Species-specific viability analysis of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus in mixed culture by flow cytometry.

BACKGROUND: Bacterial species coexist commonly in mixed communities, for instance those occurring in microbial infections of humans. Interspecies effects contribute to alterations in composition of communities with respect to species and thus, to the course and severity of infection. Therefore, knowledge concerning growth and viability of single species in medically-relevant mixed communities is of high interest to resolve complexity of interspecies dynamics and to support development of treatment strategies. In this study, a flow cytometric method was established to assess the species-specific viability in defined three-species mixed cultures. The method enables the characterization of viability of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus, which are relevant to lung infections of Cystic Fibrosis (CF) patients. The method combines fluorescence detection by antibody and lectin labeling with viability fluorescence staining using SYBRGreen I and propidium iodide. In addition, species-specific cell enumeration analysis using quantitative terminal restriction fragment length polymorphisms (qT-RFLP) was used to monitor the growth dynamics. Finally, to investigate the impact of substrate availability on growth and viability, concentrations of main substrates and metabolites released were determined. RESULTS: For each species, the time course of growth and viability during mixed culture cultivations was obtained by using qT-RFLP analysis in combination with flow cytometry. Comparison between mixed and pure cultures revealed for every species differences in growth properties, e.g. enhanced growth of P. aeruginosa in mixed culture. Differences were also observed for B. cepacia and S. aureus in the time course of viability, e.g. an early and drastic reduction of viability of S. aureus in mixed culture. Overall, P. aeruginosa clearly dominated the mixed culture with regard to obtained cell concentrations. CONCLUSIONS: In combination with qT-RFLP analysis, the methods enabled monitoring of species-specific cell concentrations and viability during co-cultivation of theses strains. Experimental findings suggest that the predominance of P. aeruginosa over B. cepacia and S. aureus in mixed culture under the chosen cultivation conditions is promoted by more efficient substrate consumption of P. aeruginosa, and antagonistic interspecies effects induced by P. aeruginosa.

Flow cytometric viability assessment of lactic acid bacteria starter cultures produced by fluidized bed drying.

For starter culture production, fluidized bed drying is an efficient and cost-effective alternative to the most frequently used freeze drying method. However, fluidized bed drying also poses damaging or lethal stress to bacteria. Therefore, investigation of impact of process variables and conditions on viability of starter cultures produced by fluidized bed drying is of major interest. Viability of bacteria is most frequently assessed by plate counting. While reproductive growth of cells can be characterized by the number of colony-forming units, it cannot provide the number of viable-but-nonculturable cells. However, in starter cultures, these cells still contribute to the fermentation during food production. In this study, flow cytometry was applied to assess viability of Lactobacillus plantarum starter cultures by membrane integrity analysis using SYBR®Green I and propidium iodide staining. The enumeration method established allowed for rapid, precise and sensitive determination of viable cell concentration, and was used to investigate effects of fluidized bed drying and storage on viability of L. plantarum. Drying caused substantial membrane damage on cells, most likely due to dehydration and oxidative stress. Nevertheless, high bacterial survival rates were obtained, and granulates contained in the average 2.7 × 10(9) viable cells/g. Furthermore, increased temperatures reduced viability of bacteria during storage. Differences in results of flow cytometry and plate counting suggested an occurrence of viable-but-nonculturable cells during storage. Overall, flow cytometric viability assessment is highly feasible for rapid routine in-process control in production of L. plantarum starter cultures, produced by fluidized bed drying.

LEKTI domain 6 displays anti-inflammatory action in vitro and in a murine atopic dermatitis model.

BACKGROUND: Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a serine protease inhibitor consisting of multiple domains. A loss of function mutation is described in Netherton patients that show severe symptoms of atopic lesions and itch. OBJECTIVES: LEKTI domain 6 (LD6) has shown strong serine protease-inhibitory action in in vitro assays and thus it was tested in vitro and in vivo for potential anti-inflammatory action in models of atopic skin disease. METHODS: Human skin equivalents were treated with LD6 and an inflammatory reaction was challenged by kallikrein-related endopeptidase 5 (KLK5). Furthermore, LD6 was tested on dorsal root ganglia cells stimulated with KLK5, SLIGRL and histamine by calcium imaging. The effect of topically administered LD6 (0.4-0.8%) in lipoderm was compared to a topical formulation of betamethasone-diproprionate (0.1%) in a therapeutic setting on atopic dermatitis-like lesions in NC/Nga mice sensitized to house dust mite antigen. Endpoints were clinical scoring of the mice as well as determination of scratching behaviour. RESULTS: KLK5 induced an upregulation of CXCL-8, CCL20 and IL-6 in skin equivalents. This upregulation was reduced by pre-incubation with LD6. KLK5 as well as histamine induced calcium influx in a population of neurons. LD6 significantly reduced the calcium response to both stimuli. When administered onto lesional skin of NC/Nga mice, both LD6 and betamethasone-dipropionate significantly reduced the inflammatory reaction. The effect on itch behaviour was less pronounced. CONCLUSION: Topical administration of LD6 might be a new therapeutic option for treatment of lesional atopic skin.

A flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture.

Mixed bacterial communities are commonly encountered in microbial infections of humans. Knowledge on the composition of species and viability of each species in these communities allows for a detailed description of the complexity of interspecies dynamics and contributes to the assessment of the severity of infections. Several assays exist for quantification of specific species in mixed communities, including analysis of quantitative terminal restriction fragment length polymorphisms. While this method allows for species-specific cell enumeration, it cannot provide viability data. In this study, flow cytometry was applied to assess the viability of Staphylococcus aureus and Burkholderia cepacia in mixed culture by membrane integrity analysis using SYBR® Green I and propidium iodide staining. Both bacteria are relevant to pulmonary infections of cystic fibrosis patients. Fluorescence staining was optimized separately for each species in pure culture due to differences between species in cell wall structure and metabolic capabilities. To determine viability of species in mixed culture, a protocol was established as a compromise between optimum conditions determined before for pure cultures. This protocol allowed the detection of viable and dead cells of both species, exhibiting an intact and a permeabilized membrane, respectively. To discriminate between S. aureus and B. cepacia, the protocol was combined with Gram-specific fluorescent staining using wheat germ agglutinin. The established three-color staining method was successfully tested for viability determination of S. aureus and B. cepacia in mixed culture cultivations. In addition, growth of both species was monitored by quantitative terminal restriction fragment length polymorphisms. The obtained data revealed alterations in viability during cultivations for different growth phases and suggest interspecies effects in mixed culture. Overall, this method allows for rapid simultaneous Gram-differentiation and viability assessment of bacterial mixed cultures and is therefore suitable for the analysis of dynamics of mixed communities of medical, environmental, and biotechnological relevance.