive and specific PCR amplicon analysis of DNA extracted from a fresh bacterial culture, as well as multiplex target analysis. Graphical abstract The flow cytometric bead system with 16S rRNA-targeted oligonucleotide probes for bacteria detection developed in this work. This system is highly specific and sensitive, with a detection limit of 180cfumL-1 bacteria.

Design, Optimization and Verification of 16S rRNA Oligonucleotide Probes of Fluorescence in-situ Hybridization for Targeting Clostridium spp. and Clostridium kluyveri.

Fluorescence in-situ hybridization (FISH) is a common and popular method used to investigate microbial communities in natural and engineered environments. In this study, two specific 16S rRNA-targeted oligonucleotide probes, CLZ and KCLZ, were designed and verified to quantify the genus Clostridium and the species Clostridium kluyveri. The optimal concentration of hybridization buffer solution for both probes was 30% (w/v). The specificity of the designed probes was high due to the use of pellets from pure reference strains. Feasibility was tested using samples of Chinese liquor from the famed Luzhou manufacturing cellar. The effectiveness of detecting target cells appears to vary widely in different environments. In pit mud, the detection effectiveness of the target cell by probes CLZ and KCLZ was 49.11% and 32.14%, respectively. Quantitative analysis by FISH technique of microbes in pit mud and fermented grains showed consistency with the results detected by qPCR and PCR-DGGE techniques, which showed that the probes CLZ and KCLZ were suitable to analyze the biomass of Clostridium spp. and C. kluyveri during liquor fermentation. Therefore, this study provides a method for quantitative analysis of Clostridium spp. and C. kluyveri and monitoring their community dynamics in microecosystems.

Visualisation of the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans in co-cultures with micro-algae by CARD-FISH

Some studies have described the isolation and 16S rRNA gene sequence-based identification of hydrocarbon-degrading bacteria living associated with marine eukaryotic phytoplankton, and thus far the direct visual observation of these bacteria on micro-algal cell surfaces (‘phycosphere’) has not yet been reported. Here, we developed two new 16S rRNA-targeted oligonucleotide probes, PCY223 and ALGAR209, to respectively detect and enumerate the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans by Catalyzed Reporter Deposition Fluorescence in situ Hybridization (CARD-FISH). To enhance the hybridization specificity with the ALGAR209 probe, a competitor probe was developed. These probes were tested and optimized using pure cultures, and then used in enrichment experiments with laboratory cultures of micro-algae exposed to phenanthrene, and with coastal water enriched with crude oil. Microscopic analysis revealed these bacteria are found in culture with the micro-algal cells, some of which were found attached to algal cells, and whose abundance increased after phenanthrene or crude oil enrichment. These new probes are a valuable tool for identifying and studying the ecology of P. algicola and A. aromaticivorans in laboratory and field samples of micro-algae, as well as opening new fields of research that could harness their ability to enhance the bioremediation of contaminated sites.

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.

Efficacy and variations in bacterial density in the gut of Litopenaeus vannamei reared in a BFT system and in clear water supplemented with a commercial probiotic mixture

The purpose of this study was to evaluate the colonization ability of a commercial probiotic mixture in the gut of Litopenaeus vannamei reared in BFT and a clear water culture and to verify the effects of the probiotic on the gut bacterial abundance of Vibrio sp. and Bacillus sp. when added to the biofloc or clear water culture system. The 45-day experiment contained four treatments: Biofloc with no Probiotic application (BFT); Biofloc with Probiotic application (BFT+P); Clear Water with no Probiotic application (CW); and Clear Water with Probiotic application (CW+P). The commercial probiotic mixture (Sanolife PRO-W® - INVE Aquaculture) was composed of Bacillus subtilis-complex and Bacillus licheniformes strains and was added to the water every 48h. The bacterial abundance in the gut of shrimp was assessed using fluorescence in situ hybridization (FISH) in which rRNA-targeted oligonucleotide probes were used to identify Bacillus sp. and Vibrio sp. Total bacterial abundance was higher in the probiotic application treatments (BFT+P and CW+P). However, the percentage of Bacillus sp. of the total bacterial abundance was higher in clear water than in BFT when the probiotic mixture was added. The percentage of Vibrio sp. was higher in the treatments without probiotic addition. The use of Sanolife PRO-W® decreased the bacterial abundance of Vibrio sp. in both the BFT and clear water culture systems, while the abundance of Bacillus increased. The results obtained in this study can contribute to our understanding of the microbial ecology of biofloc, which is currently considered a key issue of this culture system.

Direct-geneFISH: a simplified protocol for the simultaneous detection and quantification of genes and rRNA in microorganisms.

Although fluorescence in situ hybridization (FISH) with specific ribosomal RNA (rRNA)-targeted oligonucleotides is a standard method to detect and identify microorganisms, the specific detection of genes in bacteria and archaea, for example by using geneFISH, requires complicated and lengthy (> 30 h) procedures. Here we report a much improved protocol, direct-geneFISH, which allows specific gene and rRNA detection within less than 6 h. For direct-geneFISH, catalyzed amplification reporter deposition (CARD) steps are removed and fluorochrome-labelled polynucleotide gene probes and rRNA-targeted oligonucleotide probes are hybridized simultaneously. The protocol allows quantification of gene copy numbers per cell and the signal of the directly labelled probes enables a subcellular localization of the rRNA and target gene. The detection efficiencies of direct-geneFISH were first evaluated on Escherichia coli carrying the target gene on a copy-control vector. We could show that gene copy numbers correlated to the geneFISH signal within the cells. The new protocol was then applied for the detection of the sulfate thiolhydrolase (soxB) genes in cells of the gammaproteobacterial clade SUP05 in Lake Rogoznica, Croatia. Cell and gene detection efficiencies by direct-geneFISH were statistically identical to those obtained with the original geneFISH, demonstrating the suitability of the simpler and faster protocol for environmental samples.

ProbeBase--an online resource for rRNA-targeted oligonucleotide probes and primers: new features 2016.

probeBase http://www.probebase.net is a manually maintained and curated database of rRNA-targeted oligonucleotide probes and primers. Contextual information and multiple options for evaluating in silico hybridization performance against the most recent rRNA sequence databases are provided for each oligonucleotide entry, which makes probeBase an important and frequently used resource for microbiology research and diagnostics. Here we present a major update of probeBase, which was last featured in the NAR Database Issue 2007. This update describes a complete remodeling of the database architecture and environment to accommodate computationally efficient access. Improved search functions, sequence match tools and data output now extend the opportunities for finding suitable hierarchical probe sets that target an organism or taxon at different taxonomic levels. To facilitate the identification of complementary probe sets for organisms represented by short rRNA sequence reads generated by amplicon sequencing or metagenomic analysis with next generation sequencing technologies such as Illumina and IonTorrent, we introduce a novel tool that recovers surrogate near full-length rRNA sequences for short query sequences and finds matching oligonucleotides in probeBase.

Rapid and sensitive identification of marine bacteria by an improved in situ DNA hybridization chain reaction (quickHCR-FISH)

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with rRNA-targeted oligonucleotide probes has significantly improved the identification of microorganisms in various environmental samples. However, one of the major constraints of CARD-FISH is the low probe penetration due to the high molecular weight of the horseradish peroxidase (HRP) label. Recently, this limitation has been overcome by a novel signal amplification approach termed in situ DNA-hybridization chain reaction (in situ DNA-HCR). In this study, we present an improved and accelerated in situ DNA-HCR protocol (quickHCR-FISH) with increased signal intensity, which was approximately 2 times higher than that of standard in situ DNA-HCR. In addition, the amplification time was only 15min for the extension of amplifier probes from the initiator probe compared to 2h in the original protocol. The quickHCR-FISH was successfully tested for the quantification of marine bacteria with low rRNA contents in both seawater and sediment samples. It was possible to detect the same number of marine bacteria with quickHCR-FISH compared to CARD-FISH within only 3h. Thus, this newly developed protocol could be an attractive alternative to CARD-FISH for the detection and visualization of microorganisms in their environmental context.

In situ identification of the synthrophic protein fermentative Coprothermobacter spp. involved in the thermophilic anaerobic digestion process.

Thermophilic bacteria have recently attracted great attention because of their potential application in improving different biochemical processes such as anaerobic digestion of various substrates, wastewater treatment or hydrogen production. In this study we report on the design of a specific 16S rRNA-targeted oligonucleotide probe for detecting members of Coprothermobacter genus characterized by a strong protease activity to degrade proteins and peptides. The newly designed CTH485 probe and helper probes hCTH429 and hCTH439 were optimized for use in fluorescence in situ hybridization (FISH) on thermophilic anaerobic sludge samples. In situ probing revealed that thermo-adaptive mechanisms shaping the 16S rRNA gene may affect the identification of thermophilic microorganisms. The novel developed FISH probe extends the possibility to study the widespread thermophilic syntrophic interaction of Coprothermobacter spp. with hydrogenotrophic methanogenic archaea, whose establishment is a great benefit for the whole anaerobic system.

Development of a group-specific 16S rRNA-targeted probe set for the identification of Marinobacter by fluorescence in situ hybridization

Members of the Marinobacter genus play an important role in hydrocarbon degradation in the ocean – a topic of special significance in light of the recent Deepwater Horizon oil spill of 2010. The Marinobacter group has thus far lacked a genus level phylogenetic probe that would allow in situ identification of representative members. Here, we developed two new 16S rRNA-targeted oligonucleotide probes (Mrb-0625-a and Mrb-0625-b) to enumerate Marinobacter species by fluorescence in situ hybridization (FISH). In silico analysis of this probe set demonstrated 80% coverage of the Marinobacter genus. A competitor probe was developed to block hybridization by Mrb-0625-a to six Halomonas species with which it shared a one base pair mismatch. The probe set was optimized using pure cultures, and then used in an enrichment experiment with a deep sea oil plume water sample collected from the Deepwater Horizon oil spill. Marinobacter cells rapidly increased as a significant fraction of total microbial abundance in all incubations of original contaminated seawater as well as those amended with n-hexadecane, suggesting this group may be among the first microbial responders to oil pollution in the marine environment. The new probe set will provide a reliable tool for quantifying Marinobacter in the marine environment, particularly at contaminated sites where these organisms can play an important role in the biodegradation of oil pollutants.

Specificity of LSU rRNA-targeted oligonucleotide probes for Pseudo-nitzschia species tested through dot-blot hybridisation

In the scope of the development of a microarray PhyloChip for the detection of toxic phytoplankton species, we designed a large series of probes specific against targets in the nuclear large subunit (LSU) rRNA of a range of Pseudo-nitzschia species and spotted these onto the microarray. Hybridisation with rRNA extracted from monoclonal cultures and from plankton samples revealed many cross-reactions. In the present work, we tested the functionality and specificity of 23 of these probes designed against ten of the species, using a dot-blot procedure. In this case, probe specificity is tested against the target region in PCR products of the LSU rRNA gene marker region blotted on nitrocellulose filters. Each filter was incubated with a species-specific oligoprobe. Eleven of the tested probes showed specific responses, identifying seven Pseudo-nitzschia species. The other probes showed non-specific responses or did not respond at all. Results of dot-blot hybridisations are more specific than those obtained with the microarray approach and the possible reasons for this are discussed.

Assessment of the bacterial community structure in a Brazilian clay soil treated with atrazine

In the present paper, the bacterial communities in two soils, one from an agricultural sugarcane cropped field and the other from an unperturbed soil with similar geopedological characteristics, were characterized using the Fluorescence In Situ Hybridization (FISH) method. FISH consists of in situ identification of bacteria using fluorescent labeled 16S rRNA targeted oligonucleotide probes visualizable under epifluorescence microscope. In the cultivated soil, in line with agricultural practice, the pre-emergence herbicide atrazine had been regularly applied each year at a concentration of 5 L/ha. The Shannon Diversity and Evenness Indices were also calculated using the phylogenetic data obtained from the FISH analysis. Although, at the sampling time (6 months after soil atrazine treatment), no residual herbicide concentration was found, the overall bacterial community results show a lower diversity and evenness in the agricultural soil than in the unperturbed one, demonstrating how microbiological indicators are sensitive to anthropogenic disturbance. In the natural soil, the dominant groups were α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria (representing more than 50 % of the bacteria), but in the agricultural soil, their abundance decreased significantly and represented just 31 % of the bacteria domain.

Monitoring Bacterial Diversity in a full-scale Municipal Wastewater Treatment plant in Dubai by Fluorescence in situ hybridization Technique

In this study, the bacterial diversity in the activated sludge system of a full-scale municipal wastewater treatment plant in Dubai was monitored over a period of one year using ribosomal RNA (rRNA) targeted oligonucleotide probes for a defined phylogenetic group of bacteria by the Fluorescence in situ hybridization (FISH) technique. The largest fraction of the bacterial community in the sludge samples belonged to the gamma-subgroup of proteobacteria (25%) followed by gram positive bacteria of high G+C content(16%), gram positive bacteria with low G+C content (9 %), beta-proteobacteria (8%) and alpha-proteobacteria(5 %) with respect to the population percentages stained by DAPI (4,6-diamino-2-phenylindole).A specific nocardioform actinomycete, simultaneously targeted by both HGC69a and MNP1 probes, was predominantly found throughout the study period in all activated sludge mixed liquor samples. The nocardioform actinomycetes group members were detected in both branched and single cell morphotypes. Most of the previously published genus and species specific probes failed to hybridize to the sludge samples. In conclusion, the overall bacterial community populations detected by the sub-group specific 16S rRNA targeted oligonucleotide probes in FISH technique remained almost constant throughout the period of study irrespective of treatment plant conditions.

Monitoring Microbial Diversity of a Full-Scale Municipal Wastewater Treatment Plant in Dubai

In this study, fluorescence in situhybridization (FISH) technique was employedfor monitoring the microbial community in the activated sludge system of a full-scale municipal wastewater treatment plant in Dubai over a period of one year. A total of 96 activated sludge mixed liquor samples were characterized using ribosomal RNA (16S and 23S rRNA) targeted oligonucleotide probes for defined phylogenetic groups of bacteria. Several filamentous and non-filamentous bacteriawere predominantly found throughout the study period in all activated sludge mixed liquor samples. The bacterial species belonging to High G+C group were detected in both branched and single cell morphotypes. The previously published genus and species specific probes detected several of members belonging to archaea, sulphate reducing bacteria, ammonia-oxidizing bacteria, nitrobacter, nitrospira including halophilic and halotolerantnitrosomonasspp.In conclusion, the overall microbial community populations detected by the sub-group and species specific 16S and 23S rRNA targeted oligonucleotide probes found to be quite diverse and varied seasonally.