Antivirulence Treatments Research Articles

Probing the evolutionary robustness of two repurposed drugs targeting iron uptake in Pseudomonas aeruginosa.

Lay SummaryWe probed the evolutionary robustness of two antivirulence drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine in the opportunistic pathogen Pseudomonas aeruginosa. Using an experimental evolution approach in human serum, we showed that antivirulence treatments are not evolutionarily robust per se, but vary in their propensity to select for resistance.Background and objectivesTreatments that inhibit the expression or functioning of bacterial virulence factors hold great promise to be both effective and exert weaker selection for resistance than conventional antibiotics. However, the evolutionary robustness argument, based on the idea that antivirulence treatments disarm rather than kill pathogens, is controversial. Here, we probe the evolutionary robustness of two repurposed drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine of the opportunistic human pathogen Pseudomonas aeruginosa.MethodologyWe subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an ex vivo infection model. We screened evolved populations and clones for resistance phenotypes, including the restoration of growth and pyoverdine production, and the evolution of iron uptake by-passing mechanisms. We whole-genome sequenced evolved clones to identify the genetic basis of resistance.ResultsWe found that mutants resistant against antivirulence treatments readily arose, but their selective spreading varied between treatments. Flucytosine resistance quickly spread in all populations due to disruptive mutations in upp, a gene encoding an enzyme required for flucytosine activation. Conversely, resistance against gallium arose only sporadically, and was based on mutations in transcriptional regulators, upregulating pyocyanin production, a redox-active molecule promoting siderophore-independent iron acquisition. The spread of gallium resistance was presumably hampered because pyocyanin-mediated iron delivery benefits resistant and susceptible cells alike.Conclusions and implicationsOur work highlights that antivirulence treatments are not evolutionarily robust per se. Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.

Acquisition of resistance to carbapenem and macrolide-mediated quorum sensing inhibition by Pseudomonas aeruginosa via ICETn43716385

Pseudomonas aeruginosa can cause life-threatening infections in immunocompromised patients. The first-line agents to treat P. aeruginosa infections are carbapenems. However, the emergence of carbapenem-resistant P. aeruginosa strains greatly compromised the effectiveness of carbapenem treatment, which makes the surveillance on their spreading and transmission important. Here we characterized the full-length genomes of two carbapenem-resistant P. aeruginosa clinical isolates that are capable of producing New Delhi metallo-β-lactamase-1 (NDM-1). We show that blaNDM-1 is carried by a novel integrative and conjugative element (ICE) ICETn43716385, which also carries the macrolide resistance gene msr(E) and the florfenicol resistance gene floR. By exogenously expressing msr(E) in P. aeruginosa laboratory strains, we show that Msr(E) can abolish azithromycin-mediated quorum sensing inhibition in vitro and anti-Pseudomonas effect in vivo. We conclude that ICEs are important in transmitting carbapenem resistance, and that anti-virulence treatment of P. aeruginosa infections using sub-inhibitory concentrations of macrolides can be challenged by horizontal gene transfer.

Self-Assembled Colloidal Gel Using Cell Membrane-Coated Nanosponges as Building Blocks.

Colloidal gels consisting of oppositely charged nanoparticles are increasingly utilized for drug delivery and tissue engineering. Meanwhile, cell membrane-coated nanoparticles are becoming a compelling biomimetic system for innovative therapeutics. Here, we demonstrate the successful use of cell membrane-coated nanoparticles as building blocks to formulate a colloidal gel that gelates entirely based on material self-assembly without chemical cross-linking. Specifically, we prepare red blood cell membrane-coated nanosponges and mix them with an appropriate amount of cationic nanoparticles, resulting in a spontaneously formed gel-like complex. Rheological test shows that the nanosponge colloidal gel has pronounced shear-thinning property, which makes it an injectable formulation. The gel formulation not only preserves the nanosponges' toxin neutralization capability but also greatly prolongs their retention time after subcutaneous injection into mouse tissue. When tested in a mouse model of subcutaneous group A Streptococcus infection, the nanosponge colloidal gel shows significant antibacterial efficacy by markedly reducing skin lesion development. Overall, the nanosponge colloidal gel system is promising as an injectable formulation for therapeutic applications such as antivirulence treatment for local bacterial infections.

Do Bacterial “Virulence Factors” Always Increase Virulence? A Meta-Analysis of Pyoverdine Production in Pseudomonas aeruginosa As a Test Case

Bacterial traits that contribute to disease are termed “virulence factors” and there is much interest in therapeutic approaches that disrupt such traits. What remains less clear is whether a virulence factor identified as such in a particular context is also important in infections involving different host and pathogen types. Here, we address this question using a meta-analytic approach. We statistically analyzed the infection outcomes of 81 experiments associated with one well-studied virulence factor—pyoverdine, an iron-scavenging compound secreted by the opportunistic pathogen Pseudomonas aeruginosa. We found that this factor is consistently involved with virulence across different infection contexts. However, the magnitude of the effect of pyoverdine on virulence varied considerably. Moreover, its effect on virulence was relatively minor in many cases, suggesting that pyoverdine is not indispensable in infections. Our works supports theoretical models from ecology predicting that disease severity is multifactorial and context dependent, a fact that might complicate our efforts to identify the most important virulence factors. More generally, our study highlights how comparative approaches can be used to quantify the magnitude and general importance of virulence factors, key knowledge informing future anti-virulence treatment strategies.

Construction of a Multiplex Promoter Reporter Platform to Monitor Staphylococcus aureus Virulence Gene Expression and the Identification of Usnic Acid as a Potent Suppressor of psm Gene Expression.

As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm.

Bacterial fitness shapes the population dynamics of antibiotic-resistant and -susceptible bacteria in a model of combined antibiotic and anti-virulence treatment.

Bacterial resistance to antibiotic treatment is a huge concern: introduction of any new antibiotic is shortly followed by the emergence of resistant bacterial isolates in the clinic. This issue is compounded by a severe lack of new antibiotics reaching the market. The significant rise in clinical resistance to antibiotics is especially problematic in nosocomial infections, where already vulnerable patients may fail to respond to treatment, causing even greater health concern. A recent focus has been on the development of anti-virulence drugs as a second line of defence in the treatment of antibiotic-resistant infections. This treatment, which weakens bacteria by reducing their virulence rather than killing them, should allow infections to be cleared through the body׳s natural defence mechanisms. In this way there should be little to no selective pressure exerted on the organism and, as such, a predominantly resistant population should be less likely to emerge. However, before the likelihood of resistance to these novel drugs emerging can be predicted, we must first establish whether such drugs can actually be effective. Many believe that anti-virulence drugs would not be powerful enough to clear existing infections, restricting their potential application to prophylaxis. We have developed a mathematical model that provides a theoretical framework to reveal the circumstances under which anti-virulence drugs may or may not be successful. We demonstrate that by harnessing and combining the advantages of antibiotics with those provided by anti-virulence drugs, given infection-specific parameters, it is possible to identify treatment strategies that would efficiently clear bacterial infections, while preventing the emergence of antibiotic-resistant subpopulations. Our findings strongly support the continuation of research into anti-virulence drugs and demonstrate that their applicability may reach beyond infection prevention.