The conjugation of siderophores to antimicrobial molecules is an attractive strategy to overcome the low outer membrane permeability of Gram-negative bacteria. In this Trojan horse approach, the transport of drug conjugates is redirected via TonB-dependent receptors (TBDR), which are involved in the uptake of essential nutrients, including iron. Previous reports have demonstrated the involvement of the TBDRs PiuA and PirA from Pseudomonas aeruginosa and their orthologues in Acinetobacter baumannii in the uptake of siderophore-beta-lactam drug conjugates. By in silico screening, we further identified a PiuA orthologue, termed PiuD, present in clinical isolates, including strain LESB58. The piuD gene in LESB58 is located at the same genetic locus as piuA in strain PAO1. PiuD has a similar crystal structure as PiuA and is involved in the transport of the siderophore-drug conjugates BAL30072, MC-1, and cefiderocol in strain LESB58. To screen for additional siderophore-drug uptake systems, we overexpressed 28 of the 34 TBDRs of strain PAO1 and identified PfuA, OptE, OptJ, and the pyochelin receptor FptA as novel TBDRs conferring increased susceptibility to siderophore-drug conjugates. The existence of a TBDR repertoire in P. aeruginosa able to transport siderophore-drug molecules potentially decreases the likelihood of resistance emergence during therapy.

The outer membrane of Gram-negative bacteria presents an efficient barrier to the permeation of antimicrobial molecules. One strategy pursued to circumvent this obstacle is to hijack transport systems for essential nutrients, such as iron. BAL30072 and MC-1 are two monobactams conjugated to a dihydroxypyridone siderophore that are active against Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we investigated the mechanism of action of these molecules in A. baumannii. We identified two novel TonB-dependent receptors, termed Ab-PiuA and Ab-PirA, that are required for the antimicrobial activity of both agents. Deletion of either piuA or pirA in A. baumannii resulted in 4-to 8-fold-decreased susceptibility, while their overexpression in the heterologous host P. aeruginosa increased susceptibility to the two siderophore-drug conjugates by 4-to 32-fold. The crystal structures of PiuA and PirA from A. baumannii and their orthologues from P. aeruginosa were determined. The structures revealed similar architectures; however, structural differences between PirA and PiuA point to potential differences between their cognate siderophore ligands. Spontaneous mutants, selected upon exposure to BAL30072, harbored frameshift mutations in either the ExbD3 or the TonB3 protein of A. baumannii, forming the cytoplasmic-membrane complex providing the energy for the siderophore translocation process. The results of this study provide insight for the rational design of novel siderophore-drug conjugates against problematic Gramnegative pathogens.

Detrimental and beneficial interactions between co-colonizing bacteria may influence the course of infections. In cystic fibrosis (CF) airways, Staphylococcus aureus prevails in childhood, whereas Pseudomonas aeruginosa progressively predominates thereafter. While a range of interactions has been identified, it is unclear if these represent specific adaptations or correlated responses to other aspects of the environment. Here, we investigate how P. aeruginosa adapts to S. aureus by evolving P. aeruginosa in the presence and absence of S. aureus. P. aeruginosa populations that evolved for 150 generations were sequenced and compared to the ancestor strain. Mutations in the Wsp signaling system were identified in both treatments and likely occurred because of low oxygen availability. Despite showing increased killing activity, wsp mutants were less fit in the presence of S. aureus. In contrast, mutations in lipopolysaccharide (LPS) biosynthesis occurred exclusively in co-cultures with S. aureus and conferred a fitness gain in its presence. Moreover, they increased resistance towards beta-lactam antibiotics. Strikingly, both mutations in wsp and LPS genes are observed in clinical isolates from CF-patients. Our results suggest that P. aeruginosa LPS mutations are a direct consequence of S. aureus imposed selection in vitro.

BackgroundCo-colonization by Pseudomonas aeruginosa and Staphylococcus aureus is frequent in cystic fibrosis patients. Polymicrobial infections involve both detrimental and beneficial interactions between different bacterial species. Such interactions potentially indirectly impact the human host through virulence, antibiosis and immunomodulation.ResultsHere we explored the responses triggered by the encounter of these two pathogens to identify early processes that are important for survival when facing a potential competitor. Transcriptional profiles of both bacteria were obtained after 3 h co-culture and compared to the respective mono-culture using RNAseq. Global responses in both bacteria included competition for nitrogen sources, amino acids and increased tRNA levels. Both organisms also induced lysogenic mechanisms related to prophage induction (S. aureus) and R- and F- pyocin synthesis (P. aeruginosa), possibly as a response to stress resulting from nutrient limitation or cell damage. Specific responses in S. aureus included increased expression of de novo and salvation pathways for purine and pyrimidine synthesis, a switch to glucose fermentation, and decreased expression of major virulence factors and global regulators.ConclusionsTaken together, transcriptomic data indicate that early responses between P. aeruginosa and S. aureus involve competition for resources and metabolic adaptations, rather than the expression of bacteria- or host-directed virulence factors.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5398-y) contains supplementary material, which is available to authorized users.

We recently reported the discovery of antimicrobial peptide dendrimers (AMPDs) acting by a membrane‐disruptive mechanism against multidrug resistant pathogenic bacteria. Here, we combined amino acid sequence elements from different AMPDs with different activity profiles to form AMPD chimeras. By joining the outer branches of TNS18, an AMPD active against Pseudomonas aeruginosa, Acinetobacter baumannii and methicillin resistant Staphylococcus aureus, with the core of T7, another AMPD active against P. aeruginosa, A. baumannii and Klebsiella pneumoniae, we obtained AMPD chimera DC5 displaying all previously observed activities while retaining a similar mechanism of action. These experiments show that chimera design represents a useful strategy to improve the properties of AMPDs.

Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa. However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL (MW 4,531.38 Da, 15 positive charges, MIC = 8 mg/liter) showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14. Spontaneous mutants selected on Pmx-B, harboring loss of function mutations in the PhoQ sensor kinase gene, showed increased Pmx-B MICs and arnB operon expression (4-amino-l-arabinose addition to lipid A), but remained susceptible to dendrimers. Two mutants carrying a missense mutation in the periplasmic loop of the PmrB sensor kinase showed increased MICs for Pmx-B (8-fold) and G3KL (4-fold) but not for the dendrimer T7 (MW 4,885.64 Da, 16 positive charges, MIC = 8 mg/liter). The pmrB mutants showed increased expression of the arnB operon as well as of the speD2-speE2-PA4775 operon, located upstream of pmrAB, and involved in polyamine biosynthesis. Exogenous supplementation with the polyamines spermine and norspermine increased G3KL and T7 MICs in a phoQ mutant background but not in the PA14 wild type. This suggests that both addition of 4-amino-l-arabinose and secretion of polyamines are required to reduce susceptibility to dendrimers, probably neutralizing the negative charges present on the lipid A and the 2-keto-3-deoxyoctulosonic acid (KDO) sugars of the lipopolysaccharide (LPS), respectively. We further show by transcriptome analysis that the dendrimers G3KL and T7 induce adaptive responses through the CprRS two-component system in PA14.

A new family of cyclic antimicrobial peptides is reported targeting multidrug resistant Pseudomonas aeruginosa by membrane disruption.

With the increase of infections due to multidrug resistant bacterial pathogens and the shortage of antimicrobial molecules with novel targets, interest in bacteriophages as a therapeutic option has regained much attraction. Before the launch of future clinical trials, in vitro studies are required to better evaluate the efficacies and potential pitfalls of such therapies. Here we studied in an ex vivo human airway epithelial cell line model the efficacy of phage and ciprofloxacin alone and in combination to treat infection by Pseudomonas aeruginosa. The Calu-3 cell line and the isogenic CFTR knock down cell line (cftr-) infected apically with P. aeruginosa strain PAO1 showed a progressive reduction in transepithelial resistance during 24 h. Administration at 6 h p.i. of single phage, phage cocktails or ciprofloxacin alone prevented epithelial layer destruction at 24 h p.i. Bacterial regrowth, due to phage resistant mutants harboring mutations in LPS synthesis genes, occurred thereafter both in vitro and ex vivo. However, coadministration of two phages combined with ciprofloxacin efficiently prevented PAO1 regrowth and maintained epithelial cell integrity at 72 p.i. The phage/ciprofloxacin treatment did not induce an inflammatory response in the tested cell lines as determined by nanoString R gene expression analysis. We conclude that combination of phage and ciprofloxacin efficiently protects wild type and cftr-epithelial cells from infection by P. aeruginosa and emergence of phage resistant mutants without inducing an inflammatory response. Hence, phage-antibiotic combination should be a safe and promising anti-Pseudomonas therapy for future clinical trials potentially including cystic fibrosis patients.