Susceptibility profiles and resistance genomics of Pseudomonas aeruginosa isolates from European ICUs participating in the ASPIRE-ICU trial.

OBJECTIVES: To determine the susceptibility profiles and the resistome of Pseudomonas aeruginosa isolates from European ICUs during a prospective cohort study (ASPIRE-ICU). METHODS: 723 isolates from respiratory samples or perianal swabs of 402 patients from 29 sites in 11 countries were studied. MICs of 12 antibiotics were determined by broth microdilution. Horizontally acquired ß-lactamases were analysed through phenotypic and genetic assays. The first respiratory isolates from 105 patients providing such samples were analysed through WGS, including the analysis of the resistome and a previously defined genotypic resistance score. Spontaneous mutant frequencies and the genetic basis of hypermutation were assessed. RESULTS: All agents except colistin showed resistance rates above 20%, including ceftolozane/tazobactam and ceftazidime/avibactam. 24.9% of the isolates were XDR, with a wide intercountry variation (0%-62.5%). 13.2% of the isolates were classified as DTR (difficult-to-treat resistance). 21.4% of the isolates produced ESBLs (mostly PER-1) or carbapenemases (mostly NDM-1, VIM-1/2 and GES-5). WGS showed that these determinants were linked to high-risk clones (particularly ST235 and ST654). WGS revealed a wide repertoire of mutation-driven resistance mechanisms, with multiple lineage-specific mutations. The most frequently mutated genes were gyrA, parC, oprD, mexZ, nalD and parS, but only two of the isolates were hypermutable. Finally, a good accuracy of the genotypic score to predict susceptibility (91%-100%) and resistance (94%-100%) was documented. CONCLUSIONS: An overall high prevalence of resistance is documented European ICUs, but with a wide intercountry variability determined by the dissemination of XDR high-risk clones, arguing for the need to reinforce infection control measures.

Antimicrobial Peptide Dendrimers and Quorum-Sensing Inhibitors in Formulating Next-Generation Anti-Infection Cell Therapy Dressings for Burns.

Multidrug resistance infections are the main cause of failure in the pro-regenerative cell-mediated therapy of burn wounds. The collagen-based matrices for delivery of cells could be potential substrates to support bacterial growth and subsequent lysis of the collagen leading to a cell therapy loss. In this article, we report the development of a new generation of cell therapy formulations with the capacity to resist infections through the bactericidal effect of antimicrobial peptide dendrimers and the anti-virulence effect of anti-quorum sensing MvfR (PqsR) system compounds, which are incorporated into their formulation. Anti-quorum sensing compounds limit the pathogenicity and antibiotic tolerance of pathogenic bacteria involved in the burn wound infections, by inhibiting their virulence pathways. For the first time, we report a biological cell therapy dressing incorporating live progenitor cells, antimicrobial peptide dendrimers, and anti-MvfR compounds, which exhibit bactericidal and anti-virulence properties without compromising the viability of the progenitor cells.

Adaptive and Mutational Responses to Peptide Dendrimer Antimicrobials in Pseudomonas aeruginosa.

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.

Surface sensing triggers a broad-spectrum antimicrobial response in Pseudomonas aeruginosa.

Interspecies bacterial competition may occur via cell-associated or secreted determinants and is key to successful niche colonization. We previously evolved Pseudomonas aeruginosa in the presence of Staphylococcus aureus and identified mutations in the Wsp surface-sensing signalling system. Surprisingly, a ΔwspF mutant, characterized by increased c-di-GMP levels and biofilm formation capacity, showed potent killing activity towards S. aureus in its culture supernatant. Here, we used an unbiased metabolomic analysis of culture supernatants to identify rhamnolipids, alkyl quinoline N-oxides and two siderophores as members of four chemical clusters, which were more abundant in the ΔwspF mutant supernatants. Killing activities were quorum-sensing controlled but independent of c-di-GMP levels. Based on the metabolomic analysis, we formulated a synthetic cocktail of four compounds, showing broad-spectrum anti-bacterial killing, including both Gram-positive and Gram-negative bacteria. The combination of quorum-sensing-controlled killing and Wsp-system mediated biofilm formation endows P. aeruginosa with capacities essential for niche establishment and host colonization.

Synergistic Effect of Propidium Iodide and Small Molecule Antibiotics with the Antimicrobial Peptide Dendrimer G3KL against Gram-Negative Bacteria.

There is an urgent need to develop new antibiotics against multidrug-resistant bacteria. Many antimicrobial peptides (AMPs) are active against such bacteria and often act by destabilizing membranes, a mechanism that can also be used to permeabilize bacteria to other antibiotics, resulting in synergistic effects. We recently showed that G3KL, an AMP with a multibranched dendritic topology of the peptide chain, permeabilizes the inner and outer membranes of Gram-negative bacteria including multidrug-resistant strains, leading to efficient bacterial killing. Here, we show that permeabilization of the outer and inner membranes of Pseudomonas aeruginosa by G3KL, initially detected using the DNA-binding fluorogenic dye propidium iodide (PI), also leads to a synergistic effect between G3KL and PI in this bacterium. We also identify a synergistic effect between G3KL and six different antibiotics against the Gram-negative Klebsiella pneumoniae, against which G3KL is inactive.

Transcriptional profiling of Pseudomonas aeruginosa and Staphylococcus aureus during in vitro co-culture.

BACKGROUND: Co-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. RESULTS: Here 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. CONCLUSIONS: Taken 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.

Lipidated Peptide Dendrimers Killing Multidrug-Resistant Bacteria.

New antibiotics are urgently needed to address multidrug-resistant (MDR) bacteria. Herein we report that second-generation (G2) peptide dendrimers bearing a fatty acid chain at the dendrimer core efficiently kill Gram-negative bacteria including Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most problematic MDR bacteria worldwide. Our most active dendrimer TNS18 is also active against Gram-positive methicillin-resistant Staphylococcus aureus. Based on circular dichroism and molecular dynamics studies, we hypothesize that TNS18 adopts a hydrophobically collapsed conformation in water with the fatty acid chain backfolded onto the peptide dendrimer branches and that the dendrimer unfolds in contact with the membrane to expose its lipid chain and hydrophobic residues, thereby facilitating membrane disruption leading to rapid bacterial cell death. Dendrimer TNS18 shows promising in vivo activity against MDR clinical isolates of A. baumannii and Escherichia coli, suggesting that lipidated peptide dendrimers might become a new class of antibacterial agents.

TonB-Dependent Receptor Repertoire of Pseudomonas aeruginosa for Uptake of Siderophore-Drug Conjugates.

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.

Optimizing Antimicrobial Peptide Dendrimers in Chemical Space.

We used nearest-neighbor searches in chemical space to improve the activity of the antimicrobial peptide dendrimer (AMPD) G3KL and identified dendrimer T7, which has an expanded activity range against Gram-negative pathogenic bacteria including Klebsiellae pneumoniae, increased serum stability, and promising activity in an in vivo infection model against a multidrug-resistant strain of Acinetobacter baumannii. Imaging, spectroscopic studies, and a structural model from molecular dynamics simulations suggest that T7 acts through membrane disruption. These experiments provide the first example of using virtual screening in the field of dendrimers and show that dendrimer size does not limit the activity of AMPDs.

Structure and Function of the PiuA and PirA Siderophore-Drug Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii.

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 Gram-negative pathogens.

Pyomelanin-producing Pseudomonas aeruginosa selected during chronic infections have a large chromosomal deletion which confers resistance to pyocins.

When bacterial lineages make the transition from free-living to permanent association with hosts, they can undergo massive gene losses, for which the selective forces within host tissues are unknown. We identified here melanogenic clinical isolates of Pseudomonas aeruginosa with large chromosomal deletions (66 to 270 kbp) and characterized them to investigate how they were selected. When compared with their wild-type parents, melanogenic mutants (i) exhibited a lower fitness in growth conditions found in human tissues, such as hyperosmolarity and presence of aminoglycoside antibiotics, (ii) narrowed their metabolic spectrum with a growth disadvantage with particular carbon sources, including aromatic amino acids and acyclic terpenes, suggesting a reduction of metabolic flexibility. Despite an impaired fitness in rich media, melanogenic mutants can inhibit their wild-type parents and compete with them in coculture. Surprisingly, melanogenic mutants became highly resistant to two intraspecific toxins, the S-pyocins AP41 and S1. Our results suggest that pyocins produced within a population of infecting P. aeruginosa may have selected for bacterial mutants that underwent massive gene losses and that were adapted to the life in diverse bacterial communities in the human host. Intraspecific interactions may therefore be an important factor driving the continuing evolution of pathogens during host infections.

Pseudomonas aeruginosa-induced apoptosis in airway epithelial cells is mediated by gap junctional communication in a JNK-dependent manner.

Chronic infection and inflammation of the airways is a hallmark of cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The response of the CF airway epithelium to the opportunistic pathogen Pseudomonas aeruginosa is characterized by altered inflammation and apoptosis. In this study, we examined innate immune recognition and epithelial responses at the level of the gap junction protein connexin43 (Cx43) in polarized human airway epithelial cells upon infection by PAO1. We report that PAO1 activates cell surface receptors to elicit an intracellular signaling cascade leading to enhancement of gap junctional communication. Expression of Cx43 involved an opposite regulation exerted by JNK and p38 MAPKs. PAO1-induced apoptosis was increased in the presence of a JNK inhibitor, but latter effect was prevented by lentiviral expression of a Cx43-specific short hairpin RNA. Moreover, we found that JNK activity was upregulated by pharmacological inhibition of CFTR in Calu-3 cells, whereas correction of a CF airway cell line (CF15 cells) by adenoviral expression of CFTR reduced the activation of this MAPK. Interestingly, CFTR inhibition in Calu-3 cells was associated with decreased Cx43 expression and reduced apoptosis. These results indicate that Cx43 expression is a component of the response of airway epithelial cells to innate immune activation by regulating the survival/apoptosis balance. Defective CFTR could alter this equilibrium with deleterious consequences on the CF epithelial response to P. aeruginosa.

The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics.

UNLABELLED: Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. IMPORTANCE: One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the environment. These uptake systems could also be exploited by a Trojan horse strategy to facilitate the transport of antibiotics into bacterial cells. Several natural antibiotics mimic substrates of peptide uptake routes. In this study, we analyzed an ABC transporter involved in the uptake of nucleoside peptidyl antibiotics. Our data might help to design drug conjugates that may hijack this uptake system to gain access to cells.

Airway Epithelial Cell Integrity Protects from Cytotoxicity of Pseudomonas aeruginosa Quorum-Sensing Signals.

Cell-to-cell communication via gap junctions regulates airway epithelial cell homeostasis and maintains the epithelium host defense. Quorum-sensing molecules produced by Pseudomonas aeruginosa coordinate the expression of virulence factors by this respiratory pathogen. These bacterial signals may also incidentally modulate mammalian airway epithelial cell responses to the pathogen, a process called interkingdom signaling. We investigated the interactions between the P. aeruginosa N-3-oxo-dodecanoyl-L-homoserine lactone (C12) quorum-sensing molecule and human airway epithelial cell gap junctional intercellular communication (GJIC). C12 degradation and its effects on cells were monitored in various airway epithelial cell models grown under nonpolarized and polarized conditions. Its concentration was further monitored in daily tracheal aspirates of colonized intubated patients. C12 rapidly altered epithelial integrity and decreased GJIC in nonpolarized airway epithelial cells, whereas other quorum-sensing molecules had no effect. The effects of C12 were dependent on [Ca(2+)]i and could be prevented by inhibitors of Src tyrosine family and Rho-associated protein kinases. In contrast, polarized airway cells grown on Transwell filters were protected from C12 except when undergoing repair after wounding. In vivo during colonization of intubated patients, C12 did not accumulate, but it paralleled bacterial densities. In vitro C12 degradation, a reaction catalyzed by intracellular paraoxonase 2 (PON2), was impaired in nonpolarized cells, whereas PON2 expression was increased during epithelial polarization. The cytotoxicity of C12 on nonpolarized epithelial cells, combined with its impaired degradation allowing its accumulation, provides an additional pathogenic mechanism for P. aeruginosa infections.

Combining topology and sequence design for the discovery of potent antimicrobial peptide dendrimers against multidrug-resistant Pseudomonas aeruginosa.

Multidrug-resistant opportunistic bacteria, such as Pseudomonas aeruginosa, represent a major public health threat. Antimicrobial peptides (AMPs) and related peptidomimetic systems offer an attractive opportunity to control these pathogens. AMP dendrimers (AMPDs) with high activity against multidrug-resistant clinical isolates of P. aeruginosa and Acinetobacter baumannii were now identified by a systematic survey of the peptide sequences within the branches of a distinct type of third-generation peptide dendrimers. Combined topology and peptide sequence design as illustrated here represents a new and general strategy to discover new antimicrobial agents to fight multidrug-resistant bacterial pathogens.

Involvement of Fe uptake systems and AmpC ß-lactamase in susceptibility to the siderophore monosulfactam BAL30072 in Pseudomonas aeruginosa.

BAL30072 is a monosulfactam conjugated with an iron-chelating dihydroxypyridone moiety. It is active against Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa. We selected mutants with decreased susceptibilities to BAL30072 in P. aeruginosa PAO1 under a variety of conditions. Under iron-deficient conditions, mutants with overexpression of AmpC ß-lactamase predominated. These mutants were cross-resistant to aztreonam and ceftazidime. Similar mutants were obtained after selection at >16× the MIC in iron-sufficient conditions. At 4× to 8× the MIC, mutants with elevated MIC for BAL30072 but unchanged MICs for aztreonam or ciprofloxacin were selected. The expression of ampC and the major efflux pump genes were also unchanged. These BAL30072-specific mutants were characterized by transcriptome analysis, which revealed upregulation of the Fe-dicitrate operon, FecIRA. Whole-genome sequencing showed that this resulted from a single nucleotide change in the Fur-box of the fecI promoter. Overexpression of either the FecI ECF sigma factor or the FecA receptor increased BAL30072 MICs 8- to 16-fold. A fecI mutant and a fecA mutant of PAO1 were hypersusceptible to BAL30072 (MICs < 0.06 µg/ml). The most downregulated gene belonged to the pyochelin synthesis operon, although mutants in pyochelin receptor or synthesis genes had unchanged MICs. The piuC gene, coding for a Fe(II)-dependent dioxygenase located next to the piuA iron receptor gene, was also downregulated. The MICs of BAL30072 for piuC and piuA transposon mutants were increased 8- and 16-fold, respectively. We conclude that the upregulation of the Fe-dicitrate system impacts the expression of other TonB-dependent iron transporters and that PiuA and PiuC contribute to the susceptibility of P. aeruginosa PAO1 to BAL30072.

Targeting HuR-Vav3 mRNA interaction prevents Pseudomonas aeruginosa adhesion to the cystic fibrosis airway epithelium.

Cystic fibrosis (CF) is characterized by chronic bacterial infections leading to progressive bronchiectasis and respiratory failure. Pseudomonas aeruginosa (Pa) is the predominant opportunistic pathogen infecting the CF airways. The guanine nucleotide exchange factor Vav3 plays a critical role in Pa adhesion to the CF airways by inducing luminal fibronectin deposition that favors bacteria trapping. Here we report that Vav3 overexpression in CF is caused by upregulation of the mRNA-stabilizing protein HuR. We found that HuR accumulates in the cytoplasm of CF airway epithelial cells and that it binds to and stabilizes Vav3 mRNA. Interestingly, disruption of the HuR-Vav3 mRNA interaction improved the CF epithelial integrity, inhibited the formation of the fibronectin-made bacterial docking platforms, and prevented Pa adhesion to the CF airway epithelium. These findings indicate that targeting HuR represents a promising antiadhesive approach in CF that can prevent initial stages of Pa infection in a context of emergence of multidrug-resistant pathogens.

Antimicrobial Peptide-Peptoid Hybrids with and without Membrane Disruption.

Among synthetic analogues of antimicrobial peptides (AMPs) under investigation to address antimicrobial resistance, peptoids (N-alkylated oligoglycines) have been reported to act both by membrane disruption and on intracellular targets. Here we gradually introduced peptoid units into the membrane-disruptive undecapeptide KKLLKLLKLLL to test a possible transition toward intracellular targeting. We found that selected hybrids containing up to five peptoid units retained the parent AMP's α-helical folding, membrane disruption, and antimicrobial effects against Gram-negative bacteria including multidrug-resistant (MDR) strains of Pseudomonas aeruginosa and Klebsiella pneumoniae while showing reduced hemolysis and cell toxicities. Furthermore, some hybrids containing as few as three peptoid units as well as the full peptoid lost folding, membrane disruption, hemolysis, and cytotoxicity but displayed strong antibacterial activity under dilute medium conditions typical for proline-rich antimicrobial peptides (PrAMPs), pointing to intracellular targeting. These findings parallel previous reports that partially helical amphiphilic peptoids are privileged oligomers for antibiotic development.

To Fold or Not to Fold: Diastereomeric Optimization of an α-Helical Antimicrobial Peptide.

Membrane disruptive α-helical antimicrobial peptides (AMPs) offer an opportunity to address multidrug resistance; however, most AMPs are toxic and unstable in serum. These limitations can be partly overcome by introducing D-residues, which often confers protease resistance and reduces toxicity without affecting antibacterial activity, presumably due to lowered α-helicity. Here, we investigated 31 diastereomers of the α-helical AMP KKLLKLLKLLL. Three diastereomers containing two, three, and four D-residues showed increased antibacterial effects, comparable hemolysis, reduced toxicity against HEK293 cells, and excellent serum stability, while another diastereomer with four D-residues additionally displayed lower hemolysis. X-ray crystallography confirmed that high or low α-helicity as measured by circular dichroism indicated α-helical or disordered structures independently of the number of chirality switched residues. In contrast to previous reports, α-helicity across diastereomers correlated with both antibacterial activity and hemolysis and revealed a complex relationship between stereochemistry, activity, and toxicity, highlighting the potential of diastereomers for property optimization.

Personalized aerosolised bacteriophage treatment of a chronic lung infection due to multidrug-resistant Pseudomonas aeruginosa.

Bacteriophage therapy has been suggested as an alternative or complementary strategy for the treatment of multidrug resistant (MDR) bacterial infections. Here, we report the favourable clinical evolution of a 41-year-old male patient with a Kartagener syndrome complicated by a life-threatening chronic MDR Pseudomonas aeruginosa infection, who is treated successfully with iterative aerosolized phage treatments specifically directed against the patient's isolate. We follow the longitudinal evolution of both phage and bacterial loads during and after phage administration in respiratory samples. Phage titres in consecutive sputum samples indicate in patient phage replication. Phenotypic analysis and whole genome sequencing of sequential bacterial isolates reveals a clonal, but phenotypically diverse population of hypermutator strains. The MDR phenotype in the collected isolates is multifactorial and mainly due to spontaneous chromosomal mutations. All isolates recovered after phage treatment remain phage susceptible. These results demonstrate that clinically significant improvement is achievable by personalised phage therapy even in the absence of complete eradication of P. aeruginosa lung colonization.