Synergistic Activity of Colistin in Combination with Clofoctol against Colistin Resistant Gram-Negative Pathogens.

Colistin is a bactericidal antibiotic identified decades ago which is active against a number of Gram-negative pathogens. After early elimination from clinical use due to toxicity issues, colistin has been reintroduced as a last-resort treatment for antibiotic-resistant Gram-negative infections lacking other therapeutic options. Inevitably, colistin resistance has emerged among clinical isolates, making the development of colistin adjuvants extremely beneficial. Clofoctol is a synthetic antibiotic active against Gram-positive bacteria, with low toxicity and high tropism for the airways. Interestingly, clofoctol has been found to have multiple biological activities and has been proposed for the treatment of several obstructive lung diseases, including asthma, lung cancer, and SARS-CoV-2 infection. In this study, the activity of clofoctol as a colistin adjuvant was investigated in Gram-negative lung pathogens that are critical for the high prevalence of multidrug-resistant isolates, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. Clofoctol potentiated the bactericidal effect of colistin in all tested strains and reduced colistin MICs below the susceptibility breakpoint in nearly all colistin-resistant strains. Overall, this observation supports the development of inhaled clofoctol-colistin formulations for the treatment of difficult-to-treat airway infections caused by Gram-negative pathogens. IMPORTANCE Colistin is used as a last-resort antibiotic against extensively drug-resistant Gram-negative pathogens. However, colistin resistance is on the rise. Clofoctol is an antibiotic used against Gram-positive bacteria, with low toxicity and high penetration and storage in the airways. Here, a strong synergistic activity of the colistin-clofoctol combination against colistin-resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii isolates is reported, supporting the development of clofoctol-colistin formulations for the therapy of difficult-to-treat airways infections caused by these Gram-negative pathogens.

The Pseudomonas aeruginosa DksA1 protein is involved in H2O2 tolerance and within-macrophages survival and can be replaced by DksA2.

In Gram-negative pathogens, the stringent response regulator DksA controls the expression of hundreds of genes, including virulence-related genes. Interestingly, Pseudomonas aeruginosa has two functional DksA paralogs: DksA1 is constitutively expressed and has a zinc-finger motif, while DksA2 is expressed only under zinc starvation conditions and does not contain zinc. DksA1 stimulates the production of virulence factors in vitro and is required for full pathogenicity in vivo. DksA2 can replace these DksA1 functions. Here, the role of dksA paralogs in P. aeruginosa tolerance to H2O2-induced oxidative stress has been investigated. The P. aeruginosa dksA1 dksA2 mutant showed impaired H2O2 tolerance in planktonic and biofilm-growing cultures and increased susceptibility to macrophages-mediated killing compared to the wild type. Complementation with either dksA1 or dksA2 genes restored the wild type phenotypes. The DksA-dependent tolerance to oxidative stress involves, at least in part, the positive transcriptional control of both katA and katE catalase-encoding genes. These data support the hypothesis that DksA1 and DksA2 are eco-paralogs with indistinguishable function but optimal activity under different environmental conditions, and highlight their mutual contribution to P. aeruginosa virulence.

PqsE Expands and Differentially Modulates the RhlR Quorum Sensing Regulon in Pseudomonas aeruginosa.

In the opportunistic pathogen Pseudomonas aeruginosa, many virulence traits are finely regulated by quorum sensing (QS), an intercellular communication system that allows the cells of a population to coordinate gene expression in response to cell density. The key aspects underlying the functionality of the complex regulatory network governing QS in P. aeruginosa are still poorly understood, including the interplay between the effector protein PqsE and the transcriptional regulator RhlR in controlling the QS regulon. Different studies have focused on the characterization of PqsE- and RhlR-controlled genes in genetic backgrounds in which RhlR activity can be modulated by PqsE and pqsE expression is controlled by RhlR, thus hampering identification of the distinct regulons controlled by PqsE and RhlR. In this study, a P. aeruginosa PAO1 mutant strain with deletion of multiple QS elements and inducible expression of pqsE and/or rhlR was generated and validated. Transcriptomic analyses performed on this genetic background allowed us to unambiguously define the regulons controlled by PqsE and RhlR when produced alone or in combination. Transcriptomic data were validated via reverse transcription-quantitative PCR (RT-qPCR) and transcriptional fusions. Overall, our results showed that PqsE has a negligible effect on the P. aeruginosa transcriptome in the absence of RhlR, and that multiple RhlR subregulons exist with distinct dependency on PqsE. Overall, this study contributes to untangling the regulatory link between the pqs and rhl QS systems mediated by PqsE and RhlR and clarifying the impact of these QS elements on the P. aeruginosa transcriptome. IMPORTANCE The ability of Pseudomonas aeruginosa to cause difficult-to-treat infections relies on its capacity to fine-tune the expression of multiple virulence traits via the las, rhl, and pqs QS systems. Both the pqs effector protein PqsE and the rhl transcriptional regulator RhlR are required for full production of key virulence factors in vitro and pathogenicity in vivo. While it is known that PqsE can stimulate the ability of RhlR to control some virulence factors, no data are available to allow clear discrimination of the PqsE and RhlR regulons. The data produced in this study demonstrate that PqsE mainly impacts the P. aeruginosa transcriptome via an RhlR-dependent pathway and splits the RhlR regulon into PqsE-dependent and PqsE-independent subregulons. Besides contributing to untangling of the complex QS network of P. aeruginosa, our data confirm that both PqsE and RhlR are suitable targets for the development of antivirulence drugs.

In vitro Activity of Antivirulence Drugs Targeting the las or pqs Quorum Sensing Against Cystic Fibrosis Pseudomonas aeruginosa Isolates.

The chronic lung infection caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Antivirulence drugs targeting P. aeruginosa quorum sensing (QS) systems are intensively studied as antibiotics substitutes or adjuvants. Previous studies, carried out in non-CF P. aeruginosa reference strains, showed that the old drugs niclosamide and clofoctol could be successfully repurposed as antivirulence drugs targeting the las and pqs QS systems, respectively. However, frequent emergence of QS-defective mutants in the CF lung undermines the use of QS inhibitors in CF therapy. Here, QS signal production and susceptibility to niclosamide and clofoctol have been investigated in 100 P. aeruginosa CF isolates, with the aim of broadening current knowledge on the potential of anti-QS compounds in CF therapy. Results showed that 85, 78, and 69% of the CF isolates from our collection were proficient for the pqs, rhl, and las QS systems, respectively. The ability of both niclosamide and clofoctol to inhibit QS and virulence in vitro was highly variable and strain-dependent. Niclosamide showed an overall low range of activity and its negative effect on las signal production did not correlate with a decreased production of virulence factors. On the other hand, clofoctol displayed a broader QS inhibitory effect in CF isolates, with consequent reduction of the pqs-controlled virulence factor pyocyanin. Overall, this study highlights the importance of testing new antivirulence drugs against large panels of P. aeruginosa CF clinical isolates before proceeding to further pre-clinical studies and corroborates previous evidence that strains naturally resistant to QS inhibitors occur among CF isolates. However, it is also shown that resistance to pqs inhibitors is less frequent than resistance to las inhibitors, thus supporting the development of pqs inhibitors for antivirulence therapy in CF.

The two Pseudomonas aeruginosa DksA stringent response proteins are largely interchangeable at the whole transcriptome level and in the control of virulence-related traits.

The stringent response regulator DksA plays a key role in Gram negative bacteria adaptation to challenging environments. Intriguingly, the plant and human pathogen Pseudomonas aeruginosa is unique as it expresses two functional DksA paralogs: DksA1 and DksA2. However, the role of DksA2 in P. aeruginosa adaptive strategies has been poorly investigated so far. Here, RNA-Seq analysis and phenotypic assays showed that P. aeruginosa DksA1 and DksA2 proteins are largely interchangeable. Relative to wild type P. aeruginosa, transcription of 1779 genes was altered in a dksA1 dksA2 double mutant, and the wild type expression level of ≥90% of these genes was restored by in trans complementation with either dksA1 or dksA2. Interestingly, the expression of a small sub-set of genes seems to be preferentially or exclusively complemented by either dksA1 or dksA2. In addition, evidence has been provided that the DksA-dependent regulation of virulence genes expression is independent and hierarchically dominant over two major P. aeruginosa regulatory circuits, i.e., quorum sensing and cyclic-di-GMP signalling systems. Our findings support the prominent role of both DksA paralogs in P. aeruginosa environmental adaptation.