Quantitative Terminal Restriction Fragment Length Research Articles

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

BackgroundBacterial 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 SYBR®Green 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.ResultsFor 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.ConclusionsIn 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.

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.

High-Sensitivity Stable-Isotope Probing by a Quantitative Terminal Restriction Fragment Length Polymorphism Protocol

Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

Interspecies effects in a ceftazidime-treated mixed culture of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus: analysis at the single-species level

in vitro studies commonly use single bacterial isolates for testing antibiotic susceptibilities. However, interspecies effects that may arise when mixed infections are treated with antibiotics can obviously not be investigated by this approach. In the study presented here, the effect of ceftazidime against a model microbial community consisting of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus was studied in order to reveal effects that only may appear in a ceftazidime-treated mixed culture. time-kill experiments were conducted with mixed and pure cultures in a defined medium containing 30 mg/L ceftazidime. Interspecies effects were revealed by comparing growth and kill dynamics from time-kill experiments with results from untreated mixed and pure cultures. For species-specific cell enumeration, a quantitative terminal restriction fragment length polymorphism was used. Ceftazidime was measured by HPLC. P. aeruginosa showed only a lytic phase in the ceftazidime-treated mixed culture, but not in the untreated mixed culture nor in the ceftazidime-treated pure culture. On the other hand, S. aureus did not lyse in the ceftazidime-treated mixed culture, while it did in the untreated mixed culture. this finding suggests that the efficacy of ceftazidime against P. aeruginosa was increased by an interspecies effect during co-cultivation with B. cepacia and S. aureus. The latter seemed to be negatively affected by interspecies effects in mixed culture without ceftazidime. The same effect was nullified when ceftazidime was applied to the mixed culture. Further studies are required to reveal the underlying mechanisms.