Abstract
The complex spatial structure and the heterogeneity within biofilms lead to the emergence of specific social behaviors. However, the impact of resistant mutants within bacterial communities is still mostly unknown. Thus, we determined whether antibiotic resistant mutants display selfish or altruistic behaviors in mixed Pseudomonas aeruginosa biofilms exposed to antibiotics. ECFP-tagged P. aeruginosa strain PAO1 and its EYFP-tagged derivatives hyperproducing the β-lactamase AmpC or the efflux pump MexAB-OprM were used to develop single or mixed biofilms. Mature biofilms were challenged with different concentrations of β-lactams to monitor biofilm structural dynamics, using confocal laser scanning microscopy (CLSM), and population dynamics, through enumeration of viable cells. While exposure of single wild-type PAO1 biofilms to β-lactams lead to a major reduction in bacterial load, it had little effect on biofilms formed by the resistant mutants. However, the most reveling finding was that bacterial load of wild-type PAO1 was significantly increased when growing in mixed biofilms compared to single biofilms. In agreement with CFU enumeration data, CLSM images revealed the amplification of the resistant mutants and their protection of susceptible populations. These findings show that mutants expressing diverse resistance mechanisms, including β-lactamases, but also, as evidenced for the first time, efflux pumps, protect the whole biofilm community, preserving susceptible populations from the effect of antibiotics. Thus, these results are a step forward to understanding antibiotic resistance dynamics in biofilms, as well as the population biology of bacterial pathogens in chronic infections, where the coexistence of susceptible and resistant variants is a hallmark.
Highlights
Biofilms are organized bacterial communities embedded in an extracellular polymeric matrix, often attached to living or abiotic surfaces
Since that the β-lactams FEP and ATM, showed the same increase in the minimal inhibitory concentrations (MICs) with both resistant mutants, these antibiotics were chosen for the experiments
It is well known that the complex structure of biofilms along with the heterogeneity of bacterial cells in different physiological states may explain the increased tolerance to antibiotics (Costerton et al, 1999) but it is imperative to focus on the genotypic diversity of bacteria coexisting in the biofilm community
Summary
Biofilms are organized bacterial communities embedded in an extracellular polymeric matrix, often attached to living or abiotic surfaces. The development of biofilms is currently recognized as one of the most frequent modes of growth in the environment and the main driver of persistence of chronic infections. Characteristically, these bacterial communities are highly resistant to the immune system and antibiotics (Costerton et al, 1999). In addition to the physical and physiological tolerance, classical resistance mechanisms play a major role in biofilm antibiotic resistance (Høiby et al, 2010). Over the past few years many works have been focused on the effect of antimicrobial resistance mechanisms on planktonic mode of growth, but there is still a lack of knowledge about the impact of these resistance mechanisms on bacterial communities such as biofilms
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