16S rRNA-targeted Oligonucleotide Probes Research Articles

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.

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.

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.

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.

Anaerobic ammonium oxidation (Anammox) in immobilized activated sludge biofilms during the treatment of weak wastewater

This work studied the formation of molecular nitrogen by the microbial population of immobilized activated sludge of the domestic wastewater treatment plants (WWTP) that employ the technology developed by ZAO ECOS Company. The technology includes physicochemical water pretreatment and treated water recycling. A hard flexible fibrous brush carrier is used for the immobilization of microorganisms. The presence of both aerobic and anaerobic microorganisms and functioning of the methanogenic microbial community was shown in the biofilms developing on the carrier fibers and in suspended sludge. The high efficiency of nitrogen removal at a low C/N ratio was established to be due to the conjugated nitrification, denitrification, and anammox processes, whose functioning was demonstrated by laboratory cultivation methods and by studying the processes in batch and continuous reactors. Fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH) revealed bacteria belonging to the order Planctomycetales, particularly their anammox group. This work is the first evidence of the important role of the anammox process in the combined system of physicochemical and biological treatment of weak wastewater (BCDEAMOX).

Temporal and spatial distribution of Bacillus and Clostridium histolyticum in swine manure composting by fluorescent in situ hybridization (FISH)

The temporal and spatial distribution of the genus Bacillus and Clostridium histolyticum group in swine manure composting was determined by fluorescent in situ hybridization using fluorescently labeled 16S rRNA-targeted oligonucleotide probes LGC353b and Chis150, respectively. The temporal distribution of total bacteria, Bacillus and C. histolyticum, detected in each layer of the composting pile was noticeable in that the number of them detected at the high-temperature stage was higher than that of the cooling stage. The number detected at the cooling stage was higher than that of the temperature-rising stage. The number of the total bacteria distributed in three locations achieved balance at the stage of cooling. The spatial distribution of the genus Bacillus cells was that the number and the relative abundance of Bacillus cells detected in the middle layer of composting pile were the lowest at each stage of composting. However, the minimum value of the relative abundance exceeded 8%. Compared with Bacillus spp., the C. histolyticum group displayed higher relative abundance in the same layer at different stages of composting except in the top layer at the stage of high temperature. However, the characteristic of the spatial distribution was not noticeable. The detected limits of the genus Bacillus and C. histolyticum group were both found to be the high cell density of 10(6) cells g(-1) (wet weight). These results indicated that the genus Bacillus and C. histolyticum group were the predominant bacteria in the swine manure composting process and may play important role in this complex environment.

Fluorescence in situ hybridization in soil and water ecosystems: A useful method for studying the effect of xenobiotics on bacterial community structure

The ability of soil and groundwater ecosystems to recover from chemical contamination is primarily dependent on the presence of a microbial community which has the ability to remove it. Nevertheless, there has been a little research into these communities because it is strictly dependent on methods capable of identifying and characterizing their community structure and functioning. The use of molecular methods makes it possible to overcome this kind of identification limitation. In this work, we applied the fluorescence in situ hybridization (FISH) method to different samples, such as soil and groundwater contaminated with s-triazine herbicides (simazine or terbuthylazine) and surface water treated with the pharmaceutical oseltamivir carboxylate (Tamiflu). We compared the bacterial community structure in the presence/absence of these xenobiotics. The use of 16S rRNA-targeted oligonucleotide probes, designed specifically for the main phylogenetic levels (Archaea, Bacteria, α-, β-, γ-, ε-subdivision of Proteobacteria, Planctomycetes, Gram-positive bacteria with a high or low DNA G + C content, Cytophaga-Flavobacter-Bacteroides phylum, and sulfate-reducing bacteria), and a DAPI stain made it possible to assess the structure of the bacterial community and its changes in the presence of these xenobiotics in all the ecosystems studied.

Molecular Analyses of the Microbial Community Composition of an Anoxic Basin of a Municipal Wastewater Treatment Plant Reveal a Novel Lineage of Proteobacteria

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anoxic activated sludge from a municipal wastewater treatment plant. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by polymerase chain reaction using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 132 and 249 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was done using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota (93.8% of the operational taxonomic units [OTUs]) and Crenarchaeota (6.2% of the OTUs). Within the bacterial library, 84.8% of the OTUs represent novel putative phylotypes never described before and affiliated with ten divisions. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 60.4% of the OTUs, followed by Bacteroidetes (22.1%) and gram-positives (6.1%). Interestingly, we detected a novel Proteobacteria monophyletic group distinct from the five known subclasses, which we named New Lineage of Proteobacteria (NLP) lineage, and it is composed of eight clones representing 4.6% of the Proteobacteria. A new 16S rRNA-targeted hybridization probe was designed and fluorescent in situ hybridization analyses shows representatives of NLP as cocci-shaped microorganisms. The Chloroflexi, Acidobacterium, and Nitrospira phyla and TM7 candidate division are each represented by ≤3% of clone sequences. A comprehensive set of eight 16S and 23S rRNA-targeted oligonucleotide probes was used to quantify these major groups by dot blot hybridization within 12 samples. The Proteobacteria accounted for 82.5 ± 4.9%, representing the most abundant phyla. The Bacteroidetes and Planctomycetales groups accounted for 4.9 ± 1.3% and 4 ± 1.7%, respectively. Firmicutes and Actinobacteria together accounted for only 1.9 ± 0.5%. The set of probes covers 93.4 ± 14% of the total bacterial population rRNA within the anoxic basin.

Composition of the Landfill Microbial Community as Determined by Application of Domain- and Group-Specific 16S and 18S rRNA-Targeted Oligonucleotide Probes

The microbial community composition of colonized cotton and leachate samples from a landfill was quantified using small subunit (SSU) rRNA probes (quantitative rRNA hybridization). Relative quantification of bacteria, eukaryotes, and archaea revealed variations in the landfill microbial community between samples from different areas of the landfill site and indicated the presence of potentially novel archaea. Anaerobic fungi were quantified in rumen fluid samples but were not sufficiently abundant for direct detection in the landfill samples.

Quantitative analysis of the trophic groups with a fluorescence in situ hybridization (FISH) and the competitive kinetics of a propionate enriched anaerobic culture

An anaerobic culture had been enriched with propionate, which was a critical compound inhibiting the total anaerobic process when significantly accumulated. In addition, the distribution of the trophic groups was investigated using the tool of fluorescence in situ hybridization with 16S rRNA targeted oligonucleotide probes to understand the bacterial communication. Then the kinetic constants in the competitive inhibition models were obtained. The ratio of the methanogens was increased as the culture was enriched, whereas that of the acidogens was decreased. The sum of the acetoclastic and hydrogenotrophic methanogens was 34% and the methanogens was 50% at the 68th day. Approximately 16% of the cells detected by DAPI staining were unknown. The degradation of ethanol is thermodynamically more favorable. However, the substrate affinity to propionate was presumably higher than to ethanol since the culture had been acclimated to the propionate-rich environment. Ethanol and acetate did not show any inhibition effect on the methane fermentation of propionate.

Extent of Precipitation and Sorption During Copper Removal from Synthetic Wastewater in the Presence of Sulfate-Reducing Bacteria

A sulfate-reducing bacterial culture was successfully enriched from the seed sludge of anaerobic sludge digester. Sulfate-reducing bacteria (SRB) in the enriched culture (around 42%) were characterized by fluorescence in situ hybridization (FISH) with group and genus specific 16S rRNA-targeted oligonucleotide probes. Desulfosarcina, Desulfococcus, Desulfofaba, and Desulfofrigus spp. were identified as the dominant species of the enriched SRB. Subsequently, batch experiments were conducted at initial copper and sulfate concentrations of 10 and 300 mg/L, respectively, to quantify the ability of the enriched SRB in simultaneous sulfate reduction and copper removal. Sulfate reduction efficiencies of the culture in the absence (biotic system without copper, BS-1) and the presence (biotic system with copper, BS-2) of copper were 96.8 and 98.8%, respectively, after 6 days. In BS-2, 99.2% copper removal was observed after 1 day. However, 67% of copper was removed by chemical precipitation and bioaccumulation. No significant inhibition of bacterial growth was observed at the copper concentration studied, that is, 10 mg/L. Chemical precipitation as hydroxide/carbonate caused a copper removal of 44% in AS-1 (abiotic system without lactate) and 36% in AS-2 (abiotic system with lactate), after 6 days. However, the majority of copper was removed as copper sulfide well before the occurrence of copper hydroxide precipitation. Energy dispersive X-ray spectroscopy (EDS) analysis of the precipitates obtained from biotic and abiotic systems confirmed the origin of copper sulfide as the result of SRB. As a whole, the results of this study could be useful to understand the mechanisms of copper removal, i.e. chemical precipitation, bioprecipitation and biosorption/bioaccumulation during sulfate reduction by SRB.

Microbial structure of nitrifying granules and their estrogens degradation properties

It has been known that endocrine disruption compounds such as natural estrogens estrone, 17beta-estradiol, estriol, and synthetic steroid 17a-ethynylestradiol can be degraded by nitrifying bacteria. The aim of this research was to test biodegradation of estrogens by microbial granules containing nitrifying bacteria. Cultivation of microbial granules was performed in sequencing batch reactor in model wastewater with carbon to nitrogen ratio of 100:30 by weight. After the system reached the steady state, the mean diameter of granules, sludge volume index of granular biomass, and biomass concentration were 0.6 mm, 22 mL g(-1) and 7 g L(-1), respectively. Nitrite was not detected in the effluent, and conversion of the consumed NH4+-N to NO3--N was 93%. Specific NO3--N production rate was 0.12 d(-1) at the constant NH4+- loading late of 0.9 g L d(-1). The presence of ammonium-oxidizing and nitrite-oxidizing bacteria was confirmed by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes Nsm156 and Nit3. The estrogen biodegradation by the granules was conducted in batch experiments. The complete biodegradation of the mixture of estrone, 17beta-estradiol, estriol, and 17a-ethynylestradiol, 100 microg L(-1) each, by microbial granules was within 60 days. The order of biodegradation for estrogens by microbial granules was as follows: 17beta-estradiol-->estriol-->estrone-->17a-ethynylestradiol. The degradation rate constants of estrogens by microbial granules were 0.19 d(-1), 1.26 d(-1), 0.14 d(-1) and 0.11 d(-1) for estrone, 17beta-estradiol, estriol and 17a-ethynylestradiol, respectively.

Molecular analysis of the digestive microbiota in a gnotobiotic mouse model during antibiotic treatment: Influence of Saccharomyces boulardii

The probiotic Saccharomyces boulardii is a non-pathogenic yeast that has been proven efficient in the prevention of antimicrobial-associated diarrhea and of Clostridium difficile associated colitis. We evaluated the influence of the administration of S. boulardii on the composition of the fecal microbiota in a human microbiota-associated mouse model. This evaluation was run before, during and after a 7-day oral treatment with amoxicillin clavulanic acid. Predominant groups of bacteria were quantified with fluorescence in situ hybridization combined with flow cytometry using group-specific 16S rRNA targeted oligonucleotide probes designed for the Eubacteria, Bacteroides–Porphyromonas–Prevotella, Clostridium coccoides– Eubacterium rectale, Faecalibacterium prausnitzii, Clostridium histolyticum, Lactobacillus–Enterococcus and Enterobacteriaceae groups and Bifidobacterium species. S. boulardii did not quantitatively alter the total anaerobic microbiota nor the dominant bacterial groups. During the antibiotic treatment in the two groups of mice receiving the yeast or not, the level of Enterobacteriaceae and Bacteroides groups increased when the C. coccoides– E. rectale group decreased dramatically. After the antibiotic treatment was discontinued, the return to the initial level was reached more rapidly in the S. boulardii-treated mice than in the control mice ( p<0.05) for the C. coccoides– E. rectale and Bacteroides–Porphyromonas–Prevotella groups. This quicker recovery of normal intestinal microbiota equilibrium after antibiotic therapy could be a mechanism for S. boulardii preventive effect on antibiotic-associated diarrhea in humans.

Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood of honeybees (Apis mellifera)

American foulbrood (AFB) is a bacterial disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae. Although AFB and its aetiological agent are described now for more than a century, the general and molecular pathogenesis of this notifiable disease is poorly understood. We used fluorescence in situ hybridization (FISH) performed with P. larvae-specific, 16S rRNA-targeted oligonucleotide probes to analyse the early steps in the pathogenesis of American foulbrood. The following chain of events could be demonstrated: (i) the spores germinate in the midgut lumen, (ii) the vegetative bacteria massively proliferate within the midgut before, and (iii) they start to locally breach the epithelium and invade the haemocoel. The paracellular route was shown to be the main mechanism for invasion contrasting earlier hypotheses of phagocytosis of P. larvae. Invasion coincided with the death of the host implicating that the penetration of the midgut epithelium is a critical step determining the time of death.