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Pyocyanin-dependent electrochemical inhibition of biofilms is synergistic with antibiotic treatment.MBio Aug 2023biofilms are common in chronic wound infections and recalcitrant to treatment. Survival of cells within oxygen-limited regions in these biofilms is enabled by...
biofilms are common in chronic wound infections and recalcitrant to treatment. Survival of cells within oxygen-limited regions in these biofilms is enabled by extracellular electron transfer (EET), whereby small redox active molecules act as electron shuttles to access distal oxidants. Here, we report that electrochemically controlling the redox state of these electron shuttles, specifically pyocyanin (PYO), can impact cell survival within anaerobic biofilms and can act synergistically with antibiotic treatment. Prior results demonstrated that under anoxic conditions, an electrode poised at sufficiently oxidizing potential (+100 mV vs Ag/AgCl) promotes EET within biofilms by re-oxidizing PYO for reuse by the cells. Here, when a reducing potential (-400 mV vs Ag/AgCl) was used to disrupt PYO redox cycling by maintaining PYO in the reduced state, we observed a 100-fold decrease in colony forming units within these biofilms compared with those exposed to electrodes poised at +100 mV vs Ag/AgCl. Phenazine-deficient Δ* biofilms were unaffected by the potential applied to the electrode but were re-sensitized by adding PYO. The effect at -400 mV was exacerbated when biofilms were treated with sub-MICs of a range of antibiotics. Most notably, addition of the aminoglycoside gentamicin in a reductive environment almost completely eradicated wild-type biofilms but had no effect on the survival of Δ* biofilms in the absence of phenazines. These data suggest that antibiotic treatment combined with the electrochemical disruption of PYO redox cycling, either through the toxicity of accumulated reduced PYO or the disruption of EET, or both, can lead to extensive killing. IMPORTANCE Biofilms provide a protective environment but also present challenges to the cells living within them, such as overcoming nutrient and oxygen diffusion limitations. overcomes oxygen limitation by secreting soluble redox active phenazines, which act as electron shuttles to distal oxygen. Here, we show that electrochemically blocking the re-oxidation of one of these electron shuttles, pyocyanin, decreases cell survival within biofilms and acts synergistically with gentamicin to kill cells. Our results highlight the importance of the role that the redox cycling of electron shuttles fulfills within biofilms.
Topics: Biofilms; Pyocyanine; Pseudomonas aeruginosa; Anti-Bacterial Agents; Oxidation-Reduction; Electron Transport; Electrochemical Techniques
PubMed: 37314185
DOI: 10.1128/mbio.00702-23 -
BioMed Research International 2018is a major opportunistic human pathogen, responsible for nosocomial infections and infections in patients with impaired immune systems. Little data exist about the...
is a major opportunistic human pathogen, responsible for nosocomial infections and infections in patients with impaired immune systems. Little data exist about the faecal colonisation by isolates in healthy humans. The occurrence, antimicrobial resistance phenotype, virulence genotype, and genetic lineages of from faecal samples of children from two different Spanish regions were characterised. Seventy-two were isolated from 1,443 faecal samples. Low antimicrobial resistance levels were detected: ceftazidime (8%), cefepime (7%), aztreonam (7%), gentamicin (3%), ciprofloxacin (1%), and imipenem (1%); susceptibility to meropenem, amikacin, tobramycin, levofloxacin, and colistin. Four multidrug-resistant strains were found. Important differences were detected between both geographical regions. Forty-one sequence types were detected among the 48 tested strains. Virulence and quorum sensing genes were analysed and 13 virulotypes were detected, being 26 -positive strains. Alteration in protein OprD showed eight different patterns. The unique imipenem-resistant strain showed a premature stop codon in OprD. Intestinal colonisation by , mainly by international clones (as ST244, ST253, and ST274), is an important factor for the systemic infections development and the environmental dissemination. Periodic active surveillance is useful to identify these community human reservoirs and to control the evolution of antibiotic resistance and virulence activity.
Topics: Adolescent; Anti-Bacterial Agents; Child; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Humans; Microbial Sensitivity Tests; Molecular Typing; Pseudomonas Infections; Pseudomonas aeruginosa; Spain; Virulence
PubMed: 29992165
DOI: 10.1155/2018/8060178 -
Journal of Infection in Developing... May 2019Pseudomonas aeruginosa is the second most prevalent opportunistic pathogen causing nosocomial infections in Mexico. This study evaluated antibiotic resistance,...
INTRODUCTION
Pseudomonas aeruginosa is the second most prevalent opportunistic pathogen causing nosocomial infections in Mexico. This study evaluated antibiotic resistance, production of virulence factors and clonal diversity of P. aeruginosa strains isolated from patients undergoing nosocomial infections in public hospitals of northeastern Mexico.
METHODOLOGY
Ninety-two P. aeruginosa isolates from urine culture, Foley catheter, ear, wounds, respiratory tract secretions, scalp, blood culture, bronchoalveolar lavage, expectoration and cerebrospinal fluid causing nosocomial infections were analyzed. The isolates were identified by MALDI-TOF and antibiotic resistance profiles obtained by MicroScan®. The production of virulence factors was analyzed with spectrophotometric techniques and isolates genotyped by ERIC-PCR.
RESULTS
Out of the 92 isolates, 26 (28.2%) were found to be multidrug resistant (MDR); 21 (22.7%) were classified as extremely drug resistant (XDR). Highest resistance rate was found for gatifloxacin (42%) while ciprofloxacin accounted for the antibiotic with the lowest resistance rate (2%). Bronchoalveolar lavage isolates produced the highest amounts of virulence factors: biofilm (44.4% ± 2.7%), elastase (58.5% ± 4.3%), alkaline protease (60.1% ± 5.0%); except for pyocyanin production. The ERIC-PCR assay showed 83 genetic patterns (90% clonal diversity) and 13 isolates had 100% genetic similarity, forming 4 real clones, 3 of these clones were obtained from different anatomical site and/or hospital.
CONCLUSIONS
Antibiotic resistance and virulence factors production was heterogeneous among samples analyzed. Genotyping of P. aeruginosa strains showed high genetic diversity in the studied isolates.
Topics: Anti-Bacterial Agents; Cross Infection; Drug Resistance, Bacterial; Genotype; Hospitals, Public; Humans; Mexico; Microbial Sensitivity Tests; Molecular Typing; Polymerase Chain Reaction; Pseudomonas aeruginosa; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Virulence Factors
PubMed: 32053506
DOI: 10.3855/jidc.10953 -
Applied and Environmental Microbiology Jan 2021is a Gram-negative opportunistic pathogen that undergoes swarming motility in response to semisolid conditions with amino acids as a nitrogen source. With a genome...
is a Gram-negative opportunistic pathogen that undergoes swarming motility in response to semisolid conditions with amino acids as a nitrogen source. With a genome encoding hundreds of potential intergenic small RNAs (sRNAs), can easily adapt to different conditions and stresses. We previously identified 20 sRNAs that were differentially expressed (DE) under swarming conditions. Here, these sRNAs were overexpressed in strain PAO1 and were subjected to an array of phenotypic screens. Overexpression of the PrrH sRNA resulted in decreased swimming motility, whereas a Δ mutant had decreased cytotoxicity and increased pyoverdine production. Overexpression of the previously uncharacterized PA2952.1 sRNA resulted in decreased swarming and swimming motilities, increased gentamicin and tobramycin resistance under swarming conditions, and increased trimethoprim susceptibility. Transcriptome sequencing (RNA-Seq) and proteomic analysis were performed on the wild type (WT) overexpressing PA2952.1 compared to the empty vector control under swarming conditions, and these revealed the differential expression (absolute fold change [FC] ≥ 1.5) of 784 genes and the differential abundance (absolute FC ≥ 1.25) of 59 proteins. Among these were found 73 transcriptional regulators, two-component systems, and sigma and anti-sigma factors. Downstream effectors included downregulated pilus and flagellar genes, the upregulated efflux pump MexGHI-OpmD, and the upregulated operon. Genes involved in iron and zinc uptake were generally upregulated, and certain pyoverdine genes were upregulated. Overall, the sRNAs PA2952.1 and PrrH appeared to be involved in regulating virulence-related programs in , including iron acquisition and motility. Due to the rising incidence of multidrug-resistant (MDR) strains and the difficulty of eliminating infections, it is important to understand the regulatory mechanisms that allow this bacterium to adapt to and thrive under a variety of conditions. Small RNAs (sRNAs) are one regulatory mechanism that allows bacteria to change the amount of protein synthesized. In this study, we overexpressed 20 different sRNAs in order to investigate how this might affect different bacterial behaviors. We found that one of the sRNAs, PrrH, played a role in swimming motility and virulence phenotypes, indicating a potentially important role in clinical infections. Another sRNA, PA2952.1, affected other clinically relevant phenotypes, including motility and antibiotic resistance. RNA-Seq and proteomics of the strain overexpressing PA2952.1 revealed the differential expression of 784 genes and 59 proteins, with a total of 73 regulatory factors. This substantial dysregulation indicates an important role for the sRNA PA2952.1.
Topics: Bacterial Proteins; Cell Line; Cell Survival; Genes, Bacterial; Humans; Iron; Proteomics; Pseudomonas aeruginosa; RNA, Bacterial; Virulence; Zinc
PubMed: 33158897
DOI: 10.1128/AEM.02182-20 -
Microbiology Spectrum Oct 2021Pseudomonas aeruginosa, a human pathogen, causes both acute and chronic infections that are mediated by virulence factor production and biofilm formation. Since both...
Pseudomonas aeruginosa, a human pathogen, causes both acute and chronic infections that are mediated by virulence factor production and biofilm formation. Since both characteristics of P. aeruginosa are regulated by quorum sensing (QS), we screened 126 synthetic chemicals for anti-QS activity and finally selected the compounds that have both antivirulence and antibiofilm activities. To efficiently screen the chemical library, the following reporter-based bioassay systems were used: the QS- or biofilm-specific promoter- fusions (- or - for the QS activity and - for measuring the intracellular c-di-GMP levels). We also measured the production of virulence factors and biofilm formation in P. aeruginosa. A small-animal infection model using mealworms was also used for virulence analysis. From this screening, MHY1383 and MHY1387 were found to have both antivirulence and antibiofilm activities in P. aeruginosa. Most importantly, MHY1383 and MHY1387 exhibited these activities at very low concentrations, showing a significant anti-QS effect at 100 pM and an antibiofilm effect at 1 to 10 pM. By treating P. aeruginosa with these compounds, the virulence factor production and biofilm formation of P. aeruginosa were significantly reduced. These compounds can be developed as promising antipathogenic and antibiofilm drugs that can be applied in situations where such compounds must be used in an extremely low concentration. Our findings also offer a significant advantage for developing therapeutic agents with few adverse side effects. Many antibiotics are increasingly losing their efficacy due to antibiotic resistance mediated by biofilm formation. In this study, we screened a synthetic chemical library and discovered several compounds that have both antivirulence and antibiofilm effects against Pseudomonas aeruginosa, a notorious human pathogen. Two of them had these effects at extremely low concentrations and are expected not to develop resistance, unlike conventional antibiotics, because they have no effect on the growth of bacteria. Our results strongly suggest that these compounds act on the target in a noncompetitive manner, indicating that they are distinct from other previously known quorum sensing inhibitors or biofilm inhibitors. Our findings offer a significant advantage for developing therapeutic agents with few adverse side effects.
Topics: Animals; Anti-Bacterial Agents; Biofilms; Drug Evaluation, Preclinical; Pseudomonas Infections; Pseudomonas aeruginosa; Quorum Sensing; Tenebrio; Virulence; Virulence Factors
PubMed: 34494853
DOI: 10.1128/Spectrum.00249-21 -
Scientific Reports Jun 2018Pseudomonas aeruginosa evolves during chronic pulmonary infections of cystic fibrosis (CF) patients, forming pathoadapted variants that are persistent. Mucoid and rugose...
Pseudomonas aeruginosa evolves during chronic pulmonary infections of cystic fibrosis (CF) patients, forming pathoadapted variants that are persistent. Mucoid and rugose small-colony variants (RSCVs) are typically isolated from sputum of CF patients. These variants overproduce exopolysaccharides in the biofilm extracellular polymeric substance (EPS). Currently, changes to the biophysical properties of RSCV and mucoid biofilms due to variations in EPS are not well understood. This knowledge may reveal how lung infections resist host clearance mechanisms. Here, we used mechanical indentation and shear rheometry to analyse the viscoelasticity of RSCV and mucoid colony-biofilms compared to their isogenic parent at 2-, 4-, and 6-d. While the viscoelasticity of parental colony-biofilms underwent fluctuating temporal changes, in contrast, RSCV and mucoid colony-biofilms showed a gradual progression to more elastic-solid behaviour. Theoretical indices of mucociliary and cough clearance predict that mature 6-d parental and RSCV biofilms may show reduced cough clearance from the lung, while early mucoid biofilms may show reduced clearance by both mechanisms. We propose that viscoelasticity be considered a virulence property of biofilms.
Topics: Biofilms; Elasticity; Extracellular Polymeric Substance Matrix; Polysaccharides, Bacterial; Pseudomonas aeruginosa
PubMed: 29946126
DOI: 10.1038/s41598-018-28009-5 -
Current Opinion in Microbiology Dec 2015Pseudomonas aeruginosa navigates using two distinct forms of motility, swimming and twitching. A polar flagellum and Type 4 pili power these movements, respectively,... (Review)
Review
Pseudomonas aeruginosa navigates using two distinct forms of motility, swimming and twitching. A polar flagellum and Type 4 pili power these movements, respectively, allowing P. aeruginosa to attach to and colonize surfaces. Single cell imaging and particle tracking algorithms have revealed a wide range of bacterial surface behaviors which are regulated by second messengers cyclic-di-GMP and cAMP; the production of these signals is, in turn, responsive to the engagement of motility organelles with a surface. Innate immune defense systems, long known to recognize structural components of flagella, appear to respond to motility itself. The association of motility with both upregulation of virulence and induction of host defense mechanisms underlies the complex contributions of flagella and pili to P. aeruginosa pathogenesis.
Topics: Animals; Bacterial Proteins; Fimbriae, Bacterial; Flagella; Gene Expression Regulation, Bacterial; Humans; Immunity, Innate; Mice; Movement; Pseudomonas aeruginosa; Virulence
PubMed: 26476804
DOI: 10.1016/j.mib.2015.07.017 -
Antimicrobial Agents and Chemotherapy Nov 2017is a major causative agent of both acute and chronic infections. Although aminoglycoside antibiotics are very potent drugs against such infections, antibiotic failure...
is a major causative agent of both acute and chronic infections. Although aminoglycoside antibiotics are very potent drugs against such infections, antibiotic failure is steadily increasing mainly because of increasing resistance of the bacteria. Many molecular mechanisms that determine resistance, such as acquisition of genes encoding aminoglycoside-inactivating enzymes or overexpression of efflux pumps, have been elucidated. However, there are additional, less well-described mechanisms of aminoglycoside resistance. In this study, we profiled a clinical tobramycin-resistant strain that exhibited a small-colony variant (SCV) phenotype. Both the resistance and colony morphology phenotypes were lost upon passage of the isolate under rich medium conditions. Transcriptional and mutational profiling revealed that the SCV harbored activating mutations in the two-component systems AmgRS and PmrAB. Introduction of these mutations individually into type strain PA14 conferred tobramycin and colistin resistance, respectively. However, their combined introduction had an additive effect on the tobramycin resistance phenotype. Activation of the AmgRS system slightly reduced the colony size of wild-type PA14, whereas the simultaneous overexpression of , the response regulator of the GacSA two-component system, further reduced colony size. In conclusion, we uncovered combinatorial influences of two-component systems on clinically relevant phenotypes such as resistance and the expression of the SCV phenotype. Our results clearly demonstrate that the combined activation of two-component systems has pleiotropic effects with unforeseen consequences.
Topics: Aminoglycosides; Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Humans; Mutation; Phenotype; Pseudomonas aeruginosa; Tobramycin
PubMed: 28874369
DOI: 10.1128/AAC.01178-17 -
Scientific Reports Jun 2020To assess the role of core metabolism genes in bacterial virulence - independently of their effect on growth - we correlated the genome, the transcriptome and the...
To assess the role of core metabolism genes in bacterial virulence - independently of their effect on growth - we correlated the genome, the transcriptome and the pathogenicity in flies and mice of 30 fully sequenced Pseudomonas strains. Gene presence correlates robustly with pathogenicity differences among all Pseudomonas species, but not among the P. aeruginosa strains. However, gene expression differences are evident between highly and lowly pathogenic P. aeruginosa strains in multiple virulence factors and a few metabolism genes. Moreover, 16.5%, a noticeable fraction of the core metabolism genes of P. aeruginosa strain PA14 (compared to 8.5% of the non-metabolic genes tested), appear necessary for full virulence when mutated. Most of these virulence-defective core metabolism mutants are compromised in at least one key virulence mechanism independently of auxotrophy. A pathway level analysis of PA14 core metabolism, uncovers beta-oxidation and the biosynthesis of amino-acids, succinate, citramalate, and chorismate to be important for full virulence. Strikingly, the relative expression among P. aeruginosa strains of genes belonging in these metabolic pathways is indicative of their pathogenicity. Thus, P. aeruginosa strain-to-strain virulence variation, remains largely obscure at the genome level, but can be dissected at the pathway level via functional transcriptomics of core metabolism.
Topics: Animals; Gene Expression Regulation, Bacterial; Genes, Bacterial; Host-Pathogen Interactions; Male; Mutation; Pseudomonas aeruginosa; Virulence
PubMed: 32528034
DOI: 10.1038/s41598-020-66194-4 -
Proceedings of the National Academy of... Feb 2020When nutrients in their environment are exhausted, bacterial cells become arrested for growth. During these periods, a primary challenge is maintaining cellular...
When nutrients in their environment are exhausted, bacterial cells become arrested for growth. During these periods, a primary challenge is maintaining cellular integrity with a reduced capacity for renewal or repair. Here, we show that the heat-shock protease FtsH is generally required for growth arrest survival of , and that this requirement is independent of a role in regulating lipopolysaccharide synthesis, as has been suggested for We find that interacts with diverse genes during growth and overlaps functionally with the other heat-shock protease-encoding genes , , and to promote survival during growth arrest. Systematic deletion of the heat-shock protease-encoding genes reveals that the proteases function hierarchically during growth arrest, with FtsH and ClpXP having primary, nonredundant roles, and HslVU and Lon deploying a secondary response to aging stress. This hierarchy is partially conserved during growth at high temperature and alkaline pH, suggesting that heat, pH, and growth arrest effectively impose a similar type of proteostatic stress at the cellular level. In support of this inference, heat and growth arrest act synergistically to kill cells, and protein aggregation appears to occur more rapidly in protease mutants during growth arrest and correlates with the onset of cell death. Our findings suggest that protein aggregation is a major driver of aging and cell death during growth arrest, and that coordinated activity of the heat-shock response is required to ensure ongoing protein quality control in the absence of growth.
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Hot Temperature; Hydrogen-Ion Concentration; Microbial Viability; Peptide Hydrolases; Pseudomonas aeruginosa
PubMed: 32029587
DOI: 10.1073/pnas.1912082117