Pseudomonas aeruginosa Phenotypes Associated With Eradication Failure in Children With Cystic Fibrosis

Background. Pseudomonas aeruginosa is a key respiratory pathogen in people with cystic fibrosis (CF). Due to its association with lung disease progression, initial detection of P. aeruginosa in CF respiratory cultures usually results in antibiotic treatment with the goal of eradication. Pseudomonas aeruginosa exhibits many different phenotypes in vitro that could serve as useful prognostic markers, but the relative relationships between these phenotypes and failure to eradicate P. aeruginosa have not been well characterized

Pseudomonas aeruginosa in vitro Phenotypes Distinguish Cystic Fibrosis Infection Stages and Outcomes

Rationale: Pseudomonas aeruginosa undergoes phenotypic changes during cystic fibrosis (CF) lung infection. Although mucoidy is traditionally associated with transition to chronic infection, we hypothesized that additional in vitro phenotypes correlate with this transition and contribute to disease

Evidence for Induction of Integron-Based Antibiotic Resistance by the SOS Response in a Clinical Setting

Bacterial resistance to β-lactams may rely on acquired β-lactamases encoded by class 1 integron-borne genes. Rearrangement of integron cassette arrays is mediated by the integrase IntI1. It has been previously established that integrase expression can be activated by the SOS response in vitro, leading to speculation that this is an important clinical mechanism of acquiring resistance. Here we report the first in vivo evidence of the impact of SOS response activated by the antibiotic treatment given to a patient and its output in terms of resistance development. We identified a new mechanism of modulation of antibiotic resistance in integrons, based on the insertion of a genetic element, the gcuF1 cassette, upstream of the integron-borne cassette blaOXA-28 encoding an extended spectrum β-lactamase. This insertion creates the fused protein GCUF1-OXA-28 and modulates the transcription, the translation, and the secretion of the β-lactamase in a Pseudomonas aeruginosa isolate (S-Pae) susceptible to the third generation cephalosporin ceftazidime. We found that the metronidazole, not an anti-pseudomonal antibiotic given to the first patient infected with S-Pae, triggered the SOS response that subsequently activated the integrase IntI1 expression. This resulted in the rearrangement of the integron gene cassette array, through excision of the gcuF1 cassette, and the full expression the β-lactamase in an isolate (R-Pae) highly resistant to ceftazidime, which further spread to other patients within our hospital. Our results demonstrate that in human hosts, the antibiotic-induced SOS response in pathogens could play a pivotal role in adaptation process of the bacteria

Nutrient Availability as a Mechanism for Selection of Antibiotic Tolerant Pseudomonas aeruginosa within the CF Airway

Microbes are subjected to selective pressures during chronic infections of host tissues. Pseudomonas aeruginosa isolates with inactivating mutations in the transcriptional regulator LasR are frequently selected within the airways of people with cystic fibrosis (CF), and infection with these isolates has been associated with poorer lung function outcomes. The mechanisms underlying selection for lasR mutation are unknown but have been postulated to involve the abundance of specific nutrients within CF airway secretions. We characterized lasR mutant P. aeruginosa strains and isolates to identify conditions found in CF airways that select for growth of lasR mutants. Relative to wild-type P. aeruginosa, lasR mutants exhibited a dramatic metabolic shift, including decreased oxygen consumption and increased nitrate utilization, that is predicted to confer increased fitness within the nutrient conditions known to occur in CF airways. This metabolic shift exhibited by lasR mutants conferred resistance to two antibiotics used frequently in CF care, tobramycin and ciprofloxacin, even under oxygen-dependent growth conditions, yet selection for these mutants in vitro did not require preceding antibiotic exposure. The selection for loss of LasR function in vivo, and the associated adverse clinical impact, could be due to increased bacterial growth in the oxygen-poor and nitrate-rich CF airway, and from the resulting resistance to therapeutic antibiotics. The metabolic similarities among diverse chronic infection-adapted bacteria suggest a common mode of adaptation and antibiotic resistance during chronic infection that is primarily driven by bacterial metabolic shifts in response to nutrient availability within host tissues

Acquisition and Evolution of the exoU Locus in Pseudomonas aeruginosa

ExoU is a potent Pseudomonas aeruginosa cytotoxin translocated into host cells by the type III secretion system. A comparison of genomes of various P. aeruginosa strains showed that that the ExoU determinant is found in the same polymorphic region of the chromosome near a tRNA(Lys) gene, suggesting that exoU is a horizontally acquired virulence determinant. We used yeast recombinational cloning to characterize four distinct ExoU-encoding DNA segments. We then sequenced and annotated three of these four genomic regions. The sequence of the largest DNA segment, named ExoU island A, revealed many plasmid- and genomic island-associated genes, most of which have been conserved across a broad set of β- and γ-Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 tRNA(Lys)-linked genomic island, the pathogenicity islands of strain PA14, and pKLC102 of clone C strains allowed us to propose a mechanism for the origin and transmission of the ExoU determinant. The evolutionary history very likely involved transposition of the ExoU determinant onto a transmissible plasmid, followed by transfer of the plasmid into different P. aeruginosa strains. The plasmid subsequently integrated into a tRNA(Lys) gene in the chromosome of each recipient, where it acquired insertion sequences and underwent deletions and rearrangements. We have also applied yeast recombinational cloning to facilitate a targeted mutagenesis of ExoU island A, further demonstrating the utility of the specific features of the yeast capture vector for functional analyses of genes on large horizontally acquired genetic elements

c-di-GMP heterogeneity is generated by the chemotaxis machinery to regulate flagellar motility

Individual cell heterogeneity is commonly observed within populations, although its molecular basis is largely unknown. Previously, using FRET-based microscopy, we observed heterogeneity in cellular c-di-GMP levels. In this study, we show that c-di-GMP heterogeneity in Pseudomonas aeruginosa is promoted by a specific phosphodiesterase partitioned after cell division. We found that subcellular localization and reduction of c-di-GMP levels by this phosphodiesterase is dependent on the histidine kinase component of the chemotaxis machinery, CheA, and its phosphorylation state. Therefore, individual cell heterogeneity in c-di-GMP concentrations is regulated by the activity and the asymmetrical inheritance of the chemotaxis organelle after cell division. c-di-GMP heterogeneity results in a diversity of motility behaviors. The generation of diverse intracellular concentrations of c-di-GMP by asymmetric partitioning is likely important to the success and survival of bacterial populations within the environment by allowing a variety of motility behaviors.ISSN:2050-084

Metronidazole increases the emergence of ceftazidime-resistant mutants by inducing the SOS response.

<p>(A) Expression determination by RT-qPCR of <i>recA</i>, <i>lexA</i> and <i>intI1</i> with mitomycin C (a well-characterized SOS inducer used as a positive control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002778#ppat.1002778-Guerin1" target="_blank">[8]</a>), metronidazole and ceftazidime in S-<i>Pae</i>Δ<i>ampC</i> (WT), S-<i>Pae</i>Δ<i>ampC</i>Δ<i>recA</i> (Δ<i>recA</i>) and S-<i>Pae</i>Δ<i>ampC</i>Δ<i>recA</i> complemented with <i>recA</i> (<i>recA</i>⁺). Ratio of means ± SEM from at least 2 independent experiments in triplicate; two-sided Student's <i>t</i>-tests using 1.0 – dotted line – as comparator; NS, <i>P</i>>0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. (B) Frequency of emergence of ceftazidime-resistant mutant (by <i>gcuF1</i> cassette excision) in the same experimental conditions as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002778#ppat-1002778-g005" target="_blank">Figure 5A</a> (ratios of geometric means of the excision rates ± SEM, N≥3; two-sided Student's <i>t</i>-tests using 1.0 – dotted line – as comparator; NS, <i>P</i>>0.05; ***, <i>P</i><0.001).</p

<i>gcuF1</i> excision enhances the expression of <i>bla</i>_OXA-28.

<p>The mRNA levels of genes are given. Means ± SEM (<i>n</i>≥2, two-sided Student's <i>t</i>-test; NS, <i>P</i>>0.05; ***, <i>P</i><0.001). Transcripts of gene <i>ampC</i> (A) and <i>bla</i>_OXA-28 (B) were quantitatively assessed by RT-qPCR. The mRNA levels of genes were normalized to that of reference gene <i>rpsL</i> and expressed as a ratio to the levels in the R-<i>Pae</i>1 isolate (for <i>bla</i>_OXA-28) or in the wild-type PA14 (for <i>ampC</i>) in which the values were set at 1.00.</p

<i>gcuF1</i> cassette insertion modulates the resistance to ceftazidime.

<p>(A) Schematic representation of the class 1 integron carried by R-<i>Pae</i>1 and laboratory M-<i>Pae</i> mutants obtained from S-<i>Pae</i> and (B) carried by S-<i>Pae</i>, with the 319 bp <i>gcuF1</i> cassette. Open arrows represent the coding sequences and indicate the direction of transcription. The purple box indicates the LexA box. The <i>attI1</i> site is shown by the red box and colored triangles indicate the cassette <i>attC</i> sites. The black rectangles indicate the peptide signal sequences encoded in <i>bla</i>_OXA-28. The genes <i>intI1</i>, <i>aacA4 and bla</i>_OXA-28 encode the integrase IntI1, the aminoglycoside acetyltransferase AAC(6′)-Ib and the extended-spectrum oxacillinase OXA-28, respectively. The nucleotide sequences reported here appear in the EMBL/Genbank nucleotide sequence database under accession no. FJ207466 (S-<i>Pae</i>) and FJ374756 (R-<i>Pae</i>1). (C) Susceptibility to ceftazidime of <i>P. aeruginosa</i> reference strain PA14 (WT), S-<i>Pae</i>, R-<i>Pae</i>1, laboratory ceftazidime-resistant mutant obtained from S-<i>Pae</i> (M-<i>Pae</i>) and PA14Δ<i>ampC</i> carrying the empty vector pBTK27 (Vector control), cloned <i>gcuF1</i>-<i>bla</i>_OXA-28 (<i>gcuF1</i>-<i>bla</i>_OXA-28), cloned <i>bla</i>_OXA-28 (<i>bla</i>_OXA-28) and with site-directed mutations in <i>gcuF1</i>-<i>bla</i>_OXA-28: TGA stop codon inserted downstream of <i>gcuF1</i> (InsSTOP), GAAGG ribosome binding site deleted upstream of <i>bla</i>_OXA-28 (DelRBS) or substituted with a sequence with no translation initiation power (ReplRBS) and with the <i>bla</i>_OXA-28 ATG start codon replaced by GTC (ReplATG1) or GTG (ReplATG2). Means ± SEM (<i>n</i> = 3). Numbers above bars are MICs of ceftazidime. (D) Left pane: Insertion of <i>gcuF1</i> creates a new ORF. SDS-PAGE analysis of purified N-terminal His-tagged GCUF1-OXA-28 and OXA-28 from <i>E. coli</i>. Right pane: Insertion of <i>gcuF1</i> reduces the amount of processed periplasmic OXA-28. SDS-PAGE analysis of periplasmic extracts (10 µg of total proteins) from reference strain <i>P. aeruginosa</i> PA14Δ<i>ampC</i> producing C-terminal His-tagged GCUF1-OXA-28 and OXA-28 (relative band intensities are indicated under the bands). Molecular weights are indicated in kDa.</p