Social Behavior of Antibiotic Resistant Mutants Within Pseudomonas aeruginosa Biofilm Communities

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

Dynamics of Mutator and Antibiotic-Resistant Populations in a Pharmacokinetic/Pharmacodynamic Model of Pseudomonas aeruginosa Biofilm Treatment

Biofilm growth, antibiotic resistance, and mutator phenotypes are key components of chronic respiratory infections by Pseudomonas aeruginosa in cystic fibrosis patients. We examined the dynamics of mutator and antibiotic-resistant populations in P. aeruginosa flow-cell biofilms, using fluorescently tagged PAO1 and PAOMS (mutator [mutS] derivative) strains. Two-day-old biofilms were treated with ciprofloxacin (CIP) for 4 days (t4) at 2 μg/ml, which correlated with the mutant prevention concentration (MPC) and provided an AUC/MIC ratio of 384 that should predict therapeutic success. Biofilms were monitored by confocal laser scanning microscopy (CLSM), and the numbers of viable cells and resistant mutants (4- and 16-fold MICs) were determined. Despite optimized pharmacokinetic/pharmacodynamic (PK/PD) parameters, CIP treatment did not suppress resistance development in P. aeruginosa biofilms. One-step resistant mutants (MexCD-OprJ or MexEF-OprN overexpression) were selected for both strains, while two-step resistant mutants (additional GyrA or GyrB mutation) were readily selected only from the mutator strain. CLSM analysis of competition experiments revealed that PAOMS, even when inoculated at a 0.01 proportion, took over the whole biofilm after only 2 days of CIP treatment outnumbering PAO1 by 3 log at t4. Our results show that mutational mechanisms play a major role in biofilm antibiotic resistance and that theoretically optimized PK/PD parameters fail to suppress resistance development, suggesting that the increased antibiotic tolerance driven by the special biofilm physiology and architecture may raise the effective MPC, favoring gradual mutational resistance development, especially in mutator strains. Moreover, the amplification of mutator populations under antibiotic treatment by coselection with resistance mutations is for the first time demonstrated in situ for P. aeruginosa biofilms

Mechanisms of antibiotic resistance in Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa is a major cause of life-threatening acute infections and life-long lasting chronic infections. The characteristic biofilm mode of life in P. aeruginosa chronic infections severely limits the efficacy of antimicrobial therapies, as it leads to intrinsic tolerance, involving physical and physiological factors in addition to biofilm-specific genes that can confer a transient protection against antibiotics promoting the development of resistance. Indeed, a striking feature of this pathogen is the extraordinary capacity to develop resistance to nearly all available antibiotics through the selection of chromosomal mutations, evidenced by its outstanding and versatile mutational resistome. This threat is dramatically amplified in chronic infections, driven by the frequent emergence of mutator variants with enhanced spontaneous mutation rates. Thus, this mini review is focused on describing the complex interplay of antibiotic resistance mechanisms in P. aeruginosa biofilms, to provide potentially useful information for the design of effective therapeutic strategies

Antagonistic Interactions of Pseudomonas aeruginosa Antibiotic Resistance Mechanisms in Planktonic but Not Biofilm Growth▿

Pseudomonas aeruginosa has an extraordinary capacity to evade the activity of antibiotics through a complex interplay of intrinsic and mutation-driven resistance pathways, which are, unfortunately, often additive or synergistic, leading to multidrug (or even pandrug) resistance. However, we show that one of these mechanisms, overexpression of the MexCD-OprJ efflux pump (driven by inactivation of its negative regulator NfxB), causes major changes in the cell envelope physiology, impairing the backbone of P. aeruginosa intrinsic resistance, including the major constitutive (MexAB-OprM) and inducible (MexXY-OprM) efflux pumps and the inducible AmpC β-lactamase. Moreover, it also impaired the most relevant mutation-driven β-lactam resistance mechanism (constitutive AmpC overexpression), through a dramatic decrease in periplasmic β-lactamase activity, apparently produced by an abnormal permeation of AmpC out of the cell. While these results could delineate future strategies for combating antibiotic resistance in cases of acute nosocomial infections, a major drawback for the potential exploitation of the described antagonistic interaction between resistance mechanisms came from the differential bacterial physiology characteristics of biofilm growth, a hallmark of chronic infections. Although the failure to concentrate AmpC activity in the periplasm dramatically limits the protection of the targets (penicillin-binding proteins [PBPs]) of β-lactams at the individual cell level, the expected outcome for cells growing as biofilm communities, which are surrounded by a thick extracellular matrix, was less obvious. Indeed, our results showed that AmpC produced by nfxB mutants is protective in biofilm growth, suggesting that the permeation of AmpC into the matrix protects biofilm communities against β-lactams

Impacto del factor humano en el éxito de actividades de evaluación entre iguales

En este trabajo se analiza la relación entre personalidad, intención de adopción y las distintas modalidades de evaluación entre pares. El propósito de este análisis es discernir si la personalidad influye en la preferencia por una evaluación entre pares frente a una evaluación tradicional (realizada por un experto), y si existen diferencias significativas en las preferencias con respecto a la modalidad de evaluación (corrección individual, en parejas o en tríos). Para ello, se presenta un quasi-experimento realizado en la Universidad de Castilla-La Mancha y coordinado con la Universidad de Alicante con una muestra de 85 estudiantes de primer curso del grado en Ingeniería Informática. Los estudiantes realizaron tres entregas, y en cada una evaluaron a sus compañeros con una modalidad distinta, de manera que al final todos los estudiantes habían experimentado los tres modos de evaluación. Los resultados muestran que las personas más amables y/o menos neuróticas presentan más reticencias a evaluar a los compañeros. Además, los resultados indican que las modalidades de evaluación entre pares no afectan de manera significativa la intención de adopción de esta técnica

Sequential Treatment of Biofilms with Aztreonam and Tobramycin Is a Novel Strategy for Combating Pseudomonas aeruginosa Chronic Respiratory Infections

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