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

Riedele, C.; Reichl, Udo

doi:10.1093/jac/dkq394

Cited by 13 publications

(41 citation statements)

References 39 publications

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“…Interactions among P. aeruginosa and other members of the CF microbial community have been described in vitro and in vivo (14,15,(46)(47)(48). P. aeruginosa has been shown to utilize S. aureus as an iron source, contributing to S. aureus lysis in mixed culture (46).…”

Section: Discussionmentioning

confidence: 99%

“…Riedele and Reichl (15) reported differential effects of the ␤-lactam antibiotic ceftazidime on broth-grown single and mixed cultures of the CF pathogens P. aeruginosa, B. cepacia, and S. aureus; furthermore, S. aureus was protected from ceftazidime treatment in mixed culture. Coupled with the data presented here, the unintended consequence of increasing the viability of a pathogen after antibiotic treatment in mixed culture (with examples using two different classes of antibiotics) underscores the importance of studying the effects of antibiotics in the context of a polymicrobial infection.…”

Section: Discussionmentioning

confidence: 99%

“…However, deep-sequencing studies have revealed that this list of microbes underestimates and oversimplifies the polymicrobial communities that reside in the airways of patients with cystic fibrosis (CF) (4-10). Dozens of bacterial genera have been identified in a single sputum sample, including organisms not generally identified by conventional culturing, such as Streptococcus spp., and anaerobes, including Prevotella (4-14).Interspecies interactions within polymicrobial communities can be cooperative and competitive and can be influenced by the presence of antibiotics (15)(16)(17). Of particular interest in the CF lung is the potential for interspecies interactions between P. aeruginosa and Streptococcus species.…”

mentioning

confidence: 99%

“…Interspecies interactions within polymicrobial communities can be cooperative and competitive and can be influenced by the presence of antibiotics (15)(16)(17). Of particular interest in the CF lung is the potential for interspecies interactions between P. aeruginosa and Streptococcus species.…”

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confidence: 99%

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Tobramycin-Treated Pseudomonas aeruginosa PA14 Enhances Streptococcus constellatus 7155 Biofilm Formation in a Cystic Fibrosis Model System

et al. 2016

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Cystic fibrosis (CF) is a human genetic disorder which results in a lung environment that is highly conducive to chronic microbial infection. Over the past decade, deep-sequencing studies have demonstrated that the CF lung can harbor a highly diverse polymicrobial community. We expanded our existing in vitro model of Pseudomonas aeruginosa biofilm formation on CF-derived airway cells to include this broader set of CF airway colonizers to investigate their contributions to CF lung disease, particularly as they relate to the antibiotic response of the population. Using this system, we identified an interspecies interaction between P. aeruginosa, a bacterium associated with declining lung function and worsening disease, and Streptococcus constellatus, a bacterium correlated with the onset of pulmonary exacerbations in CF patients. The growth rate and cytotoxicity of S. constellatus 7155 and P. aeruginosa PA14 were unchanged when grown together as mixed biofilms in the absence of antibiotics. However, the addition of tobramycin, the frontline maintenance therapy antibiotic for individuals with CF, to a mixed biofilm of S. constellatus 7155 and P. aeruginosa PA14 resulted in enhanced S. constellatus biofilm formation. Through a candidate genetic approach, we showed that P. aeruginosa rhamnolipids were reduced upon tobramycin exposure, allowing for S. constellatus 7155 biofilm enhancement, and monorhamnolipids were sufficient to reduce S. constellatus 7155 biofilm viability in the absence of tobramycin. While the findings presented here are specific to a biofilm of S. constellatus 7155 and P. aeruginosa PA14, they highlight the potential of polymicrobial interactions to impact antibiotic tolerance in unanticipated ways. IMPORTANCEDeep-sequencing studies have demonstrated that the CF lung can harbor a diverse polymicrobial community. By recapitulating the polymicrobial communities observed in the CF lung and identifying mechanisms of interspecies interactions, we have the potential to select the best therapy for a given bacterial community and reveal potential opportunities for novel therapeutic interventions. Using an in vitro model of bacterial infection on CF airway cells, we tested how a particular polymicrobial community grows, damages human cells, and responds to antibiotics in single and mixed infections. We describe here the mechanism of an interspecies interaction between two pathogens in the CF lung, P. aeruginosa and S. constellatus, which is potentiated by a commonly prescribed antibiotic, tobramycin. C ystic fibrosis is a human genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mutations in the CFTR gene result in a wide array of deleterious effects throughout the body, including chronic lifelong respiratory infections, the primary cause of morbidity and mortality in patients with cystic fibrosis. Historically, chronic lung infections have been attributed to relatively few organisms, including Pseudomonas aeruginosa, Staphylococcus aureus, Ha...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Tobramycin-Treated Pseudomonas aeruginosa PA14 Enhances Streptococcus constellatus 7155 Biofilm Formation in a Cystic Fibrosis Model System

et al. 2016

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“…Furthermore, the authors characterized the growth of these species in a defined mixed culture in chemostat cultivations, and a mathematical chemostat model was established to identify interspecies effects [9]. Finally, Riedele et al used the qT-RFLP protocol to study the efficacy of the antibiotic ceftazidime on the growth of these species in mixed culture [10]. While qT-RFLP method allows to discriminate between species and to quantify cell concentration, viability is not determined.…”

Section: Introductionmentioning

confidence: 99%

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

2014

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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.

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

et al. 2012

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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.

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

Cited by 13 publications

References 39 publications

Tobramycin-Treated Pseudomonas aeruginosa PA14 Enhances Streptococcus constellatus 7155 Biofilm Formation in a Cystic Fibrosis Model System

Tobramycin-Treated Pseudomonas aeruginosa PA14 Enhances Streptococcus constellatus 7155 Biofilm Formation in a Cystic Fibrosis Model System

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

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

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