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

Rüger, Marc; Ackermann, Mandy; Reichl, Udo

doi:10.1186/1471-2180-14-56

Cited by 33 publications

(18 citation statements)

References 54 publications

(75 reference statements)

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“…Flow cytometry can also be applied to quantify viability of different bacterial species after resuspension of multispecies biofilms. As an example, the viability of P. aeruginosa , B. cepacia , and S. aureus in a mixed culture was quantified by means of fluorescence detection using multifluorescent labeling with antibody, lectins, SYBR Green and propidium iodide ( Rüger et al, 2014 ). This method has also been applied to P. aeruginosa axenic biofilms in order to separate active and dormant cell populations and compare their phenotypes and resistance to various antimicrobial agents ( Kim et al, 2009 ).…”

Section: Experimental Methods To Study Multispecies Biofilms and Theimentioning

confidence: 99%

Pathogens protection against the action of disinfectants in multispecies biofilms

et al. 2015

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Biofilms constitute the prevalent way of life for microorganisms in both natural and man-made environments. Biofilm-dwelling cells display greater tolerance to antimicrobial agents than those that are free-living, and the mechanisms by which this occurs have been investigated extensively using single-strain axenic models. However, there is growing evidence that interspecies interactions may profoundly alter the response of the community to such toxic exposure. In this paper, we propose an overview of the studies dealing with multispecies biofilms resistance to biocides, with particular reference to the protection of pathogenic species by resident surface flora when subjected to disinfectants treatments. The mechanisms involved in such protection include interspecies signaling, interference between biocides molecules and public goods in the matrix, or the physiology and genetic plasticity associated with a structural spatial arrangement. After describing these different mechanisms, we will discuss the experimental methods available for their analysis in the context of complex multispecies biofilms.

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Section: Experimental Methods To Study Multispecies Biofilms and Theimentioning

confidence: 99%

Pathogens protection against the action of disinfectants in multispecies biofilms

et al. 2015

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“…Therefore, several offline methods have been applied offering a lower information density in comparison to an online signal. This includes the utilization of molecular methods such as quantitative terminal restriction fragment length polymorphism (Rüger, Ackermann, & Reichl, 2014) and quantitative real time polymerase chain reaction (Pérez et al, 2015) for the biomass measurement.…”

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confidence: 99%

Noninvasive tool for optical online monitoring of individual biomass concentrations in a defined coculture

Geinitz

Rehmann

Büchs

et al. 2020

Biotech & Bioengineering

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Cocultures bear great potential in the conversion of complex substrates and process intensification, as well as, in the formation of unique components only available due to inter‐species interactions. Dynamic data of coculture composition is necessary for understanding and optimizing coculture systems. However, most standard online determined parameters measure the sum of all species in the reactor system. The kinetic behavior of the individual species remains unknown. Up to now, different offline methods are available to determine the culture composition, as well as the online measurement of fluorescence of genetically modified organisms. To avoid any genetic modification, a noninvasive online monitoring tool based on the scattered light spectrum was developed for microtiter plate cultivations. To demonstrate the potential, a coculture consisting of the bacterium Lactococcus lactis and the yeast Kluyveromyces marxianus was cultivated. Via partial least squares regression of scattered light spectra, the online determination of the individual biomass concentrations without further sampling and analyses is possible. The results were successfully validated by a Coulter counter‐analysis, taking advantage of the different cell sizes of both organisms. The findings prove the applicability of the new method to follow in detail the dynamics of a coculture.

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“…This striking negative clinical correlation between S. aureus and P. aeruginosa during teenage years and young adulthood has driven several in vitro and in vivo studies geared toward characterizing the interbacterial interactions of these two organisms (36)(37)(38)(39)(40)(41)(42)(43)(44). These previous studies have found that P. aeruginosa secretes various antistaphylococcal products and proteases, such as LasA, that can cause both biofilm dispersion and cell lysis of S. aureus (42,45).…”

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Coculture of Staphylococcus aureus with Pseudomonas aeruginosa Drives S. aureus towards Fermentative Metabolism and Reduced Viability in a Cystic Fibrosis Model

et al. 2015

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The airways of patients with cystic fibrosis are colonized with diverse bacterial communities that change dynamically during pediatric years and early adulthood. Staphylococcus aureus is the most prevalent pathogen during early childhood, but during late teens and early adulthood, a shift in microbial composition occurs leading to Pseudomonas aeruginosa community predominance in ϳ50% of adults. We developed a robust dual-bacterial in vitro coculture system of P. aeruginosa and S. aureus on monolayers of human bronchial epithelial cells homozygous for the ⌬F508 cystic fibrosis transmembrane conductance regulator (CFTR) mutation to better model the mechanisms of this interaction. We show that P. aeruginosa drives the S. aureus expression profile from that of aerobic respiration to fermentation. This shift is dependent on the production of both 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) and siderophores by P. aeruginosa. Furthermore, S. aureus-produced lactate is a carbon source that P. aeruginosa preferentially consumes over medium-supplied glucose. We find that initially S. aureus and P. aeruginosa coexist; however, over extended coculture P. aeruginosa reduces S. aureus viability, also in an HQNO-and P. aeruginosa siderophore-dependent manner. Interestingly, S. aureus small-colony-variant (SCV) genetic mutant strains, which have defects in their electron transport chain, experience reduced killing by P. aeruginosa compared to their wild-type parent strains; thus, SCVs may provide a mechanism for persistence of S. aureus in the presence of P. aeruginosa. We propose that the mechanism of P. aeruginosa-mediated killing of S. aureus is multifactorial, requiring HQNO and P. aeruginosa siderophores as well as additional genetic, environmental, and nutritional factors. IMPORTANCEIn individuals with cystic fibrosis, Staphylococcus aureus is the primary respiratory pathogen during childhood. During adulthood, Pseudomonas aeruginosa predominates and correlates with worse patient outcome. The mechanism(s) by which P. aeruginosa outcompetes or kills S. aureus is not well understood. We describe an in vitro dual-bacterial species coculture system on cystic fibrosis-derived airway cells, which models interactions relevant to patients with cystic fibrosis. Further, we show that molecules produced by P. aeruginosa additively induce a transition of S. aureus metabolism from aerobic respiration to fermentation and eventually lead to loss of S. aureus viability. Elucidating the molecular mechanisms of P. aeruginosa community predominance can provide new therapeutic targets and approaches to impede this microbial community transition and subsequent patient worsening. C omplex polymicrobial communities colonize the airways of cystic fibrosis (CF) patients within the first month of life (1). Culture-independent studies have revealed the simultaneous presence of numerous bacterial taxa, fungi, and viruses in respiratory samples from CF patients at all stages of life (2-8). This abundance of microbes colonizing the respiratory ...

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

Cited by 33 publications

References 54 publications

Pathogens protection against the action of disinfectants in multispecies biofilms

Pathogens protection against the action of disinfectants in multispecies biofilms

Noninvasive tool for optical online monitoring of individual biomass concentrations in a defined coculture

Coculture of Staphylococcus aureus with Pseudomonas aeruginosa Drives S. aureus towards Fermentative Metabolism and Reduced Viability in a Cystic Fibrosis Model

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