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.
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.
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.
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
and their orthologues in
in the uptake of siderophore-beta-lactam drug conjugates. By
screening, we further identified a PiuA orthologue, termed PiuD, present in clinical isolates, including strain LESB58. The
gene in LESB58 is located at the same genetic locus as
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
able to transport siderophore-drug molecules potentially decreases the likelihood of resistance emergence during therapy.
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.
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.
Machine learning (ML) consists of the recognition of patterns from training data and offers the opportunity to exploit large structure-activity databases for drug design. In the area of peptide ...drugs, ML is mostly being tested to design antimicrobial peptides (AMPs), a class of biomolecules potentially useful to fight multidrug-resistant bacteria. ML models have successfully identified membrane disruptive amphiphilic AMPs, however mostly without addressing the associated toxicity to human red blood cells. Here we trained recurrent neural networks (RNN) with data from DBAASP (Database of Antimicrobial Activity and Structure of Peptides) to design short non-hemolytic AMPs. Synthesis and testing of 28 generated peptides, each at least 5 mutations away from training data, allowed us to identify eight new non-hemolytic AMPs against
Pseudomonas aeruginosa
,
Acinetobacter baumannii
, and methicillin-resistant
Staphylococcus aureus
(MRSA). These results show that machine learning (ML) can be used to design new non-hemolytic AMPs.
Machine learning models trained with experimental data for antimicrobial activity and hemolysis are shown to produce new non-hemolytic antimicrobial peptides active against multidrug-resistant bacteria.
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
and
Here, we investigated the mechanism of action of these molecules in
We identified two novel TonB-dependent receptors, termed
-PiuA and
-PirA, that are required for the antimicrobial activity of both agents. Deletion of either
or
in
resulted in 4- to 8-fold-decreased susceptibility, while their overexpression in the heterologous host
increased susceptibility to the two siderophore-drug conjugates by 4- to 32-fold. The crystal structures of PiuA and PirA from
and their orthologues from
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
, 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.
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.
Multidrug‐resistant bacteria, such as Pseudomonas aeruginosa and Acinetobacter baumannii, are a major public health threat. By combining topology and amino acid sequence design, new antimicrobial peptide dendrimers with high activity against these pathogens were found, which represents a new and general approach for the discovery of antimicrobial agents.
Bacteria communicate and cooperate to perform a wide range of social behaviors including production of extracellular products (public goods) that are crucial for growth and virulence. Their ...expression may be switched on by the detection of threshold densities of diffusible signals Quorum-Sensing (QS). Studies using the opportunistic pathogen Pseudomonas aeruginosa suggest that QS "cheats"--individuals that don't respond to the QS signal, but are still able to use public goods produced by others--have a selective advantage in the presence of QS cooperators. It is, however, unclear whether this type of social exploitation is relevant in clinical contexts. Here, we report the evolutionary dynamics and virulence of P. aeruginosa populations during lung colonization of mechanically ventilated patients in the absence of antimicrobial treatments. We observed a large diversity of QS phenotypes among initial colonizing isolates. This diversity decreased over a matter of days, concomitant with a gradual increase in the proportion of QS cheating mutants (lasR mutants), which were found in 80% of the patients after 9 days of colonization. These mutants often evolved from initial wild-type genotypes. The fitness advantage of the lasR mutants is almost certainly due to social exploitation, because this advantage was only apparent in the presence of QS wild-type cells. Crucially, ventilator-associated pneumonia occurred significantly earlier in patients predominantly colonized by QS wild-type populations, highlighting the importance of QS in this clinical situation. These results demonstrate that social interactions can shape the short-term evolution and virulence of bacterial pathogens in humans, providing novel opportunities for therapy.
Microbial populations which are resistant to antibiotics are an emerging environmental concern with potentially serious implications for public health. Thus, there is a growing concern in exploring ...the occurrence of antibiotic resistance in the environment with no limitations to the factors that contribute to their emergence. The aquatic environment is considered to be a hot-spot for the acquisition and spread of antibiotic resistance due to pollution with emerging contaminants derived from anthropogenic activities. In this study, we report on the isolation and characterization of 141 Pseudomonas spp. from aquatic sediments receiving partially (un)treated hospital and communal effluents from three distinct geographical locations: Democratic Republic of the Congo (DRC), India (IN), and Switzerland (CH). P. putida (42%) and P. aeruginosa (39%) were the dominant Pseudomonas species. The highest frequency of antibiotic resistance against eight anti-pseudomonas agents was found among IN isolates (35–60%), followed by DRC (18–50%) and CH (12–54%). CTX-M was the most frequent β-lactamase found in CH (47% of isolates), while VIM-1 was dominant in isolates from DRC (61%) and IN (29%). NDM-1 was found in 29% of the total IN isolates and surprisingly also in 6% of CH isolates. Chromosomally-encoded efflux mechanisms were overexpressed in P. aeruginosa isolates from all three geographic locations. In vitro conjugative transfers of antibiotic resistance plasmids occurred more frequently under tropical temperatures (30 and 37 °C) than under temperate conditions (10 °C). The presence of Extended Spectrum β-lactamases (ESBLs) and Metallo β-lactamases (MBLs) in the isolates from environmental samples has important implications for humans who depend on public water supply and sanitation facilities. To our knowledge, this is the first study to demonstrate a comparison between treated/untreated effluents from urban and hospital settings as a source of microbial resistance by evaluating the aquatic ecosystems sediments from tropical and temperate climate conditions. Taken together, our findings demonstrate a widespread occurrence of antibiotic resistance in aquatic ecosystems sediments receiving untreated/treated wastewater and how these contemporary sources of contamination, contribute to the spread of microbial resistance in the aquatic environment. This research presents also useful tools to evaluate sediment quality in the receiving river/reservoir systems which can be applied to similar environments.
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•Prevalence of MDR Pseudomonas spp., from sediments receiving (un)treated effluents.•Wide spread of ESBLs and MBLs in communal settings – result of co-selection.•Tropical climate may favor the exchange of genetic material among bacteria.•Need for the requirement of surveillance of resistance in the aquatic ecosystem.
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