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Developing selective media for quantification of multispecies biofilms following antibiotic treatment.

ABSTRACT: The lungs of cystic fibrosis (CF) patients are chronically colonized by a polymicrobial biofilm community, leading to difficult-to-treat infections. To combat these infections, CF patients are commonly treated with a variety of antibiotics. Understanding the dynamics of polymicrobial community composition in response to antibiotic therapy is essential in the search for novel therapies. Culture-dependent quantification of individual bacteria from defined multispecies biofilms is frequently carried out by plating on selective media. However, the influence of the selective agents in these media on quantitative recovery before or after antibiotic treatment is often unknown. In the present study we developed selective media for six bacterial species that are frequently co-isolated from the CF lung, i.e. Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, and Gemella haemolysans. We show that certain supplementations to selective media strongly influence quantitative recovery of (un)treated biofilms. Hence, the developed media were optimized for selectivity and quantitative recovery before or after treatment with antibiotics of four major classes, i.e. ceftazidime, ciprofloxacin, colistin, or tobramycin. Finally, in a proof of concept experiment the novel selective media were applied to determine the community composition of multispecies biofilms before and after treatment with tobramycin.

SUBMITTER: Vandeplassche E

PROVIDER: S-EPMC5679531 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Developing selective media for quantification of multispecies biofilms following antibiotic treatment.

Vandeplassche Eva E Coenye Tom T Crabbé Aurélie A

PloS one 20171109 11

The lungs of cystic fibrosis (CF) patients are chronically colonized by a polymicrobial biofilm community, leading to difficult-to-treat infections. To combat these infections, CF patients are commonly treated with a variety of antibiotics. Understanding the dynamics of polymicrobial community composition in response to antibiotic therapy is essential in the search for novel therapies. Culture-dependent quantification of individual bacteria from defined multispecies biofilms is frequently carrie ...[more]

PMID: 29121069

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Project description:Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance.IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.

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Developing selective media for quantification of multispecies biofilms following antibiotic treatment.

Publications

Developing selective media for quantification of multispecies biofilms following antibiotic treatment.

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