Immunomodulation of monocytes by probiotic and selected lactic Acid bacteria.

Some lactic acid bacteria (LAB), especially bacteria belonging to the genus Lactobacillus, are recognized as common inhabitants of the human gastrointestinal tract and have received considerable attention in the last decades due to their postulated health-promoting effects. LAB and probiotic bacteria can modulate the host immune response. However, much is unknown about the mediators and mechanisms responsible for their immunological effect. Here, we present a study using cytokine secretion from the monocytic cell line THP-1 and NF-κB activation in the monocytic cell line U937-3xkB-LUC to elucidate immune stimulating abilities of LAB in vitro. In this study, we investigate both commercially available and potential probiotic LAB strains, and the role of putative surface proteins of L. reuteri using mutants. L. reuteri strains induced the highest cytokine secretion and the highest NF-κB activation, whereas L. plantarum strains and L. rhamnosus GG were low inducers/activators. One of the putative L. reuteri surface proteins, Hmpref0536_10802, appeared to be of importance for the stimulation of THP-1 cells and the activation of NF-κB in U937-3xkB-LUC cells. Live and UV-inactivated preparations resulted in different responses for two of the strains investigated. Our results add to the complexity in the interaction between LAB and human cells and suggest the possible involvement of secreted pro- and anti-inflammatory mediators of LAB. It is likely that it is the sum of bacterial surface proteins and bacterial metabolites and/or secreted proteins that induce cytokine secretion in THP-1 cells and activate NF-κB in U937-3xkB-LUC cells in this study.

Explorative screening of complex microbial communities by real-time 16S rDNA restriction fragment melting curve analyses.

We have developed restriction fragment melting curve analyses (RFMCA), which is a novel method for the real-time analysis of microbial communities. The major advantage of RFMCA compared to, for example, terminal restriction fragment length polymorphism (T-RFLP) or temperature/denaturing gradient gel electrophoresis (TGGE/DGGE) is that the physical separation of DNA fragments is avoided. The RFMCA detection is done by melting point analyses in closed tube systems, which enables high-throughput applications. The robustness of RFMCA was demonstrated by analyzing both mixtures of known samples and the microbial communities in the cecal content of poultry. Our conclusions are that RFMCA is robust, gives a relatively high resolution, and has the potential for high-throughput explorative screenings of microbial communities and large clone libraries.

Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples.

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log(10) was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.

Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR.

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.