Different Optima for Viability Staining of Each Species
SYBR Green I and PI were applied for flow cytometric determination of viability of S. aureus and B. cepacia for membrane integrity analysis. Fluorescence staining was optimized separately for each species in pure culture with respect to different physiological states during growth, thereby considering differences between the bacteria of interest in cell wall structure and metabolic capabilities. Cells from exponential and stationary growth phases were tested at different dye concentrations. The MFI of SYBR Green I stained bacteria increased with dye concentration either until the fluorescence signal was saturated (S. aureus) or a maximum level was achieved (B. cepacia), suggesting maximal binding of this dye to nucleic acids. Due to relatively high dye concentrations, it can be assumed that binding of SYBR Green I is highly selective to ds DNA (17). Hence, a contribution of SYBR Green I bound to RNA or single-stranded DNA to overall fluorescence can be largely neglected. The decrease of MFI observed at high concentrations of dye as shown for B. cepacia might reflect an efflux of SYBR Green I from cells through active extrusion pumps. Another reason might be the quenching of emitted fluorescence by bound SYBR Green I molecules due to their close proximity on DNA strands. The clear difference in uptake of SYBR Green I observed between S. aureus and B. cepacia may be caused by differences in cell wall composition (15), particularly due to the outer membrane of B. cepacia, which acts as a barrier for penetration of hydrophobic dyes (31). Only staining with GTA in buffer resulted in satisfying fluorescence staining of B. cepacia. This is most likely due to the fact that GTA increases the permeability of the outer membrane to hydrophobic SYBR Green I by crosslinking outer membrane proteins through hydrophilic free amino groups (32, 33). Similar findings were reported by Morono et al. (34), who showed that fluorescence staining with hydrophobic carboxyfluorescein diacetate can be improved considerably by using GTA.
Although maximum MFIs were comparable for both species, the concentration of dye required for optimal staining of B. cepacia was much higher than that needed for S. aureus. This phenomenon may be explained by an efflux of SYBR Green I from B. cepacia. Another possible explanation would be differences in total DNA content and in base composition of DNA of both species (35) since SYBR Green I is known to preferentially bind adenine-thymine (AT) rich regions (17). Genomic data for each species obtained through an Integrated Microbial Genome (IMG) database search supports this hypothesis. B. cepacia (IMG Project ID Gc00309) exhibit a larger genome than S. aureus (IMG Project ID Gc01218). Yet, its DNA contains a comparatively low number of AT bases.
Remarkably, for B. cepacia, SYBR Green I staining was dependent on the physiological state of cells. Exponentially growing cells showed higher MFIs than stationary cells. This might be due to the high proliferation activity of exponentially growing cells exhibiting more than one genome in average, whereas stationary cells contain only the minimum number of genomes because of starvation (36).
For each species, simultaneous staining of tested bacteria with SYBR Green I and PI resulted reproducibly in a characteristic viability pattern. For both species, dead and viable cells were detected with permeabilized and intact cytoplasmic membranes, respectively. These subpopulations could clearly be discriminated from each other due to a fluorescence resonance energy transfer (FRET) from SYBR Green I to PI: If cells are double-stained, green fluorescence is quenched and red fluorescence is increased (29). Concomitantly, displacement of SYBR Green I by PI may play an additional role in the discrimination of viable and dead cells due to a higher binding affinity of PI to nucleic acids as reported by Stocks (37) for a similar staining protocol using the commercially available Baclight™ kit. PI concentration testing revealed optimal PI concentrations, which had no effect on membrane integrity and enabled complete FRET from SYBR Green I to PI for discrimination of dead from viable cells. For S. aureus, an additional subpopulation exhibiting intense SYBR Green I and PI fluorescence was detected. Taking into account the findings of other authors (13, 20), these events were referred to as damaged cells, i.e., cells with slightly damaged membranes. Here, a lower amount of PI entered cells resulting in an incomplete FRET from SYBR Green I to PI (29). Even though a loss of membrane integrity is generally associated with absence of reproductive growth and metabolic activity, the membrane of growing cells can also be perforated during cell division and cell wall synthesis (15). Staining would then result in false PI positive cells.
Staining Protocol for Viability Determination in Mixed Culture
In order to determine the viability of S. aureus and B.cepacia in mixed cultures, the staining protocol described above was modified and extended to enable species discrimination.
For detection of species-specific viability, Gram-specific staining of S. aureus using fluorescently labeled WGA was successfully employed in combination with SYBR Green I and PI using the mixed culture staining protocol. WGA is known for its specific binding to the peptidoglycan layer of Gram-positive bacteria, specifically to N-acetylglucosamine and N-acetylneuroaminic acid residues (38). In this study, WGA did not bind to B. cepacia, which is in agreement with results reported by Sizemore et al. (39), who explained this finding by the presence of the outer membrane of Gram-negative bacteria, which covers the peptidoglycan layer. In contrast to this, reports from other authors showed WGA binding to Gram-negative bacteria, in particular for Escherichia coli, where the enterobacterial common antigen was labeled (40, 41). On the other hand, flow cytometric studies of Holm and Jespersen (42) demonstrated no WGA binding to E. coli among other Gram-negative bacteria. Therefore, it was important to test the specificity of WGA for the species and the physiological state of interest. WGA staining of S. aureus was highly efficient and reproducible. More than 97% of S. aureus could be stained irrespective of growth phase and membrane integrity. In addition, the binding capacity of the lectin did not decrease significantly over the time period covered in our cultivations as observed by Heine et al. (43) for labeling of yeast cells with concanavalin A during brewing processes.
In contrast to widely used fluorescence in situ hybridization-based techniques used for specific detection of bacterial species or taxa in mixed communities involving fluorescently labeled single stranded nucleic acid probes, no permeabilization of cytoplasmic membrane is needed for specific labeling by WGA. Furthermore, the specificity of WGA is superior compared to commercially available fluorescence-based Gram-staining kits involving hexidium iodide. These kits stain not only Gram-positive bacteria (44) but also Gram-negative bacteria if lipopolysaccharides are destabilized (45). Therefore, WGA staining can generally be employed for flow cytometry in combination with different viability staining techniques.
Viability During Growth in Pure and Mixed Culture
The optimized three-color staining protocol was successfully applied for the assessment of viability of S. aureus and B.cepacia during growth in mixed culture over a cultivation period of 32 h. To compare pure and mixed culture cultivations, viability was determined additionally in pure culture. Furthermore, for each culture, qT-RFLP analysis was performed to analyze growth. In mixed culture, the growth of both species was not affected significantly by the presence of the other species. Even though it was expected, neither growth inhibition of S. aureus due to substrate competition nor an growth advantage of B. cepacia due to an existing food chain could be observed as had previously been reported for CF-relevant mixed communities (10, 46). This may be due to differences in species composition as Riedele and Reichl (10) characterized three-species communities or differences in mixed culture conditions as Hesseler et al. (46) described competition in chemostat cultivations. However, growth of both species was improved in mixed culture in this study. However, this is due to batch-to-batch variations or an interspecies effect remained to be clarified.
Viability of S. aureus was initially high for pure as well as for mixed culture but decreased within the first 1 h or 2 h of growth, respectively. Concomitantly, the frequency of damaged S. aureus cells increased. This may be caused by PI entry mediated by cell wall perforation encountered during cell division and cell wall synthesis (15, 47), especially for exponentially growing cells. Accordingly, PI uptake in highly reproductive bacteria during exponential growth was reported in several studies (47, 48). Furthermore, some of the events here classified as damaged might be associated with aggregation of viable and dead S. aureus cells, since it had previously been shown that Staphylococci clump during growth (49). This was also observed in our experiments by increased FSC and SSC signals during growth (data not shown). After 3 h, the frequency of viable S. aureus increased again to high levels (above the initial values) and remained relatively constant until stationary phase was reached. This may be explained by increased cell concentrations during this phase as indicated by cell doublings observed during qT-RFLP analysis. Furthermore, cells grew slower after exponential growth phase due to glucose limitation (data not shown) and, therefore, reduced sensitivity to PI staining (48). The decrease of viability of S. aureus during stationary growth phase in pure and mixed cultures could probably be attributed to the exhaustion of nutrients. As a result, metabolic activity was reduced and impairment of active transports led eventually to membrane permeabilization (14). The tremendous increase in frequency of dead cells toward the end of cultivation was in agreement with results reported by other authors (50, 51), which suggested the loss of membrane integrity as the last detectable cellular event after damage. Furthermore, this may explain the existence of damaged S. aureus cells, which reflects a transient state during loss of membrane integrity. Furthermore, this suggests a slower damage process of the cytoplasmic membrane in comparison to B. cepacia.
The time course of viability of B. cepacia during exponential growth phase in pure and mixed culture was comparable to S. aureus. The correlated increase and decrease in the frequency of dead cells is probably due to the same mechanisms as discussed for S. aureus. In contrast, the viability of B. cepacia remained relatively constant during stationary growth phase until the end of cultivation. This may imply the ability of B. cepacia to maintain membrane integrity during stationary phase, despite of nutrient limitations. For S. aureus, during stationary growth phase clear differences in the time courses of viability in mixed and pure culture were found. In mixed culture, viability dropped earlier and decreased faster compared to pure culture, especially toward the end of cultivation. These observations suggest interspecies effects with B. cepacia in mixed culture. This could probably be attributed to lytic activity of B. cepacia against S. aureus inducing membrane damage, e.g., by peptidoglycan hydrolases (52). However, obtained qT-RFLP data did not indicate any cell lysis. Furthermore, proteomic analyses of a three-species mixed community of relevance did not reveal staphylolytic activity of B. cepacia (53). The mechanism of the observed interspecies effect remained to be clarified. In contrast to S. aureus, viability dynamics of B. cepacia were comparable in mixed and pure culture, which suggest that no negative interspecies effects were induced by the presence of S. aureus. This is in line with results reported by Riedele and Reichl (10).
In conclusion, a novel flow cytometric three-color staining method using SYBR Green I, PI, and fluorescently labeled WGA was established, validated and successfully tested for assessment of viability of S. aureus and B. cepacia in pure and mixed cultures. The method allowed rapid simultaneous Gram-differentiation and viability monitoring of bacterial mixed cultures and is therefore recommended for quantitative analysis of dynamics of mixed cultures, not only for medical research but also for biotechnological applications, food processing, and environmental studies. In combination with methods for species-specific enumeration, in particular qT-RFLP assays, a rapid and reproducible analysis of mixed culture dynamics is achieved.