Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds

James, Garth A.; Zhao, Alice Ge; Usui, Marcia L.; Underwood, Robert; Nguyen, Hung D.; Beyenal, Haluk; Pulcini, Elinor deLancey; Hunt, Alessandra Agostinho; Bernstein, Hans C.; Fleckman, Philip; Olerud, John E.; Williamson, Kerry S.; Franklin, Michael J.; Stewart, Philip S.

doi:10.1111/wrr.12401

Cited by 99 publications

(75 citation statements)

References 39 publications

(58 reference statements)

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“…3a). Oxygen was quickly depleted throughout most of the biofilm, except near the biofilm-air interface where an aerobic region was established as observed experimentally [81]. Similarly, glucose was rapidly depleted in all regions except near the tissue-biofilm interface where a glucose rich region was maintained.…”

Section: Resultssupporting

confidence: 52%

Metabolic modeling of a chronic wound biofilm consortium predicts spatial partitioning of bacterial species

et al. 2016

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BackgroundChronic wounds are often colonized by consortia comprised of different bacterial species growing as biofilms on a complex mixture of wound exudate. Bacteria growing in biofilms exhibit phenotypes distinct from planktonic growth, often rendering the application of antibacterial compounds ineffective. Computational modeling represents a complementary tool to experimentation for generating fundamental knowledge and developing more effective treatment strategies for chronic wound biofilm consortia.ResultsWe developed spatiotemporal models to investigate the multispecies metabolism of a biofilm consortium comprised of two common chronic wound isolates: the aerobe Pseudomonas aeruginosa and the facultative anaerobe Staphylococcus aureus. By combining genome-scale metabolic reconstructions with partial differential equations for metabolite diffusion, the models were able to provide both temporal and spatial predictions with genome-scale resolution. The models were used to analyze the metabolic differences between single species and two species biofilms and to demonstrate the tendency of the two bacteria to spatially partition in the multispecies biofilm as observed experimentally. Nutrient gradients imposed by supplying glucose at the bottom and oxygen at the top of the biofilm induced spatial partitioning of the two species, with S. aureus most concentrated in the anaerobic region and P. aeruginosa present only in the aerobic region. The two species system was predicted to support a maximum biofilm thickness much greater than P. aeruginosa alone but slightly less than S. aureus alone, suggesting an antagonistic metabolic effect of P. aeruginosa on S. aureus. When each species was allowed to enhance its growth through consumption of secreted metabolic byproducts assuming identical uptake kinetics, the competitiveness of P. aeruginosa was further reduced due primarily to the more efficient lactate metabolism of S. aureus. Lysis of S. aureus by a small molecule inhibitor secreted from P. aeruginosa and/or P. aeruginosa aerotaxis were predicted to substantially increase P. aeruginosa competitiveness in the aerobic region, consistent with in vitro experimental studies.ConclusionsOur biofilm modeling approach allows the prediction of individual species metabolism and interspecies interactions in both time and space with genome-scale resolution. This study yielded new insights into the multispecies metabolism of a chronic wound biofilm, in particular metabolic factors that may lead to spatial partitioning of the two bacterial species. We believe that P. aeruginosa lysis of S. aureus combined with nutrient competition is a particularly relevant scenario for which model predictions could be tested experimentally.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0334-8) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 52%

Metabolic modeling of a chronic wound biofilm consortium predicts spatial partitioning of bacterial species

et al. 2016

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“…7A and B), which is also a recognized feature in chronic infections (18, 27–29). The aggregates formed primarily in the outermost ∼100 μm of the beads (Fig.…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo -Like Characteristics

Sønderholm

Kragh

Koren

et al. 2017

Appl Environ Microbiol

121

113

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Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 μm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3− as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3− as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment.IMPORTANCE Pseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.

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“…7) and therefore represents a common feature of biofilm infections, where multiple lines of evidence point to hypoxic conditions (29)(30)(31)(32)(33). Hypoxia within the gel biofilm is also indicated by localized expression of the lactate dehydrogenase gene, which is expressed in the transition to anaerobiosis (Fig.…”

Section: Discussionmentioning

confidence: 99%

Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit Growth in Dense Aggregates, Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression

Pabst

Pitts

Lauchnor

et al. 2016

Antimicrob Agents Chemother

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An experimental model that mimicked the structure and characteristics of in vivo biofilm infections, such as those occurring in the lung or in dermal wounds where no biomaterial surface is present, was developed. In these infections, microbial biofilm forms as cell aggregates interspersed in a layer of mucus or host matrix material. This structure was modeled by filling glass capillary tubes with an agarose gel that had been seeded with Staphylococcus aureus bacteria and then incubating the gel biofilm in medium for up to 30 h. Confocal microscopy showed that the bacteria formed in discrete pockets distributed throughout the gel matrix. These aggregates enlarged over time and also developed a size gradient, with the clusters being larger near the nutrientand oxygen-supplied interface and smaller at greater depths. Bacteria entrapped in gels for 24 h grew slowly (specific growth rate, 0.06 h ؊1 ) and were much less susceptible to oxacillin, minocycline, or ciprofloxacin than planktonic cells. Microelectrode measurements showed that the oxygen concentration decreased with depth into the gel biofilm, falling to values less than 3% of air saturation at depths of 500 m. An anaerobiosis-responsive green fluorescent protein reporter gene for lactate dehydrogenase was induced in the region of the gel where the measured oxygen concentrations were low, confirming biologically relevant hypoxia. These results show that the gel biofilm model captures key features of biofilm infection in mucus or compromised tissue: formation of dense, distinct aggregates, reduced specific growth rates, local hypoxia, and antibiotic tolerance.

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Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds

Cited by 99 publications

References 39 publications

Metabolic modeling of a chronic wound biofilm consortium predicts spatial partitioning of bacterial species

Metabolic modeling of a chronic wound biofilm consortium predicts spatial partitioning of bacterial species

Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo -Like Characteristics

Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit Growth in Dense Aggregates, Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression

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