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

Abstract

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.

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

“…Therefore, nucleic acid double-staining (NADS) was applied, comprising PI for identification of dead cells and SYBR Green I for total cell staining. Staining conditions (5 μg/mL PI; 5 × 10 3 SYBR Green I dilution; NaCl phosphate buffer containing 0.05 mg/mL GTA) were chosen according to an optimized method for viability analysis of S. aureus and B. cepacia in mixed culture published recently [26]. However, since PI staining of P. aeruginosa in NaCl phosphate buffer (NaCl-P) led to a total loss of membrane integrity (Figure 1(A)), this buffer was replaced by Ringer solution containing GTA.…”

“…The existence of dead cells was verified by staining of cells treated with 70% (v/v) isopropanol (positive controls), which resulted in identical fluorescence signals compared to the subpopulation of dead cells in untreated samples. For S. aureus , an additional subpopulation was detected, which was referred to as “cells with slightly damaged membranes” (damaged) as described previously [26]. In comparison to the staining method established previously in NaCl phosphate buffer [26], similar values were determined for the frequency of viable and dead cells of B. cepacia (Figure 3(A)) as well as viable, damaged and dead cells of S. aureus (Figure 3(B)) in pure culture using Ringer solution.…”

“…Therefore, fluorescence detection enables discrimination between viable and dead cells exhibiting an intact or permeabilized membrane, respectively. In contrast to PMA-T-RFLP, detection of cells with a slightly damaged membrane can be obtained after optimization of staining conditions, as recently demonstrated for S. aureus [26]. For determination of species-specific viability by flow cytometry, fluorescence species-specific detection is a key prerequisite.…”

Anti-methicillin Resistant Staphylococcus aureus Compound Isolation from Halophilic Bacillus amyloliquefaciens MHB1 and Determination of Its Mode of Action Using Electron Microscope and Flow Cytometry Analysis

“…Flow cytometry is a sensitive Fig. 7 In vitro anti-MRSA activity of crude and purified compound by disk diffusion method technique that allows us to determine the viability of bacteria on a single cell level using fluorescent probes [30]. Many investigators have used this method to study the interaction between antibacterial agents and bacteria.…”

Aptamers: Novel Molecules as Diagnostic Markers in Bacterial and Viral Infections?

“…In this sense the discovery of novel technologies and moreover their subsequent applications have become important in laboratory and field studies. As an example, innovations in high-throughput single cell analysis for diagnostic purposes but also for functional analysis and drug discovery are today available in order to analyze pathogenic organisms such as the viruses Hepatitis C [1], influenza [2], or HIV [3] as well as bacteria like Mycobacterium tuberculosis [4] or Staphylococcus aureus [5]. The SELEX technique (Systematic Evolution of Ligands by Exponential Enrichment) was originally discovered by Gold and Szostak [6, 7].…”