Adela M. Luján

Mucoidy, Quorum Sensing, Mismatch Repair and Antibiotic Resistance in Pseudomonas aeruginosa from Cystic Fibrosis Chronic Airways Infections

Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the mutational dynamics that lead to the adaptation of P. aeruginosa to the CF lung.

Coexistence and Within-Host Evolution of Diversified Lineages of Hypermutable Pseudomonas aeruginosa in Long-term Cystic Fibrosis Infections

The advent of high-throughput sequencing techniques has made it possible to follow the genomic evolution of pathogenic bacteria by comparing longitudinally collected bacteria sampled from human hosts. Such studies in the context of chronic airway infections by Pseudomonas aeruginosa in cystic fibrosis (CF) patients have indicated high bacterial population diversity. Such diversity may be driven by hypermutability resulting from DNA mismatch repair system (MRS) deficiency, a common trait evolved by P. aeruginosa strains in CF infections. No studies to date have utilized whole-genome sequencing to investigate within-host population diversity or long-term evolution of mutators in CF airways. We sequenced the genomes of 13 and 14 isolates of P. aeruginosa mutator populations from an Argentinian and a Danish CF patient, respectively. Our collection of isolates spanned 6 and 20 years of patient infection history, respectively. We sequenced 11 isolates from a single sample from each patient to allow in-depth analysis of population diversity. Each patient was infected by clonal populations of bacteria that were dominated by mutators. The in vivo mutation rate of the populations was ∼100 SNPs/year–∼40-fold higher than rates in normo-mutable populations. Comparison of the genomes of 11 isolates from the same sample showed extensive within-patient genomic diversification; the populations were composed of different sub-lineages that had coexisted for many years since the initial colonization of the patient. Analysis of the mutations identified genes that underwent convergent evolution across lineages and sub-lineages, suggesting that the genes were targeted by mutation to optimize pathogenic fitness. Parallel evolution was observed in reduction of overall catabolic capacity of the populations. These findings are useful for understanding the evolution of pathogen populations and identifying new targets for control of chronic infections.

Evolution and Adaptation in Pseudomonas aeruginosa Biofilms Driven by Mismatch Repair System-Deficient Mutators

Pseudomonas aeruginosa is an important opportunistic pathogen causing chronic airway infections, especially in cystic fibrosis (CF) patients. The majority of the CF patients acquire P. aeruginosa during early childhood, and most of them develop chronic infections resulting in severe lung disease, which are rarely eradicated despite intensive antibiotic therapy. Current knowledge indicates that three major adaptive strategies, biofilm development, phenotypic diversification, and mutator phenotypes [driven by a defective mismatch repair system (MRS)], play important roles in P. aeruginosa chronic infections, but the relationship between these strategies is still poorly understood. We have used the flow-cell biofilm model system to investigate the impact of the mutS associated mutator phenotype on development, dynamics, diversification and adaptation of P. aeruginosa biofilms. Through competition experiments we demonstrate for the first time that P. aeruginosa MRS-deficient mutators had enhanced adaptability over wild-type strains when grown in structured biofilms but not as planktonic cells. This advantage was associated with enhanced micro-colony development and increased rates of phenotypic diversification, evidenced by biofilm architecture features and by a wider range and proportion of morphotypic colony variants, respectively. Additionally, morphotypic variants generated in mutator biofilms showed increased competitiveness, providing further evidence for mutator-driven adaptive evolution in the biofilm mode of growth. This work helps to understand the basis for the specific high proportion and role of mutators in chronic infections, where P. aeruginosa develops in biofilm communities.

MutS deficiency and activity of the error-prone DNA polymerase IV are crucial for determining mucA as the main target for mucoid conversion in Pseudomonas aeruginosa.

Pseudomonas aeruginosa colonizes the respiratory tract of cystic fibrosis (CF) patients, where mutators along with mucoid variants emerge leading to chronic infection. Mucoid conversion generally involves mutations inactivating the mucA gene. This study correlates the frequency and nature of mucA mutations with the activity of factors determining the mutation rate, such as MutS and polymerase IV (Pol IV). Results show that: (i) the emergence frequency of mucoid variants was higher in isolates arising from mutS populations compared with the wild‐type strain; (ii) in both strains mucoid conversion occurred mainly by mucA mutations; (iii) however, the mutator strain harboured mostly mucA22 (a common allele in CF isolates), while the wild type showed a wider spectrum of mucA mutations with low incidence of mucA22; (iv) disruption of dinB in the wild‐type and mutS strains decreased drastically the emergence frequency of mucoid variants; (v) furthermore, the incidence of mucA mutations diminished in the mutS dinB double mutant strain which consisted only in mucA22; (vi) finally, the mucoid isolates obtained from the dinB strain showed an unexpected absence of mucA mutations. Taken together results demonstrate the implication of both MutS and Pol IV in determining mucA as the main target for conversion to mucoidy.

Is genotyping of single isolates sufficient for population structure analysis of Pseudomonas aeruginosa in cystic fibrosis airways

BackgroundThe primary cause of morbidity and mortality in cystic fibrosis (CF) patients is lung infection by Pseudomonas aeruginosa. Therefore much work has been done to understand the adaptation and evolution of P. aeruginosa in the CF lung. However, many of these studies have focused on longitudinally collected single isolates, and only few have included cross-sectional analyses of entire P. aeruginosa populations in sputum samples. To date only few studies have used the approach of metagenomic analysis for the purpose of investigating P. aeruginosa populations in CF airways.ResultsWe analysed five metagenomes together with longitudinally collected single isolates from four recently chronically infected CF patients. With this approach we were able to link the clone type and the majority of SNP profiles of the single isolates to that of the metagenome(s) for each individual patient.ConclusionBased on our analysis we find that when having access to comprehensive collections of longitudinal single isolates it is possible to rediscover the genotypes of the single isolates in the metagenomic samples. This suggests that information gained from genome sequencing of comprehensive collections of single isolates is satisfactory for many investigations of adaptation and evolution of P. aeruginosa to the CF airways.

Ecological selection of siderophore-producing microbial taxa in response to heavy metal contamination

Abstract Some microbial public goods can provide both individual and community‐wide benefits, and are open to exploitation by non‐producing species. One such example is the production of metal‐detoxifying siderophores. Here, we investigate whether conflicting selection pressures on siderophore production by heavy metals – a detoxifying effect of siderophores, and exploitation of this detoxifying effect – result in a net increase or decrease. We show that the proportion of siderophore‐producing taxa increases along a natural heavy metal gradient. A causal link between metal contamination and siderophore production was subsequently demonstrated in a microcosm experiment in compost, in which we observed changes in community composition towards taxa that produce relatively more siderophores following copper contamination. We confirmed the selective benefit of siderophores by showing that taxa producing large amounts of siderophore suffered less growth inhibition in toxic copper. Our results suggest that ecological selection will favour siderophore‐mediated decontamination, with important consequences for potential remediation strategies.

Adaptation to public goods cheats in Pseudomonas aeruginosa

Cooperation in nature is ubiquitous, but is susceptible to social cheats who pay little or no cost of cooperation yet reap the benefits. The effect such cheats have on reducing population productivity suggests that there is selection for cooperators to mitigate the adverse effects of cheats. While mechanisms have been elucidated for scenarios involving a direct association between producer and cooperative product, it is less clear how cooperators may suppress cheating in an anonymous public goods scenario, where cheats cannot be directly identified. Here, we investigate the real-time evolutionary response of cooperators to cheats when cooperation is mediated by a diffusible public good: the production of iron-scavenging siderophores by Pseudomonas aeruginosa. We find that siderophore producers evolved in the presence of a high frequency of non-producing cheats were fitter in the presence of cheats, at no obvious cost to population productivity. A novel morphotype independently evolved and reached higher frequencies in cheat-adapted versus control populations, exhibiting reduced siderophore production but increased production of pyocyanin—an extracellular toxin that can also increase the availability of soluble iron. This suggests that cooperators may have mitigated the negative effects of cheats by downregulating siderophore production and upregulating an alternative iron-acquisition public good. More generally, the study emphasizes that cooperating organisms can rapidly adapt to the presence of anonymous cheats without necessarily incurring fitness costs in the environment they evolve in.

No effect of intraspecific relatedness on public goods cooperation in a complex community.

Many organisms—notably microbes—are embedded within complex communities where cooperative behaviors in the form of excreted public goods can benefit other species. Under such circumstances, intraspecific interactions are likely to be less important in driving the evolution of cooperation. We first illustrate this idea with a simple theoretical model, showing that relatedness—the extent to which individuals with the same cooperative alleles interact with each other—has a reduced impact on the evolution of cooperation when public goods are shared between species. We test this empirically using strain of Pseudomonas aeruginosa that vary in their production of metal‐chelating siderophores in copper contaminated compost (an interspecific public good). We show that nonsiderophore producers grow poorly relative to producers under high relatedness, but this cost can be alleviated by the presence of the isogenic producer (low relatedness) and/or the compost microbial community. Hence, relatedness can become unimportant when public goods provide interspecific benefits.

Draft Genome Sequence of Pseudomonas aeruginosa Strain Hex1T Isolated from Soils Contaminated with Used Lubricating Oil in Argentina.

ABSTRACT Pseudomonas aeruginosa Hex1T was isolated from soils contaminated with used lubricating oil from a garage in Córdoba, Argentina. This strain is capable of utilizing this pollutant as the sole carbon and energy source. Here, we present the 6.9-Mb draft genome sequence of Hex1T, which contains many heavy metal-resistance genes.

WS19.6 Diversity of Pseudomonas aeruginosa in cystic fibrosis airways

Objectives Previous investigations of the population dynamics of P. aeruginosa in the CF airways have been based on longitudinally collected single isolates, but recently this approach has been questioned. The aim of this study is to determine whether genotypic characterization of single isolates can represent the adaptation of P. aeruginosa populations in CF airways. Methods Five metagenomes from sputum samples from four CF patients were sequenced without pre-culturing. The metagenomes were analysed and compared with single isolates from the same patients. Results For all the investigated patients the most recent clone type identified among the single isolates was compared with the metagenome of the specific patient. In all cases the metagenomes were linked to a significantly higher degree of the mutations found in the single isolates sampled from the same patient, than to single isolates sampled from other patients(p Conclusion Our results document that single isolates represent the dominant clone type in the population of P. aeruginosa in CF lungs, as observed by the comparison between single isolate genomes and corresponding metagenomes. Furthermore, single isolates also show the same mutational patterns as metagenomes sampled from the same patient, and is different from metagenomes and single isolates sampled from other patients. The evolutionary dynamics of the population represented by single isolates is supported by the dominant SNP alleles from metagenomes. According to these results, the genomic information from longitudinally collected single isolates is sufficient for investigations of the dynamics of P. aeruginosa populations in the CF airways.