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Title: Fitness Landscape of Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms}
Article:
Abstract:
Bacteria in biofilms have higher antibiotic tolerance than their planktonic counterparts. A major outstanding question is the degree to which the biofilm-specific cellular state and its constituent genetic determinants contribute to this hyper-tolerant phenotype. Here, we used genome-wide functional profiling of a complex, heterogeneous mutant population of Pseudomonas aeruginosa MPAO1 in biofilm and planktonic growth conditions with and without tobramycin to systematically quantify the contribution of each locus to antibiotic tolerance under these two states. We identified large sets of mutations that contribute to antibiotic tolerance predominantly in the biofilm or planktonic setting only, offering global insights into the differences and similarities between biofilm and planktonic antibiotic tolerance. Our mixed population-based experimental design recapitulated the complexity of natural biofilms and, unlike previous studies, revealed clinically observed behaviors including the emergence of quorum sensing-deficient mutants. Our study revealed a substantial contribution of the cellular state to the antibiotic tolerance of biofilms, providing a rational foundation for the development of novel therapeutics against P. aeruginosa biofilm-associated infections.
Introduction:
Biofilms are ubiquitous in nature, and the majority of human bacterial infections involve biofilms[1],[2]. While biofilms contain cells with a heterogeneous range of states[3], on average, bacteria in biofilms have a much higher—up to 1000-fold— antibiotic tolerance than their planktonic counterparts[4]. A case in point is P. aeruginosa, the major cause of morbidity in cystic fibrosis patients[5] and a frequent cause of nosocomial infections[6]. In the lungs of cystic fibrosis patients, P. aeruginosa persists as a biofilm, which further enhances the organism's inherently high antibiotic tolerance[7]. Aerosolized tobramycin, an aminoglycoside, is commonly prescribed to combat P. aeruginosa infections in cystic fibrosis patients[8]. However, during the course of treatment, the drug's efficacy typically decreases as adaptive mutations accumulate leading to the emergence of hyper-tolerant mutants[9],[10]. In an attempt to combat the problem, aminoglycoside tolerance, and more specifically tobramycin tolerance, has been studied extensively in both the biofilm and planktonic states in P. aeruginosa. A number of factors are thought to be involved including oxidative phosphorylation[11],[12],[13], lipopolysaccharide( LPS) composition[11], cyclic di-guanosine monophosphate( c-di-GMP) levels[14], quorum sensing[15], and membrane permeability[11]. In spite of the vast amount of work on the subject, our understanding of the connection between biofilms and antibiotic tolerance remains incomplete. For example, while bacteria in biofilms are generally known to be more tolerant of antimicrobial agents, it is still not clear if strains that are better at biofilm formation necessarily have higher antibiotic tolerance. To date, several loci have been linked to aminoglycoside tolerance in Pseudomonas. Some, including ndvB, pvrR, arr, and the PA1874-PA1877 efflux pump genes, modulate aminoglycoside tolerance only in the biofilm state[14],[16],[17],[18] and others such as amgRS, mexXY-oprM, and the pel locus have a general impact on tolerance independent of the cellular state[19],[20],[21],[22]. The identified genes, many of which are strain specific[17],[19], belong to a variety of different biological processes including efflux pumping( PA1874–7 and mexXY-oprM), polysaccharide biosynthesis( pel and ndvB), and signaling( arr, pvrR, amgRS). These examples, however, do not provide a comprehensive perspective of antibiotic tolerance in different cellular states, and the extent to which planktonic and biofilm antibiotic tolerances share similar mechanisms and genetic components has not been systematically explored. Additionally, the most commonly used antibiotic sensitivity assays, which are carried out in monocultures of homogenous mutants, do not capture the complex interactions between mutants and the heterogeneous populations from which they emerge. In order to address these shortcomings, we designed an experimental approach capable of identifying mutants with enhanced antibiotic tolerance in the context of a diverse population. To this end, we adapted and optimized a transposon mutagenesis and genetic footprinting technology[23] for P. aeruginosa and used it to quantify the contribution of each P. aeruginosa locus to tobramycin tolerance in the biofilm and planktonic states. Our novel experimental design recapitulated many behaviors observed in clinical isolates, such as the high fitness of quorum sensing-deficient mutants[24]. Our results indicate that large sets of loci contribute to antibiotic tolerance predominantly in the biofilm or planktonic setting only and reveal how the cellular state and multi-cellular interactions combine to impact the response to an antibiotic challenge.
Discussion:
To better understand the role of cellular state and gene-environment interactions in antibiotic tolerance, we examined the relative importance of each gene in P. aeruginosa to fitness in the presence of tobramycin as a function of whether the bacterium is living in a biofilm or growing planktonically. Several previous studies identified a small number of P. aeruginosa genes whose contribution to antibiotic tolerance depends on whether the cells are in a biofilm or planktonic state[16],[17],[18], but this work represents a substantially more comprehensive and systematic examination of the question. Here, we competed the mutants in a transposon library en masse in each of four conditions: biofilms with and without tobramycin, and planktonic growth with and without tobramycin. We then characterized the changes in the population by genetic-footprinting and microarray hybridization. All the biofilms were formed on plastic slides under static conditions with limited oxygen availability, likely creating micro-aerobic conditions. Cellular physiology in oxygen-limited biofilms is clinically relevant as during chronic, late-stage cystic fibrosis, P. aeruginosa grows under reduced oxygen tension and is capable of respiring anaerobically within the thickened airway mucus[48]. Although it is well-known that the biofilm state increases drug tolerance[4], we find that the strains that are most fit in biofilm environments do not necessarily have higher antibiotic tolerance. In fact, our population-level data shows that the set of mutants with high fitness in biofilms( not exposed to tobramycin) has minimal overlap with the set of mutants with high fitness in biofilms in the presence of tobramycin. This implies that the general resistance provided by a biofilm against antibiotics does not protect all members equally and that genetic factors contribute to fitness in the biofilm context. Moreover, while there is a considerable overlap between the loci that modulate antibiotic tolerance in biofilm and planktonic cells, the relative importance of most genes is state-specific. For example, the vast majority of mutants with the strongest fitness advantages in the biofilm state also have only a weak to moderate advantage in the planktonic state. Such strains, however, perform poorly in planktonic tobramycin challenges when in complex mutant populations due to the presence of a myriad of other strains, such as NADH dehydrogenase mutants, with more pronounced planktonic drug tolerance capacities. Similarly, while NADH dehydrogenase mutants are among the fittest strains in planktonic tobramycin challenges, these mutants exhibit only a comparatively moderate advantage in biofilms exposed to tobramycin. We expect that such differential fitness reflects not only the physiological state of the cells but also environmental differences, such as lower oxygen availability. To better understand the pathways contributing to tobramycin tolerance in biofilms, we undertook a broad characterization of a subset of the strains that demonstrated a fitness advantage in biofilms in the presence of tobramycin and whose role in antibiotic tolerance had not been previously identified. We found mutants with changes in membrane permeability, quorum sensing, efflux pump abundance, and oxidative respiration activity—changes previously associated with planktonic antibiotic tolerance[11],[12],[13],[15]. Some mutants, however, did not show changes in any of the above pathways, suggesting that additional mechanisms are at play( Figure 6). One such mechanism that we did not explore is conversion to the RSCV( Rough Small-Colony Variant) state, which is associated with hyper-adherence to solid surfaces and higher antibiotic tolerance[17],[49]. As P. aeruginosa cells in microcolonies have elevated mutation rates[50] and many clinical isolates of P. aeruginosa are hypermutable[51], each population explores a large part of the fitness landscape. In the course of this real-time evolution, each new mutant starts as a minority and competes against the pre-existing population. In this study, we attempted to capture some elements of natural conditions by analyzing our library of mutants as a heterogeneous pool rather than as homogeneous cultures of individual mutants as has been done more commonly[11],[16]. Each individual mutant was present at low abundance and was tested for a fitness advantage or disadvantage within a diverse population that was expected to function collectively as a wild-type proxy. Our choice of experimental paradigm leads to some important discrepancies with previous works. For example, while Bjarnsholt et al. showed that quorum sensing enhances tobramycin tolerance in P. aeruginosa[15], our results indicate that some quorum sensing-defective mutants have a fitness advantage in the presence of tobramycin in competition with a quorum sensing-capable strain. The cheating behavior of quorum-sensing mutants in a mixed population[41] can explain the incongruity, and, indeed, quorum sensing-deficient mutants, specifically lasR mutants, have been frequently isolated from Pseudomonas–associated infections[24]. Hence, the mixed population approach utilized here, in both the initial selections and the follow-up competitions, appears to capture some real-world, biological phenomena not observed in homogeneous cultures. Efforts to develop effective therapeutic strategies against P. aeruginosa infections can benefit from a thorough understanding of how each gene contributes to the organism's antibiotic tolerance in the range of microenvironments present within an infection. We hope that this work and future studies using similar tools in other natural and clinical isolates will contribute to that effort.
Biofilms , matrix-enclosed surface-colonized communities of bacteria , are extremely resistant to antimicrobial agents , withstanding concentrations of antibiotics orders of magnitude higher compared to free-swimming planktonic cells . This is a well-established characteristic of infections caused by the opportunistic pathogen Pseudomonas aeruginosa , the major cause of morbidity in cystic fibrosis patients and a frequent cause of nosocomial infections , and Pseudomonas infections generally persist despite the use of long-term antibiotic therapy . Nonetheless , the genetic basis of the hyper-tolerance of biofilms to antimicrobial agents is poorly understood . In this study , we use a genome-wide genetic footprinting technology to systematically quantify the contribution of each locus in P . aeruginosa to antibiotic tolerance in both biofilm and planktonic states . Comparing and contrasting the genome-wide genetic profile of these two physiological states revealed that large sets of genes modulate antibiotic tolerance as a function of the cellular state .