Most microbes live in heterogeneous, multi-species communities in close association with each other. Microbial consortia are dynamic, interacting communities of two or more microbial groups. Synergistic interspecies associations can provide complex functional capabilities to the consortium, far greater than that achievable with monocultures (1). Biofilms are surface-associated, structured, multi-species microbial consortia that colonize a wide range of substrates of medical importance, posing a serious clinical and public health concern (2). There is an imperative need for innovative strategies to overcome biofilm resistance. Harnessing the potential of individual cell populations and their interactions could serve as a novel therapeutic approach to combat antibiotic resistance in biofilms.
When wild-type Pseudomonas aeruginosa PA14 cells are deposited on a background of antibiotic-resistant mutant cells overlaid on antibiotic-containing agar (tobramycin or gentamicin 8 mg/ml), we observe inhibition of mutants in the region of wild-type deposition.
Inhibition in a spatially-structured system. Wild-type Pseudomonas aeruginosa PA14 cells (left) deposited on the filter-disc inhibit antibiotic-resistant mutant lawns (tobramycin 8 mg/ml agar) in their region of deposition. No inhibition (right) is seen when Luria-Bertani culture medium without cells is deposited. Pictures taken after one day of incubation at 37°C. Scale bar = 5 mm.
Seventeen clinical cystic fibrosis isolates , PAO1, P. fluorescens, Pseudomonas sandgrass isolate, E. coli, Methicillin-resistant S. aureus, Burkholderia cepacia and Serratia marcescens also produce inhibition of antibiotic-resistant mutant lawns. We find that antibiotic is not needed for the production of the inhibitory factor but is needed for inhibition of antibiotic-resistant mutants. Further, nutrient depletion, reactive oxygen species and pyocins do not appear to play a role in mediating inhibition. Based on these observations, we believe that the inhibitory factor is a small, diffusible, metabolic product that is produced in the absence of antibiotic but inhibits antibiotic-resistant mutants in synergy with antibiotic.
To observe inhibition at biologically-relevant spatial scales, constant numbers of antibiotic-resistant mutants were mixed with different wild-type cell densities, plated on antibiotic-containing agar (tobramycin 8 mg/ml) and the number of surviving mutant colonies were counted. The probability of mutant survival shows a non-monotonic dependence on the wild-type cell density. At low densities wild-type cells appear to protect mutants. However, at high cell densities, wild-type inhibition of mutants is seen to predominate, indicating that this phenomenon of inhibition influences antibiotic-resistant mutant survival even at biologically-relevant spatial scales.
Protection and inhibition in a spatially-mixed system. Constant numbers of antibiotic-resistant mutants were mixed with different densities of wild-type PA14 cells and plated on tobramycin 8 mg/ml agar. Antibiotic-resistant mutants show a non-monotonic survival trend where at low densities, wild-type cells appear to protect mutants and at high densities, wild-type inhibition of mutants predominates.
We have identified a clinically-significant microbial interaction whereby bacterial populations otherwise resistant to antibiotics can be inhibited in the presence of a bacterial product synergistic with antibiotics. This work could open a continuum of research directed at exploiting interactions in microbial consortia to overcome antibiotic resistance. Further, it highlights the possibility that the lifetime of current antibiotics can be extended with the use of natural bioproducts as synergistic agents. We are currently working to refine our models to capture the physical characteristics of inhibition and identify the isolatable inhibitory factor.
1. Brenner K, You L, Arnold FH. Engineering microbial consortia: a new frontier in synthetic biology. Trends in Biotechnology (2008): 26; 483-489.
2. Costerton JW, Lewandowski Z. Microbial Biofilms. Ann. Rev. Microbiol (1995): 49; 711-745.