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1. Altering the interfacial rheology of Pseudomonas aeruginosa and Staphylococcus aureus with N-acetyl cysteine and cysteamine

   https://doi.org/10.3389/fcimb.2023.1338477  

   Balmuri, Sricharani Rao; Noaman, Sena; Usman, Huda; Niepa, Tagbo H. (January 2024, Frontiers in Cellular and Infection Microbiology)

   IntroductionChronic lung infection due to bacterial biofilms is one of the leading causes of mortality in cystic fibrosis (CF) patients. Among many species colonizing the lung airways,Pseudomonas aeruginosaandStaphylococcus aureusare two virulent pathogens involved in mechanically robust biofilms that are difficult to eradicate using airway clearance techniques like lung lavage. To remove such biological materials, glycoside hydrolase-based compounds are commonly employed for targeting and breaking down the biofilm matrix, and subsequently increasing cell susceptibility to antibiotics. Materials and methodsIn this study, we evaluate the effects of N-acetyl cysteine (NAC) and Cysteamine (CYST) in disrupting interfacial bacterial films, targeting different components of the extracellular polymeric substances (EPS). We characterize the mechanics and structural integrity of the interfacial bacterial films using pendant drop elastometry and scanning electron microscopy. Results and discussionOur results show that the film architectures are compromised by treatment with disrupting agents for 6 h, which reduces film elasticity significantly. These effects are profound in the wild type and mucoidP. aeruginosa, compared toS. aureus. We further assess the effects of competition and cooperation betweenS. aureusandP. aeruginosaon the mechanics of composite interfacial films. Films ofS. aureusand wild-typeP. aeruginosacocultures lose mechanical strength while those ofS. aureusand mucoidP. aeruginosaexhibit improved storage modulus. Treatment with NAC and CYST reduces the elastic property of both composite films, owing to the drugs’ ability to disintegrate their EPS matrix. Overall, our results provide new insights into methods for assessing the efficacy of mucolytic agents against interfacial biofilms relevant to cystic fibrosis infection. 

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2. High prevalence of lipopolysaccharide mutants and R2-pyocin susceptible variants in Pseudomonas aeruginosa populations sourced from cystic fibrosis lung infections

   https://doi.org/10.1128/spectrum.01773-23  

   Mei, Madeline; Pheng, Preston; Kurzeja-Edwards, Detriana; Diggle, Stephen P (December 2023, Microbiology Spectrum)

   Van_Tyne, Daria (Ed.)

   ABSTRACT Chronic, highly antibiotic-resistant infections in cystic fibrosis (CF) lungs contribute to increasing morbidity and mortality.Pseudomonas aeruginosa, a common CF pathogen, exhibits resistance to multiple antibiotics, contributing to antimicrobial resistance (AMR). These bacterial populations display genetic and phenotypic diversity, but it is unclear how this diversity affects susceptibility to bacteriocins. R-pyocins, i.e., bacteriocins produced byP. aeruginosa, are phage-tail-like antimicrobials. R-pyocins have potential as antimicrobials, however, recent research suggests the diversity ofP. aeruginosavariants within CF lung infections leads to varying susceptibility to R-pyocins. This variation may be linked to changes in lipopolysaccharide (LPS), acting as the R-pyocin receptor. Currently, it is unknown how frequently R-pyocin-susceptible strains are in chronic CF lung infection, particularly when considering the heterogeneity within these strains. In this study, we tested the R2-pyocin susceptibility of 139P.aeruginosavariants from 17 sputum samples of 7 CF patients and analyzed LPS phenotypes. We found that 83% of sputum samples did not have R2-pyocin-resistant variants, while nearly all samples contained susceptible variants. There was no correlation between LPS phenotype and R2-pyocin susceptibility, though we estimate that about 76% of sputum-derived variants lack an O-specific antigen, 40% lack a common antigen, and 24% have altered LPS cores. The absence of a correlation between LPS phenotype and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Our research supports the potential of R-pyocins as therapeutic agents, as many infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations. IMPORTANCECystic fibrosis (CF) patients often experience chronic, debilitating lung infections caused by antibiotic-resistantPseudomonas aeruginosa, contributing to antimicrobial resistance (AMR). The genetic and phenotypic diversity ofP. aeruginosapopulations in CF lungs raises questions about their susceptibility to non-traditional antimicrobials, like bacteriocins. In this study, we focused on R-pyocins, a type of bacteriocin with high potency and a narrow killing spectrum. Our findings indicate that a large number of infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations, supporting their potential use as therapeutic agents. The absence of a clear correlation between lipopolysaccharide (LPS) phenotypes and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Understanding the relationship between LPS phenotypes and R-pyocin susceptibility is crucial for developing effective treatments for these chronic infections. 

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3. Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa

   https://doi.org/10.1073/pnas.2105370118  

   Koeppen, Katja; Nymon, Amanda; Barnaby, Roxanna; Bashor, Laura; Li, Zhongyou; Hampton, Thomas H.; Liefeld, Amanda E.; Kolling, Fred W.; LaCroix, Ian S.; Gerber, Scott A.; et al (July 2021, Proceedings of the National Academy of Sciences)

   Pseudomonas aeruginosais an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown thatP. aeruginosasecretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA–containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation byP. aeruginosa. Specifically, human EVs deliver miRNA let-7b-5p toP. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC inP. aeruginosabut also the corresponding orthologs inBurkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited toP. aeruginosa. Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7–family miRNAs are in clinical trials to reduce inflammation and because chronicP. aeruginosalung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistantP. aeruginosainfections. 

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4. Responses to chemical cross-talk between the Mycobacterium ulcerans toxin, mycolactone, and Staphylococcus aureus

   https://doi.org/10.1038/s41598-021-89177-5  

   Dhungel, Laxmi; Burcham, Lindsey; Park, Joo Youn; Sampathkumar, Harshini Devi; Cudjoe, Albert; Seo, Keun Seok; Jordan, Heather (December 2021, Scientific Reports)

   null (Ed.)

   Abstract Buruli ulcer is a neglected tropical disease caused by the environmental pathogen, Mycobacterium ulcerans whose major virulence factor is mycolactone, a lipid cytotoxic molecule. Buruli ulcer has high morbidity, particularly in rural West Africa where the disease is endemic. Data have shown that infected lesions of Buruli ulcer patients can be colonized by quorum sensing bacteria such as Staphylococcus aureus, S. epidermidis, and Pseudomonas aeruginosa , but without typical pathology associated with those pathogens’ colonization. M. ulcerans pathogenesis may not only be an individual act but may also be dependent on synergistic or antagonistic mechanisms within a polymicrobial network. Furthermore, co-colonization by these pathogens may promote delayed wound healing, especially after the initiation of antibiotic therapy. Hence, it is important to understand the interaction of M. ulcerans with other bacteria encountered during skin infection. We added mycolactone to S. aureus and incubated for 3, 6 and 24 h. At each timepoint, S. aureus growth and hemolytic activity was measured, and RNA was isolated to measure virulence gene expression through qPCR and RNASeq analyses. Results showed that mycolactone reduced S. aureus hemolytic activity, suppressed hla promoter activity, and attenuated virulence genes, but did not affect S. aureus growth . RNASeq data showed mycolactone greatly impacted S. aureus metabolism. These data are relevant and significant as mycolactone and S. aureus sensing and response at the transcriptional, translational and regulation levels will provide insight into biological mechanisms of interspecific interactions that may play a role in regulation of responses such as effects between M. ulcerans , mycolactone, and S. aureus virulence that will be useful for treatment and prevention. 

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5. Contribution of Pseudomonas aeruginosa Exopolysaccharides Pel and Psl to Wound Infections

   https://doi.org/10.3389/fcimb.2022.835754  

   Fleming, Derek; Niese, Brandon; Redman, Whitni; Vanderpool, Emily; Gordon, Vernita; Rumbaugh, Kendra P. (April 2022, Frontiers in Cellular and Infection Microbiology)

   Biofilms are the cause of most chronic bacterial infections. Living within the biofilm matrix, which is made of extracellular substances, including polysaccharides, proteins, eDNA, lipids and other molecules, provides microorganisms protection from antimicrobials and the host immune response. Exopolysaccharides are major structural components of bacterial biofilms and are thought to be vital to numerous aspects of biofilm formation and persistence, including adherence to surfaces, coherence with other biofilm-associated cells, mechanical stability, protection against desiccation, binding of enzymes, and nutrient acquisition and storage, as well as protection against antimicrobials, host immune cells and molecules, and environmental stressors. However, the contribution of specific exopolysaccharide types to the pathogenesis of biofilm infection is not well understood. In this study we examined whether the absence of the two main exopolysaccharides produced by the biofilm former Pseudomonas aeruginosa would affect wound infection in a mouse model. Using P. aeruginosa mutants that do not produce the exopolysaccharides Pel and/or Psl we observed that the severity of wound infections was not grossly affected; both the bacterial load in the wounds and the wound closure rates were unchanged. However, the size and spatial distribution of biofilm aggregates in the wound tissue were significantly different when Pel and Psl were not produced, and the ability of the mutants to survive antibiotic treatment was also impaired. Taken together, our data suggest that while the production of Pel and Psl do not appear to affect P. aeruginosa pathogenesis in mouse wound infections, they may have an important implication for bacterial persistence in vivo. 

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