Specific cultivation-independent enumeration of viable cells in probiotic products using a combination of fluorescence in situ hybridization and flow cytometry.

This study introduces an optimized integration of flow cytometry and fluorescence in situ hybridization (Flow-FISH) as an approach for the specific enumeration of gram-positive bacteria in probiotic products, overcoming the limitations of conventional methods. The enhanced Flow-FISH technique synergizes the rapid and automated capabilities of flow cytometry with the high specificity of FISH, facilitating the differentiation of viable cells at the species level within probiotic blends. By analyzing lyophilized samples of Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, and Bifidobacterium animalis subsp. lactis, and a commercial product, the study highlights the optimized Flow-FISH protocol's advantages, including reduced hybridization times to 1.5 h and elimination of centrifugation steps. Comparative evaluations with the widely accepted enumeration methods plate count and Live/Dead (L/D) staining were conducted. The study revealed that Flow-FISH produces higher viable cell counts than plate count, thereby challenging the traditional "gold standard" by highlighting its predisposition to underestimate actual viable cell numbers. Against L/D staining, Flow-FISH achieved comparable results, which, despite the different foundational premises of each technique, confirms the accuracy and reliability of our method. In conclusion, the optimized Flow-FISH protocol represents a significant leap forward in probiotic research and quality control. This method provides a rapid, robust, and highly specific alternative for the enumeration of probiotic bacteria, surpassing traditional methodologies. Its ability to enable a more detailed and reliable analysis of probiotic products paves the way for precise quality control and research insights, underscoring its potential to improve the field significantly.

Symbioflor2® Escherichia coli Genotypes Enhance Ileal and Colonic Gene Expression Associated with Mucosal Defense in Gnotobiotic Mice.

Symbioflor2® is a probiotic product composed of six Escherichia coli genotypes, which has a beneficial effect on irritable bowel syndrome. Our objective was to understand the individual impact of each of the six genotypes on the host, together with the combined impact of the six in the compound Symbioflor2®. Gnotobiotic mice were mono-associated with one of the six genotypes or associated with the compound product. Ileal and colonic gene expression profiling was carried out, and data were compared between the different groups of gnotobiotic mice, along with that obtained from conventional (CV) mice and mice colonized with the probiotic E. coli Nissle 1917. We show that Symbioflor2® genotypes induce intestinal transcriptional responses involved in defense and immune mechanisms. Using mice associated with Symbioflor2®, we reveal that the product elicits a balanced response from the host without any predominance of a single genotype. The Nissle strain and the six bacterial genotypes have different effects on the intestinal gene expression, suggesting that the impacts of these probiotics are not redundant. Our data show the effect of the Symbioflor2® genotypes at the molecular level in the digestive tract, which further highlights their beneficial action on several aspects of intestinal physiology.

Evaluation of the applicability of the aquatic ascomycete Phoma sp. UHH 5-1-03 for the removal of pharmaceutically active compounds from municipal wastewaters using membrane bioreactors.

Membrane bioreactors (MBRs) augmented with terrestrial white-rot basidiomycetes have already been tested for the removal of pharmaceutically active compounds (PhACs) from wastewaters. Within the present study, an aquatic ascomycete (Phoma sp.) was initially demonstrated to efficiently remove several PhACs at their real environmental trace concentrations from nonsterile municipal wastewater on a laboratory scale. Then, a pilot MBR was bioaugmented with Phoma sp. and successively operated in two configurations (first treating full-scale MBR effluent as a posttreatment, and then treating raw municipal wastewater). Treatment of influent wastewater by the Phoma-bioaugmented pilot MBR was more efficient than influent treatment by a concomitantly operated full-scale MBR lacking Phoma sp and posttreatment of full-scale MBR permeate using the pilot MBR. A stable removal of the PhACs carbamazepine (CBZ) and diclofenac (DF) (39 and 34% on average, respectively) could be achieved throughout the pilot MBR influent treatment period of 51 days, without the need for additional nutrient supplementation (full-scale MBR: on average, 15% DF but no CBZ removed during 108 days). The long-term presence of Phoma sp. in the pilot MBR could be demonstrated using fluorescence in situ hybridization analysis, but still open questions regarding its long-term activity maintenance remain to be answered.

A Review of Research Conducted with Probiotic E. coli Marketed as Symbioflor.

This review article summarizes the scientific literature that is currently available about a probiotic E. coli that is known under the name Symbioflor E. coli. The probiotic is marketed for human use and has been subjected to over 20 years of scientific research. As is presented here, the available literature not only contains multiple works to investigate and analyse the probiotic activity of this E. coli, but also describes a variety of other research experiments, dealing with a surprising and interesting range of subjects. By compiling all these works into one review article, more insights into this interesting probiotic E. coli were obtained.

Virulence genes in a probiotic E. coli product with a recorded long history of safe use.

The probiotic product Symbioflor2 (DSM 17252) is a bacterial concentrate of six different Escherichia coli genotypes, whose complete genome sequences are compared here, between each other as well as to other E. coli genomes. The genome sequences of Symbioflor2 E. coli components contained a number of virulence-associated genes. Their presence seems to be in conflict with a recorded history of safe use, and with the observed low frequency of adverse effects over a period of more than 6 years. The genome sequences were used to identify unique sequences for each component, for which strain-specific hybridization probes were designed. A colonization study was conducted whereby five volunteers were exposed to an exceptionally high single dose. The results showed that the probiotic E. coli could be detected for 3 months or longer in their stools, and this was in particular the case for those components containing higher numbers of virulence-associated genes. Adverse effects from this long-term colonization were absent. Thus, the presence of the identified virulence genes does not result in a pathogenic phenotype in the genetic background of these probiotic E. coli.

Lab scale experiments using a submerged MBR under thermophilic aerobic conditions for the treatment of paper mill deinking wastewater.

This paper describes the results of laboratory experiments using a thermophilic aerobic MBR (TMBR) at 50 °C. An innovative use of submerged flat-sheet MBR modules to treat circuit wastewater from the paper industry was studied. Two experiments were conducted with a flux of 8-13 L/m(2)/h without chemical membrane cleaning. COD and BOD(5) elimination rates were 83% and 99%, respectively. Calcium was reduced from 110 to 180 mg/L in the inflow to 35-60 mg/L in the permeate. However, only negligible membrane scaling occurred. The observed sludge yield was very low and amounted to 0.07-0.29 g MLSS/g COD(eliminated). Consequently, the nutrient supply of ammonia and phosphate can be lower compared to a mesophilic process. Molecular-biological FISH analysis revealed a likewise high diversity of microorganisms in the TMBR compared to the mesophilic sludge used for start-up. Furthermore, ammonia-oxidising bacteria were detected at thermophilic operation.

Development and validation of a FISH-based method for the detection and quantification of E. coli and coliform bacteria in water samples.

Monitoring of microbiological contaminants in water supplies requires fast and sensitive methods for the specific detection of indicator organisms or pathogens. We developed a protocol for the simultaneous detection of E. coli and coliform bacteria based on the Fluorescence in situ Hybridization (FISH) technology. This protocol consists of two approaches. The first allows the direct detection of single E. coli and coliform bacterial cells on the filter membranes. The second approach includes incubation of the filter membranes on a nutrient agar plate and subsequent detection of the grown micro-colonies. Both approaches were validated using drinking water samples spiked with pure cultures and naturally contaminated water samples. The effects of heat, chlorine and UV disinfection were also investigated. The micro-colony approach yielded very good results for all samples and conditions tested, and thus can be thoroughly recommended for usage as an alternative method to detect E. coli and coliform bacteria in water samples. However, during this study, some limitations became visible for the single cell approach. The method cannot be applied for water samples which have been disinfected by UV irradiation. In addition, our results indicated that green fluorescent dyes are not suitable to be used with chlorine disinfected samples.

Monitoring structure and activity of nitrifying bacterial biofilm in an automatic biodetector of water toxicity.

Automatic biodetector of water toxicity is a biosensor based on monitoring of catalytic activity of the nitrifying bacteria. To create a standardized biosensing system, development of the biofilm must be characterized to determine the prerequisites for its biological (biocatalytic) stability. In this paper, growth of biofilm comprising ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the open cellular polyurethane material polyurethane sponge bioreactor has been investigated. Dynamics of the biofilm formation was estimated using AOB and NOB metabolic activity and the volume occupied by these two types of bacteria in the biofilm. Spectrophotometry liquid ion chromatography and image cytometry were used, respectively, for these measurements. A mathematical model of the dynamics of biofilm formation was established. These data indicate that open cellular polyurethane material is a good basis for the immobilization of nitrifying bacteria. Moreover, growth of the biofilm leads to its stable structural form, whose biocatalytic activity (12.29 for AOB and 6.84 micromol min(-1) for NOB) is constant in the long term.

Optimization of conditions for profiling bacterial populations in food by culture-independent methods.

In this study we used culture-independent methods to profile bacterial populations in food products. Denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH) were employed in order to identify bacterial species without the need of isolation and biochemical identification. The protocols used to extract the DNA, subsequently subjected to PCR amplification for DGGE, as well as the hybridization procedure for FISH, were optimised. Moreover, an extensive study on the primers and probes to be used for the direct detection and identification of microorganisms commonly found in food, was carried out. Meat and cheese samples, fresh or processed, were subjected to DGGE and FISH analysis and the results obtained highlighted how the processing in food industry is decreasing the bacterial biodiversity. Not only processed cheese or meat but also fermented products were dominated by only one or few species. Lactobacillus sakei, Lactobacillus curvatus and Brochothrix thermosphacta were the main species found in meat products, while in cheese(s) Lactococcus lactis, Streptococcus thermophilus and Leuconostoc spp. were repeatedly detected. The results obtained by the two culture-independent methods used always correlated well.

Filamentous Alphaproteobacteria associated with bulking in industrial wastewater treatment plants.

The phylogeny and distribution of filamentous Alphaproteobacteria, morphologically similar to "Nostocoida limicola" and Eikelboom Type 021N that cause the solids separation problem of bulking in industrial activated sludge plants is described here. A combination of culture-dependent and culture-independent molecular methods has characterized 5 novel species. 16S rRNA targeted oligonucleotide probes were designed for their in situ identification by fluorescence in situ hybridisation (FISH) and used to monitor their presence in 86 WWTPs treating different industrial effluents in four European countries. The involvement of these bacteria in bulking in these plants was confirmed. Filaments hybridising with the ALF-968 probe for the Alphaproteobacteria were present in 65% of the WWTPs examined. They were dominant and therefore probably responsible for bulking in 25.5% of them. The heterogeneous filamentous alphaproteobacterial populations in these communities could be completely identified after application of the oligonucleotide probes used in this study in 91% of the plants containing them. The only filamentous Alphaproteobacteria retrieved in pure culture was isolated from three different industrial WWTPs plants. None of these isolates could grow anaerobically on glucose or denitrify, but all grew aerobically and heterotrophically on a range of carbon sources. Although morphologically similar to the Eikelboom Type 021N morphotype, they were not involved in sulphur metabolism. These bacteria accumulated lipidic storage granules that were associated with their presence under the unbalanced growth conditions existing in these plants.

Fluorescence based rRNA sensor systems for detection of whole cells of Saccharomonospora spp. and Thermoactinomyces spp.

Airborne thermophilic actinomycetes (TPAs) are a growing hygienic challenge in different occupational situations e.g. large scale composting. This study describes first results of a new approach for highly specific and rapid detection of organisms of this group using fluorescently labelled oligonucleotide probes as sensors for whole cells. Three genus-specific 16S rRNA-targeted probes, two for Saccharomonospora spp. and one for Thermoactinomyces spp. were developed and evaluated in a fluorescence in situ hybridisation (FISH) format with agar-grown whole cells. For optimal sensitivity and specificity of FISH, conditions for cell wall permeabilisation and hybridisation stringency were evaluated independently for both genera. Performing specified pretreatment protocols, all three probes yielded strong fluorescence signals. However, the relative fraction of detectable cells or spores clearly depended on the single bacterial species. The probes can serve as cell sensors for direct detection of TPAs in natural samples.